Secretory Immunoglobulins1

Secretory Immunoglobulins’ T H O M A S B . TOMASI. JR., AND J O H N BIENENSTOCK State Universify of New York. Buffalo. New York

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I I1 111 IV

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Introduction . . . . . . . . . . . . . Historical Aspects of Secretory Immunoglobulins and Local Immunity Evidence for the Existence of a Secretory Immunoglobulin System . Technical Problems Encountered in the Analysis of Secretory . . . . . . . . . . . Inununoglobulins . Secretions Involved in the Secretory Immunoglobulin System . . A . Digestive Tract . . . . . . . . . . . B. Respiratory Tract . . . . . . . . . . . C . Genital Tract . . . . . . . . . . . D. Urinary Tract . . . . . . . . . . . E . Mammary Gland . . . . . . . . . . . Chemical and Immunological Characteristics of Secretory . . . . . . . . . . . Immunoglobulins . A. Isolation . . . . . . . . . . . . B . Immunological Properties . . . . . . . . . C. Chemical Properties of Secretory Immunoglobulins . . . D. Three-Dimensional Conformation . . . . . . . E . Isolation and Characteristics of the Secretory “Piece” . . . Sites of Synthesis of Secretory Immunoglobulins . . . . . A. Immunological Studies . . . . . . . . . B Immunofluorescent Studies . . . . . . . . . C. In Vioo Radioactive Tracer Studies . . . . . . . D. Tissue Culture . . . . . . . . . . . E . Possible Mechanisms of Secretion of yA . . . . . . Biological Properties of Secretory yA . . . . . . . A. “Natural” Secretory Antibodies . . . . . . . . B . Secretory Antibodies Following Immunization . . . . C . Complement Fixation . . . . . . . . . . Secretory Immunoglobulins in Disease . . . . . . . A. Antibody Deficiency States . . . . . . . . B. Gastrointestinal Diseases . . . . . . . . . C. Rheumatoid and Antinuclear Factors . . . . . . D. Secretory Immunoglobulins in Respiratory Allergies . . . Secretory Immunoglobulins in Animals . . . . . . . A. Rabbit . . . . . . . . . . . . . R . Cow and Sheep . . . . . . . . . . . C . Dog . . . . . . . . . . . . .

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by U S . Public Health Service Grant AM 10419.

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

D. Mouse . E. Conclusions XI. Summary . References .

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Introduction

Early work in immunology was devoted in large part to investigations involving the serological responses of man and animals to infections, and immunization with a variety of antigens. This serological emphasis has to some extent persisted so that serum titers of antibody are commonly considered to be representative of the efficacy of immunization. Although in some cases there appears to be a good correlation between serum antibody and resistance to infection, certain observations, which will be reviewed later in some detail, suggest that factors other than the level of serum antibody may be involved in recovery from and resistance to infections. These observations, as well as others, have suggested the concept of a local immune response and its possible importance in the resistance of the organism to infection. This review will attempt to summarize current knowledge concerning the antibodies found in various nonvascular secretions and their potential relationship to local immunity. Attention will, on the whole, be given to those secretions in which some characterization of antibodies involved has been undertaken. Although an enormous body of literature now exists concerning various antibacterial properties of whole secretions, in many cases the antibacterial activity has not been clearly shown to be due to antibody, and these reports will not be reviewed here. II.

Historical Aspects of Secretory Immunoglobulins and local Immunity

Besredka, in 1919, as a result of studies on experimentally induced oral infections by enterobacteriaceae and skin infections with Bucillus unthrucis, postulated that local immunity could be established independently of systemic immunity and that this was of great importance in the general resistance of the organism to infections originating in the gastrointestinal (GI) tract and skin. Davies, in 1922, demonstrated that specific antibodies could be found in the feces of patients suffering from dysentery, and similar findings on experimentally induced infections in animals suggested an important protective role for local GI immunity and fecal antibody in infections. In the late 1940’s, Burrows and coworkers (1947; Burrows and Havens, 1948) in a classic series of experiments investigating the effect of cholera vaccine in guinea pigs,

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3

demonstrated a correlation between local fecal antibody ( coproantibody) and protection against experimental infection. They were unable, however, to find a good correlation between serum antibody titers and resistance to oral infection. Those animals having high serum titers following parenteral immunization showed systemic immunity as demonstrated by resistance to intracerebral challenge with the organism but were not resistant to oral infection unless coproantibody was present. In addition, these investigators showed that coproantibody and urinary antibody titers appeared to be independent of serum titers and, therefore, were probably not derived from serum directly by simple transudation. The independence of serum and local antibody is well demonstrated in Fig. 1. Studies in irradiated animals further highlighted the independent behavior of serum and fecal antibodies by demonstrating that following an appropriate dose of irradiation, fecal antibody response to cholera vaccine was inhibited whereas serum antibody levels remained essentially unchanged (Burrows et al., 1950a,b). Antibody mediated immunity in saliva was demonstrated by the presence of diphtheria antitoxin in the saliva both of naturally immune individuals and following immunization with toxoid (Sugg and Neill, 1931). In this report it was concluded that the antitoxin concentration in saliva was directly related to the concentration in the blood. Schubert ( 1938), however, in systematic studies involving both man and rabbits, concluded that the salivary antitoxin was independent of that in the serum as shown in Fig. 1. Moreover, Schubert (1938) found that the concentration of antitoxin in the saliva was of suEcient magnitude to be of potential importance in immunity against diphtheria and that the presence of antitoxin in human saliva correlated well with the Schick test-only Schick-negative individuals had significant concentrations of salivary antitoxin. Bull and McKee, in 1929, called attention to the possible participation of local antibody (mucoantibody) in the resistance of the respiratory tract to infection by reporting that rabbits could be rendered resistant to pneumococcal respiratory infections by prior intranasal immunization with the same strain. Resistance occurred in some animals in whom serum antibody could not be detected, and these authors concluded that this constituted “another instance of the resistance of tissues apparently local to invasion of bacteria in the absence of demonstrable antibodies in the blood serum.” Walsh and Cannon (1936, 1933) confirmed these findings and, in addition, showed that animals in whom serum antibody could not be detected, nevertheless, had acquired significant immunity. Extensive studies on the origin of bronchial mucoanti-

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THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCK

r 200

20001

a w

1500-

'150

z z

2

I-

1000- FECAL

'100

500-

-50 J

3

-I

w

LL

I IP

I

2

O t

IP

6

DAYS

SERUM/ I

+

TOXOID

{ TOXOID

0

I

4

5

I0

I5

20

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25

60

DAYS

FIG. 1. Studies illustrating independence of serum and secretory antibody titers. (Top) Serum and fecal cholera agglutinins in guinea pigs; (bottom) Diphtheria antitoxin content of serum and saliva of immunized rabbits. [Cholera data redrawn from Burrows et al. (1950a); saliva data redrawn from Schubert (1938).]

bodies have been carried out by Fazekas de St. Groth and his colleagues (1951; Fazekas de St. Groth, 1951) on experimentally induced influenza virus infection in mice. These workers showed that following intranasal instillation of the virus there was a simultaneous appearance of antibody in both serum and mucus and that the proportion of mucoantibody to serum antibody was over 10 times that produced by subcutaneous immunization. By the intranasal inoculation of an unrelated virus or

SECRETORY IMMUNOGLOBULINS

5

certain astringents (e.g., tannic acid), a high proportion of bronchial antibody could also be induced in mice vaccinated via the intraperitoneal route. This phenomenon called “pathotopic potentiation” is thought to result from a local increasc in capillary or tissue permeability caused by the inflammatory effect of these agents. This important work emphasized that a rise in mucoantibody titer may reflect either local antibody production or the extravasation of humoral antibody due to alterations in capillary permeability. There has been great interest in veterinary medicine in regard to the diagnostic significance of antibodies which appear in the secretions of the reproductive tract. Most extensively investigated have been local immunity to the three major infectious diseases causing infertility in cattle (trichomoniasis, vibriosis, and brucellosis). Byrne and Nelson (1939) showed in a study of experimental trichomoniasis in rabbits that acquired resistance to reinfection did not coincide with circulating antibody and that intravenous immunization, which was followed by the appearance of humoral antibody, did not confer immunity. Similarly, in cattle, antibodies to Trichomonas foetus in vaginal mucin ( mucoantibodies ) occur in the absence of circulating antibodies, whereas parenteral administration of antigen produced circulating but no mucoantibodies (Kerr and Robertson, 1953). In the studies of Kerr ( 1955) Brucella abortus antibodies were absent in the vaginal and uterine secretions in animals with circulating titers as high as 1:2560 when immunization was carried out by parenteral administration of antigen. However, the intrauterine instillation of Brucella abortus vaccine resulted in vagina1 titers of 1:280 with serum titers 1 : l O or less. Batty and Warrack ( 1955) using the Oakley technique (Oakley et al., 1955) have also shown the local production of diphtheria and tetanus antibodies in the vagina and uterus of rabbits. The appearance of antibody was correlated with the accumulation of plasma cells in the vaginal mucosa. The studies discussed above, as well as other early reports reviewed in more detail by Pierce ( 1959) and Tomasi ( 1965), have suggested the following. In certain infections, circulating antibody has relatively little significance or is only indirectly related to resistance to infection. Local immunity and mucoantibodies may be of primary importance in the defense against certain infections particularly those which are noninvasive and confhed to the mucous surfaces. In some cases, mucoantibodies may have a diagnostic significance which is superior to that of serum antibodies. The effectiveness of immunization may not always be assayed by measurement of humoral antibody.

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

More recent developments in the concept of local immunity and the existence of a secretory system have depended on the recognition of the heterogeneity of antibodies and the definition of the various immunoglobulin classes (for review, see Cohen and Milstein, 1967). Most important was the discovery by Heremans et al. (1959) of the yA class of antibodies which is now known to be the predominant class in external secretions. Gugler et al. (1958) appear to have first described by immunoelectrophoresis the occurrence of yA in the external secretion, colostrum. The systematic studies of Hanson (1961) clearly demonstrated the complexity of the immunoglobulins in human colostrum and the quantitative predominance of yA in this fluid. The observation that many different external secretions contain by quantitative measurement a marked predominance of yA (Chodirker and Tomasi, 1963), together with the fhding of a predominance of yA-containing plasma cells locally in a secretory gland (Tomasi et al., 1965), formed the basis for the suggestion of the existence of a more or less distinct immunological system characteristic of external secretions ( Tomasi et al., 1965). Other observations pertinent to this thesis are reviewed below. 111.

Evidence for the Existence of a Secretory Immunoglobulin System

Secretions can be conveniently classified into two groups-internal and external according to their immunoglobulin content ( Fig. 2). External secretions bathe the mucous membrane surfaces which are in communication with the external environment, such as the secretions of

YM Internal

YG

YA Y M Externol

FIG. 2. Relative concentrations of immunoglobulin classes-serum on left and external secretion on right. In external secretions the ratios of yG/yA vary with different secretions but are generally less than 1. In internal secretions the ratios of yG/yA are similar to that of serum (approx. 6 : l ) . yM is either undetectable or present in small amounts relative to serum. Secretions, characterized by immunoglobulin content, are listed below. Those secretions in which secretory yA was identified are starred. INTERNAL aqueous humor, cerebrospinal fluid, synovial, amniotic, pleural, peritoneal

EXTERNAL 'parotid, 'submaxillary, 'lacrimal, 'nasal, * tracheo-bronchial, 'gastric, 'small intestinal, large intestinal, bile, seminal, 'urine

SECRETORY IMMUNOGLOBULINS

7

the GI and respiratory tracts. Internal secretions have immunoglobulin contents showing a quantitative distribution similar to that seen in normal serum with a ratio of yG/yA of approximately 6: 1. In this group are included the aqueous humor of the eye, cerebrospinal fluid, synovial fluid, and pleural, amniotic, and peritoneal fluid, all of which occur in cavities having no direct continuity with the outside environment. The external secretions are produced by or bathe mucous membranes and are, therefore, mostly related to cuboidal and columnar-type epithelia. In contrast with the internal secretions, the immunoglobulin which predominates in the external secretions is yA with the result that the rG/yA ratio approaches unity and is commonly less than 1. The relative predominance of immunoglobulins in normal external secretions is mainly in the order: yA, yM, yG. However considerable variation in the relative amounts of yM vs. yG are found in different fluids. A major characteristic of all external secretions examined to date has been the predominance of yA in a special secretory form that differs in several of its physicochemical characteristics from the yA of serum. Under external secretions are now included parotid and submaxillary secretions, colostrum, lacrimal fluid, nasal secretions, bronchial secretions, intestinal fluids ( gastric, small and large intestinal), bile, urine, and seminal plasma. Justification for considering the secretory system as more or less distinct from that responsible for the production of circulating antibody is based on evidence which is briefly summarized below; a detailed discussion of the individual points will be Ieft for subsequent sections. The evidence for the existence of the secretory systems stems primarily from the folIowing observations. 1. In certain external secretions such as saliva, the albumin-toglobulin ratio differs markedly from that of serum, suggesting that plasma proteins are not present simply as a result of transudation from serum. 2. In the majority of external secretions thus far studied, yA is the predominant immunoglobulin class ( approximately 60-1W of the total immunoglobulins present), whereas this immunoglobulin is a minor component (10-20%) in serum. 3. The yA of external secretions differs chemically and antigenically from serum yA. 4. There is evidence that in certain external fluids, secretory yA is produced locally. The evidence for this comes primarily (although not solely) from studies involving the fluorescent antibody technique and the demonstration that y A-producing plasma cells predominate in the area just below the mucous membranes of the respiratory and GI tracts.

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THOMAS B. TOMASI, JR. AND JOHN BLENENSTOCK

5. The apparent lack of correlation between the concentration of yA in serum and secretions in certain pathological conditions and during development following birth. 6. The frequent finding of a dissociation between both the levels and class of antibody activity in serum and secretions of healthy individuals and following infections or immunization. Recovery from certain infections, such as viral respiratory diseases, is better correlated with the levels of local antibody than with serum antibody. Likewise, evidence is available that resistance to certain infections following immunization is best correlated with secretory rather than serum antibody.

IV.

Technical Problems Encountered in the Analysis of Secretory Immunoglobulins

The concentration of an immunoglobulin in a secretion is best expressed in milligrams per 100 ml. of unconcentrated fluid. In many secretions the total protein concentration varies widely within the same individual, depending both upon the rate of fluid secretion as well as other factors. It is desirable, therefore, to measure the total protein content of a secretion as well as the volume collected over a given time period. Immunoglobulin concentration can then be compared with the total concentration of protein in the sample, or expressed, as in the case of the urine, as milligrams excreted per 1000 ml. or per 24 hours. Measurement of total protein on whole secretions by most of the commonly used techniques involving colorimetric reactions (such as the Folin or biuret) does not give absolute concentrations because of the technical difficulties encountered with color development ( Kabat and Mayer, 1987) and also because of the standards employed. Since secretions are complex mixtures of proteins, many of which have not been isolated, use of a single standard such as albumin or y-globulin leads to results which are at best relative. A technique based on staining of paper strips, partially avoids some of these difficulties (Heremans, 1958). In some fluids such as bile the inherent color of the fluid prevents the use of colorimetric procedures, and other techniques such as the paper strip or refractive index methods must be used. It is important to realize, therefore, that none of the available methods provide absolute values for the total protein content of a secretion. Most of the more commonly used immunological techniques for quantitating immunoglobulins in secretions are not sufficiently sensitive to be used directly on the unconcentrated fluid and, therefore, preliminary concentration is often necessary. Many of the methods available, such as the collodion bag negative pressure dialysis, pre-evaporation, and

SECRETORY IMMUNOGLOBULINS

9

lyophilization techniques, incur considerable loss at the surface of the membrane or can result in significant denaturation of proteins. Secretory yA antibody activity does, however, at least partially survive lyophilization and myeloma yA preparations can be lyophilized and still retain the majority of their antigenicity. Concentration procedures currently thought to be most effective in dealing with secretions are negative pressure dialysis in Visking s/&-in. membranes or the use of artificial membranes and positive pressure.2 Quantitation of immunoglobulins is most frequently performed by quantitative radial diffusion in which ring diameters obtained with the concentrated fluids (usually 10-50 times) are compared with the diameters produced by purified standards. For quantitation of yA in secretions, it is necessary to use a homogeneous, 11 S, secretory yA as a standard. Marked differences result if a 7 S serum yA standard is used. For example, a salivary specimen, which gives a ring diameter on radial diffusion of 8 mm. when read from an 11S secretory yA standard curve, gives a concentration of 18 mg.% With a serum 7 s yA standard the concentration is 5.4 mg.% Although use of an 11S yA standard is considerably more accurate in view of the fact that this is the predominant molecular size in most external secretions, absolute figures for yA concentrations are not obtained since, as discussed below, many secretions contain 7 S and polymeric (18-20 S ) yA in varying proportions. Complement fixation techniques for measuring yA in large part avoid the problem of size heterogeneity, but other technical problems particularly anticomplementary activity are encountered with certain fluids. A new technique, electroimmunodiffusion, has been successfully applied to the quantitation of immunoglobulins in secretions by Merrill et al. (1967). This procedure also requires an 11S yA standard but has the advantage that it is sufficiently sensitive to be used directly on unconcentrated fluids such as saliva. Accurate quantitation of *the other immunoglobulins such as yG and yM is compIicated by the presence of degradation products of these molecules due to proteolytic activity. For example, GI secretions, urine, as well as other secretions, frequently contain Fc and F’c fragments. Thus, the standard used cannot be representative of the whole range of molecular sizes to be found in these fluids which are still reactive with the antisera. If the standard used has a molecular size corresponding to the largest size to be found in that particular biological fluid, then the

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

concentration obtained can be considered to be representative of the maximum possible amount of the immunoglobulin present. Other technical problems using precipitation techniques involve the presence of nonspecific precipitin lines not due to specific immunological reactions. Gastrointestinal secretions and bile often give several nonspecific rings on radial diffusion plates. If normal serum is diffused against normal concentrated GI juice, two or three precipitin lines can sometimes be detected. Such reactions can cause great difficulty in quantitation and in other studies involving antigenic analysis on these fluids. In studies on biological fluids using antisera raised against the concentrated whole secretion and absorbed with normal human serum, claims have often been made of the demonstration of secretion-specific antigens not found in normal human serum. A major pitfall in this kind of interpretation results if the substance is present in small amounts in serum but in relatively high concentration in the secretion. An example of this is free L chains which can be readily detected in urine since they are selectively concentrated by the kidney, but are very difficult to detect in normal serum, although recent evidence has clearly shown their presence ( Berggard, 1961) . A major problem in immunological studies of the secretory system has been the preparation of specific secretory yA antisera. Preparations of 11s secretory yA which are homogeneous by all criteria applied, including Ouchterlony analysis with potent antiwhole serum and antiwhole secretion antisera, when injected into animals often result in antisera containing antibodies against one or more secretion-specific components. We have examined some twelve different secretory yA antisera and all, except one bleeding from one animal, contained antibodies against contaminants when carefully analyzed against highly concentrated secretions. The most common contaminants are lactoferrin and the macromolecular component ( MMC) shown in Fig. 3. Such antisera, unless properly absorbed to render them specific for secretory yA, are useless for fluorescent and other studies involving sensitive techniques (such as complement hation) for the detection of secretory YA. Another problem, which arises in attempting to evaluate in various diseases whether an antibody is produced locally or is derived from serum, involves the effect of the inflammation itself on the extravasation of antibody from serum-so-called pathotopic potentiation. It is necessary in attempting to distinguish between the local formation of antibody and pathotopic potentiation to measure the antibody activity in the

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SECRETORY IhlMUNOGLOBULlNS MMC

Secretory y A

~

n

~ ~ Locloferrin

~

~

MMC

-

~

~

b

~“~iece“

+

FIG. 3. Schematic representation of some commonly encountered immunological reactions with secretion specific antigens. Antisera against apparently homogeneous preparations of secretory yA often contain antibodies against “impurities” such as lactoferrin and macromolecular component (MMC) or both in addition to anti-0-chain and antisecretory “piece” antibodies.

secretion against an unrelated “marker” antigen and, in the case of local immunization studies, to administer two antigens, as suggested by Oakley et al. (1955). It seems likely that, in the presence of high serum titers, pathotopic potentiation may obscure a relatively small contribution from local formation. Evidence for antibody activity in a given immunoglobulin class in external secretions has frequently been obtained by the use of indirect methods. For example, the distribution of antibody activity has been shown to parallel the distribution of a particular immunoglobulin class by gel filtration or density gradient ultracentrifugation. The demonstration of antibody activity in an apparently homogeneous immunoglobulin preparation, unless in very high titer compared with the whole secretion, is not su5cient evidence in itself to establish the immunoglobulin class of the antibody. The inactivation of antibody activity by reducing agents, such as P-mercaptoethanol, has often been used as a measure of yM antibodies. However, since serum yA polymers and in some species other immunoglobulins (Onoue et a?., 1966; Franklin, 1962) are sensitive to reduction, this criterion is not absolute. Secretory yA antibody activity is relatively resistant to reduction (see Section VI). These types of indirect evidence when taken together is what is referred to in Table VIII as suggestive evidence that an antibody activity is in a given immunoglobulin class. More direct evidence can be obtained employing indirect hemagglutination techniques utilizing antisera directed against immunoglobulin heavy chains, or b y absorption studies with specific antisera showing the removal of antibody activity subsequent to absorption of a particular immunoglobulin from thc secretion. Other direct methods involve the use of the indirect immunofluorescent technique and radioimmunoelectrophoresis. In the latter methods the antigen is L I S L I ~labeled ~ with I3’I, although other isotopes may be used. For

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

example, in the ingenious experiments of Hodes et al. (1964) the poliovirus, which is apparently small enough to diffuse through the agar gel, was internally labeled with 32Pincorporated into the viral ribonucleic acid (RNA). All the above mentioned “direct” techniques involve the use of specific antisera to demonstrate the immunoglobulin class of the antibody. In addition to utilizing immunoglobulin H-chain-specific antisera one can also use secretory “piece” (SP) -specific antisera in order to demonstrate whether SP is involved in a given antibody activity. This type of experiment is demonstrated in Fig. 14. V.

Secretions Involved in the Secretory Immunoglobulin System

A. DIGESTIVE TRACT In 1960, Gab1 and Pastner demonstrated the presence of several serum proteins including 7-globulins in saliva. Similar studies were reported in 1960 by Ellison, Mashimo, and Mandel. Kraus and Sirisinha, in 1962, suggested that an individually regulated transfer of serum proteins occurred into the oral cavity and based this hypothesis on a quantitative comparison of 7-globulins in saliva and plasma. A more recent study (Kraus and Konno, 1963) demonstrated a lack of quantitative correlation between titers of antibody in plasma and saliva. Investigations of the types of y-globulin found in saliva produced evidence that in common with other external secretions the yG/yA ratio in parotid secretions was less than unity ( Chodirker and Tomasi, 1963). Moreover, the fact that the 7-globulin/albumin ratio was 6 times that found in normal human serum (Tomasi and Zigelbaum, 1963) suggested that transudation was not the sole mechanism of secretion. Further confirmation that the yA immunoglobulin class predominated in human saliva was produced by Simons et al. (1964). Tomasi et al. in 1965 demonstrated that the salivary yA has a sedimentation coefficient of 11S and possesses unique antigenic properties. In retrospect, Patton and Pigman who in 1959 performed analytical ultracentrifugal studies on parotid, submaxillary, and sublingual secretions were the first to demonstrate the 11S component in all of these secretions, subsequently shown to be secretory yA. Parotid fluid has been the salivary secretion most studied because of the ease of obtaining samples free of contamination from other secretions. Mean concentrations of yA in parotid saliva of approximately 3 mg.% have been reported (Tomasi and Zigelbaum, 1963), however, the standard used for calibration was serum 7 S yA which would underestimate ( approximately threefold) that actually present. More recently,

SECRETORY IMMUNOGLOBULINS

13

Claman et (11. ( 1967) have reported a median value of 9.5 mg.% in normal healthy adults and have demonstrated little clifftwnce between the concentrations of salivary yA in samples taken sirnultaneously frorn right and Icft parotid glands in the same intlivitlual. However, considerable variation exists from day to day in the same individual and depends in part on whether collections are obtained postpraiidially and the type of stimulation employed. Mixed submaxillary and sublingual samples contain a Iittle more than half the concentration of yA found in parotid secretions. The concentration of salivary yA found in whole saliva, which represents the normal admixture of parotid, submaxillary, sublingual, buccal, and gingival secretions, is apparently similar to that found for parotid secretions (Lehner et aZ., 1967). However yG can be regularly detected in whole saliva, whereas the concentration of yG in most normal parotid fluids is very low, and like yM and yD can only be found if extreme concentration of samples is undertaken ( Claman et al., 1967). Immunofluorescent studies of salivary glands (Tomasi et al., 1965) have demonstrated the predominance of ykcontaining plasma cells in the interstitial areas between the glandular acini (see Table I ) . The average volume of whole saliva produced per day in a 70-kg. man is approximately 1000-1500 ml. (Best and Taylor, 1966). Assuming a yA concentration of 5 to 15 mg.X, then the amount of yA secreted per day in whole saliva is between 50 and 250 mg. This represents approximately 5 1 0 %of the total yA synthesized per day. It has been recognized by oral physiologists that the gingival pocket surrounding the tooth contributes a small volume of fluid to the oral cavity and, therefore, to whole saliva. This crevicular fluid has been shown to contain predominantly yG and the yG/yA ratio in the secretion is 8 : 1 similar to that found in normal serum (Brandtzaeg, 1965). The crevicular fluid should, therefore, not be considered as a typical external secretion although it does communicate with the oral cavity. It is pertinent, however, that the epithelium lining the gingival pocket is squamous in type and not the typical mucous membrane as seen with most other external secretions. In addition, the subepithelial plasma cells around the gingiva have been shown by the immunofluorescent studies of Brandtzaeg and Kraus (1965) to stain mainly with anti yG antisera. Immunoelectrophoretic studies of gastric fluids have demonstrated the presence of yG and yA immunoglobulins (Hirsch-Marie and Burtin, 1964; Tenerova et al., 1961; Hurlimann, 1963). Hirsch-Marie and Burtin showed that the principal y-globulins of gastric juice are yG and yA; y M was not found in this study. The yA in gastric juice was thought to be derived from saliva, and studies with antigastric juice antiserum ab-

TABLE I TISSUELOCALIZATION OF SECRETORY IMMUKOGLOBULINS AND SECRETORY “PIECE”BY IMMUNOFLUORESCESCE

Structure

Relative cellular predominance

Quant itat.ive ratios Distribution of ?Acontaining plasma yA y M yG cells Interstitial bet,ween acini

Parotid gland

yh

yM

yG

Submaxillary gland Gingival tissue

yA

yM

yG

yG

yA

no yM

Nasopharyngeal tonsil

yG

yA

yM = yD

Nasalmucosa

yA

yG

yM

3-8

Bronchial mucosa

?A

= yG

yM

5

Epithelial yA a

Epithelial ‘‘piece”b

-

+

Tomasi et nl. (1963)

+

Gelzayd et al. (1967a) Rossen et al. (196ic)

Occasional acinar

+

-

Lymphoid follicles Occasional stained with all antisew 1 Submucosal in Faint intercellular glandular areas and along secretory ducts

5

1

+-

Serous acinar

Reference

Brandtzaeg and Kraus (1965) Crabbe and Heremans (1967a) Brandtzaeg et al. (196’7)

+ + +

Rossen et al. (1967~) Martinez-Tello and Blanc (1968) Itossen et al. (1967~)

-

Lamina propria

Faint, intercellular Duodenum mid jejunum

yA

Appendix

yA

=

yh1

yG

yG

yM

22

3 . 3 1 Lamina propria

-

-

23

Lymphoid follicles stained with all antisera 1 . 1 1 Lamina propria Lamina propria

15.6 1 . 7

Lacrimal gland a

yA

yM

yG

?A

yM

yG

1 Lamina propria

Lamina propria

Determined by use of fluorescent antiserum yA antisera. fluorescent antisecretory “piece” specific antisera.

* Determined by use of

Apical epithelial

+ mucus

Apical epkhelinl mucus

+

Jeffries and Sleisenger (1965); Crabb6 and Heremans ( 1 9 6 6 ~ ) Brandtzaeg et nl. (1967) Rubin et al. (1965); Crabbe et al. (1965); Crabbe and Heremans (1966r) Crabbe (1967)

Crabbe and Heremans ( 1 9 6 6 ~;) Gelzayd et al. (1967b) Gelzayd et al. (1967a) Crabbe and Heremans ( 1 9 6 6 ~;)Gelxayd et al. (1967aj Gelzagd et al. (1967s)

Brandtzaeg et al. (1967)

16

THOMAS B. TOMASI, JR. AND JOHN BENENSTOCK

sorbed with normal human serum demonstrated a precipitin arc in the yA region on immunoelectrophoresis of saliva and gastric juice. In retrospect, these findings were probably due to the specific secretory yA determinants now known to be characteristic of this molecule. Because of the difficulties in distinguishing the salivary vs. gastric origin of the immunoglobulins found in gastric juice, studies have been carried out on tissue extracts from gastric mucosa. Rapp et al. (1964) found a quantitative predominance of yA in gastric extracts. Havez et al. ( 1966b) have reported the identification of an antigenically unique form of yA in gastric extracts, probably representing secretory yA, although further studies are necessary to establish this with certainty. The predominance of yA-containing plasma cells in the lamina propria of the gastric mucosa (see Table I ) is also consistent with the hypothesis that gastric juice is part of the secretory system, in keeping with similar findings with the large and small intestine. Adult, small intestinal fluids have been reported to have a rG/ yA ratio of 3.811 (Chodirker and Tomasi, 1963). This ratio was obtained using a serum 7s yA standard. Moreover, because of the presence of H-chain fragments of y G (Fc- and F’c-like material) the concentration of yG is probably overestimated (see Section IV). For these reasons the ratio of yG/yA is probably lower than 3.811, although its true value remains to be established. The nature, origin, and biological significance of the immunoglobulin fragments found in normal intestinal juice have not been investigated. Presumably the majority of the fragments arise from proteolytic degradation in the lumen following secretion and are of little biological significance. However, it has not been ruled out that the more resistant Fab- and F(ab),-like units with their antibodycombining sites do possess important biological activities. In all investigations of fluids from the GI tract, efforts must be made to retard proteolysis and subsequent degradation of immunoglobulins in order to obtain accurate estimates of the quantities of intact immunoglobulins present. Alkalinization, collection in ice, and addition of trypsin inhibitors have met with only partial control of proteolysis. Plaut et al. (1968) have reported that heating of GI juice to 56°C. for 30 minutes appears markedly to inhibit subsequent proteolysis and degradation of exogenous added immunoglobulins. In normal small intestinal juice ( Plaut, 1968) and cholera stool, Northmp et al. (1968) have shown that yA predominates as determined by immunoclectrophoresis. Secretory yA can be identified although a significant fraction of the yA detected has a sedimentation coefficient lower than 11 S. Apparently intact yG and y M can be identified in small amounts in these secretions. Likewise, the intestinal

SECRETORY IMMUNOGLOBULINS

17

secretions of children have been shown to contain yA, yM, and yG ( Nusslt. et al., 1962) although their absolute concentrations have not been determined. Gastrointestinal secretions have also been shown to contain specific yA polio antibody and yA anti-Escherichia coli antibody often in higher titers and with a different distribution among the immunoglobulin classes than found in serum (Berger et al., 1967; Tourville et al., 1968a). Immunofluorescent studies have also demonstrated the predominance of YA-containing plasma cells in the lamina propria of the small intestine (see Table I ) . The major immunoglobulin class found in colonic and rectal washings appears to be yA (Bull, Bienenstock, and Tomasi, unpublished), and villous tumors of the rectum secrete a fluid rich in yA (Masson et al., 1966). Immunofluorescent studies of the plasma cells of colon and rectum support these findings. Several investigators have demonstrated the presence in human bile of yG immunoglobulin (Hardwicke et al., 1964; Russell and Burnett, 1963; Rawson, 1962). Chodirker and Tomasi (1963) reported a yG/yA ratio for bile of 3:1, and Schultze and Heremans (1966) state that the concentration of yA is as high or higher than that of yG in many, although not all, samples of bile. However, it has been suggested that, on the basis of the studies of Hardwicke et al. (1964) and from the demonstration by Rouiller (1956) of communications between the space of Disse and bile canaliculi, the majority of the plasma proteins found in bile originate in the serum. Further studies involving careful quantitation of immunoglobulins and determination of the physicochemical nature of the biliary yA and its cellular origin are needed.

B.

RESPIRATORY

T R A ~

Although the capacity of human nasal secretions to inactive viruses was described as long ago as 1917 by Amoss and Taylor, the first demonstrations that such activity was due to specific antibody was furnished by Francis (1943; Francis et al., 1943). Remington et al. (1964) demonstrated that the yA immunoglobulin class predominated in nasal fluid and that yG was present in only trace amounts in normal secretions. Similar conclusions were reported by Artenstein et al. (1964) who also showed by absorption studies with specific antisera that there were both yA and yG antibodies to several types of viruses in nasal secretions. Ample confirmation and extension of the studies quoted above have been furnished by the careful investigation of nasal secretions by Rossen et al. ( 1965, 1966a,b) and Bellanti et al. (1965). These studies have demonstrated that about 80%of the yA found in nasal secretions is of the 11 S secretory type both by sedimentation and anti-

18

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCX

FIG.4. Immunofluorescent studies on bronchial biopsies. (A) Bronchial glands stained with an anti serum yA antiserum (low power); ( B ) bronchial glands stained with an anti secretory “piece” antiserum (low power); ( C ) high-power view of interstitial plasma cells stained with an anti serum yA antiserum [A and B from Tourville, Bienenstock, Adler, and Tomasi (unpublished ); C from Martinez-Tello and Blanc (1968).1

SECRETORY IMMUNOGLOBULINS

19

genic nnalysis. Approximatt.ly 20% of nasal yA appcws to have a sedimentation coefficient of 7 S and is aiitigenically identical to serum 7 S yA. A surprising feature of one of these studies (Rossen et a]., 1966b) was the apparent demonstration that nasal yA was negative for InV factors despite the presence of K light chains. This finding does not correspond to data obtained for colostral yA (Tomasi et al., 1965) showing both K and h chains and InV determinants. Rossen et al. (1966b) have also suggested that there may be differences in yG subclass distribution between serum and nasal washings. However, because of small amounts of yG present and thc. technical d w i e s involved in quantitating subclasses, these results are difficult to interpret and further studies are necessary before significant differences in the distribution of subclasses can be accepted. In keeping with the other glandular structures so far discussed, Brandtzaeg et al. ( 1967) have demonstrated a predominance of plasma cells containing yA particularly marked along the nasal glandular secretory ducts. The plasma cell yA/yG ratio varied according to the section studied (Table I ) but the proportions of plasma cells containing yA and yG were in good accord with the ratios of these immunoglobulins in nasal secretions. There is uniform agreement in the literature that yA is the major immunoglobulin class to be found in bronchial fluids ( Anzai et al., 1963a; Ibayashi et al., 1963; Keimowitz, 1964; Dennis et al., 1964; Masson et al., 1965; Chodirker and Tomasi, 1963). Keimowitz, Chodirker, Masson and colleagues demonstrated that yA was the major type of immunoglobulin in tracheobronchial washings, whereas the other investigators studied sputum which of necessity is contaminated by saliva. Using antisera raised against bronchial mucus or tracheobronchial washings, none of these investigators were able to show specific antigenic characteristics of the yA molecule. This, however, may be due to technical problems since SP determinants have been recently demonstrated (Hanson and Johansson, 1967; Havez et al., 1966a) in fluids derived from the bronchial tree in normal individuals and in patients lacking serum yA. Moreover, fluorescent studies of normal bronchial mucosa show a predominance of yA cells (Martinez-Tello and Blanc, 1968) and staining of mucosal glandular epithelial cells occurs with SP-specific antisera (Fig. 4). Dennis et al. (1964) demonstrated that with infection and during acute asthmatic attacks as well as in pulmonary tuberculosis, the yG/yA ratio of sputum more nearly approached that of serum, whereas, in normal individuals it is approximately 1. Anzai et al. (1963a) conclusively demonstrated that yA is part of the insoluble miicous gel of sputum and resists limited hydrolysis. Masson and Heremans (1966) showed that the

20

THOMAS B. TOMASI, J l i . AND JOHN BIENENSTOCK

sedimentation coefficient of yA from sputum corresponded to that of saliva, i.e., 11.3 S; however, the sputum was probably contaminated to a small extent by saliva. Investigations on lacrimal fluids have shown that yA is the major immunoglobulin class found in this secretion (Chodirker and Tomasi, 1963; Josephson and Lockwood, 1964; Settipane et aZ., 1965); yG is found inconsistently in trace amounts and y M can usually not be detected by Ouchterlony analysis. Passage of several serum proteins not normally found in tears into lacrimal fluid occurs upon mild irritation of the conjunctiva produced, for example, by rubbing the eye firmly several times (Josephson and Lockwood, 1961). Antigenic analysis of lacrimal yA has shown it to be antigenically identical to secretory yA in parotid and nasal secretions (Rossen et aZ., 1966b). These workers have also shown that the majority of lacrimal yA has a sedimentation coefficient of 11 s. C. GENITALTRACT Much of the work that has been done with regard to immunoglobulins and antibody activity in the reproductive tract has been carried out in animals and is reviewed briefly in Section 11 (also see Pierce, 1959). Isohemagglutinins have been described in the cervical mucus with no significant correlation between secretions and serum titers (Solish et uZ., 1961). The occurrence of isohemagglutinins in cervical mucus was found in this study to be unrelated to blood group secretory status. In cervical mucus, yG immunoglobulin has been detected (Moghissi et al., 1960), and immunoglobulins of all three classes, yA, yG, and yM, have been shown to be present on immunoelectrophoresis of cervical mucus by Moghissi and Neuhaus (1962) and Anzai et al. (1963b). The former investigators concluded that, on the basis of inspection of immunoelectrophoretic patterns, yG was present in greater amounts than the other two classes of immunoglobulins. However, accurate quantitation of the immunoglobulin classes in cervical secretions does not appear to have been performed. Fennell and Vazquez ( 1960), in an immunohistochemical study of the human and rabbit vagina, have showed 7-globulin staining in the intermediate zone of the stratified squamous epithelial cells and also in exfoliated cells. Further studies on the cervix and endometrium (Fennell and Vazquez, 1962) led the authors to suggest that the cytoplasmic staining was due to nonspecific absorption of serum proteins diffusing from the capillary bed through the epithelium. No fluorescence of endometrial glands was noted. There appears to be little information available on immunoglobulins in human tubular fluid or

SECRETORY IMMUNOGLOBULINS

21

uterine secretions apart from the studies on the cervical mucus. Hervk et al. (1965) in an investigation of cervical mucus have noted differences in immunoelectrophoretic patterns depending on the stage in the ovarian cycle. Little or no protein was detected in normal cervical mucus. However, at ovulation on day 14 of the cycle, a pattern similar to that of serum was obtained. Analysis of fluid from a ruptured Graafian follicle suggested to these authors that the follicular fluid was similar in protein content to normal serum. In patients with obstruction of their Fallopian tubes, “plasma” proteins were absent in midcycle. Only yG was identified with certainty in this study. However, Schumacher et al. (1965) found y c in all samples of cervical mucus regardless of the stage of the cycle; yA was also found but only between the fifteenth and eighteenth days. Further study is evidently needed in this area of investigation especially regarding the possible influence of hormones on the protein content of secretions of the female genital tract. Little definitive work on immunoglobulins in prostatic and seminal vesicle secretions has appeared in the literature. Leithoff and Leithoff (1961) have demonstrated yG, yA, and yM immunoglobulins in seminal plasma, and Chodirker and Tomasi (1963) reported that prostatic fluid obtained by massage showed no difference in y C f y A ratios compared with that of serum. However, because these studies were performed on patients with prostatic disease the amount of yG might be expected to increase as serum components appeared secondary to the inflammation. Acharya et al. (1968) have recently demonstrated that yA appears to be the predominant immunoglobulin class in normal seminal plasma and that it might be in high-molecular-weight (11S ) form although this was not conclusively proven.

D. URIIVARYTRACX The yA in both male and female urine is of high molecular weight (Tomasi and Zigelbaum, 1963; Turner and Rowe, 1964, 1967) having a sedimentation coefficient of 11S and is antigenically indistinguishable from the secretory yA molecule characteristic of other external secretions (Bienenstock and Tomasi, 1968). A yG/yA ratio of 3 : l has been reported. However, because of the technical problems encountered with quantitation of yG fragments this probably represents a maximum figure and 11 S yA and 7 S yG are probably present in approximately equal concentrations in normal u1inc (about 1 to 2 mg./24 hours). Special problems are encountered in iiivcstigation of the urine which are not met in the other secretions. Large volumes of urine are produced and only about 80 mg. of protein per day is found in normal bladder urine.

22

THOMAS B.

TOMASI,

JR. AND JOHN BIENENSTOCK

Of this only about 1 mg. is yA (Bienenstock and Tomasi, 1968). Slight variations in glomerular permeability, or in tubular reabsorption or secretion mechanisms, or even alterations lower in the urinary tract can cause considerable differences both quantitatively and qualitatively in the proteins found in bladder urine. In normal urine, yM is not found although yD can be detected depending on the degree of concentration. Immunoglobulin fragments, such as L chains and Fc and F’c fragments, are routinely found in normal urine, and small fragments with a molecular weight of about 12,500 have been repeatedly reported to be present in urine and have biological activity against a variety of immunizing antigens (Merler, 1966; Hanson and Tan, 1965; Merler et al., 1963).

E. MAMMARYGLAND Interest in the immune globulins in colostrum has attracted the attention of a large number of investigators, and some of this work has been reviewed briefly in Section 11. Similar work in the cow, goat, rabbit, sheep, dog, and mouse will be reviewed in Section X. Gugler at al. (1958) appeared to have first described the predominance of yA in human colostrum and this was confirmed by Havez and Biserte (1959) and Montreuil et al. (1960). Further careful immunological studies suggested not only the predominance of yA but a very complex pattern of immunoglobulins in human colostrum including the presence of yM and yG (Hanson, 1961; Hanson and Berggard, 1962). The possibility of specific antigenic determinants present on colostral yA molecule not shared by serum yA was mentioned by Hanson (1961). In retrospect this may have represented secretory yA, but since definitive studies were not done, it is difficult to say with certainty. Chodirker and Tomasi (1963) showed by quantitative measurement the predominance of yA in colostrum. Tomasi et al. (1965) isolated yA from colostrum and found that approximately 80% was of the secretory type and was identical immunologically with that isolated from other secretions; 20% had a sedimentation coefficient of 7 S and was identical immunologically with serum yA. Investigations by Havez et al. (1967) have confirmed the secretory nature of yA in colostrum. A secretory yA molecule has been identsed in rabbit colostrum very similar in most parameters so far investigated to human secretory yA (Sell, 1967; Cebra and Robbins, 1966; Cebra and Small, 1967). This is the only animal which has so far been conclusively shown to have a secretory yA system quite analogous to man. As pointed out in Table 11, witches’ milk, the mammary secretion of newborns, does not contain yA but free unattached secretory

23

SECRETORY IMMUNOGLOBULINS

Parotid yA-Deficiency states (includes newborns, first 1 P 2 0 days) Early childhood (1-12 mo.), some adults Most normal adults Lacrimal Nasal Tracheobronchial yA-Deficiency states Normal adults Colostrum yA-Deficiency states, Witches’ milk Normal Gastric Small intestinal Urine yA-Deficiency states Normal adults Sweat,

+ + -

+ +

+ + + +

T r Tr Tr Tr -

+ +

-

+

+

+

+

+

-

+ + + -

+ +

+ + + + + + +

-20%

+ + + - + - +

“piece” is present ( Hanson and Johansson, 1967). In these respects this fluid resembles other external fluids of the newborn as well as the secretions of patients with dysproteinemias characterized by a deficiency of yA. From the above discussion concerning the character of the immunoglobulins of fluids bathing the mucous membranes it seems quite clear that there is some type of relationship (as yet undefined) between the epithelial cell and the yA immunoglobulin system. Although the morphological appearance of the epithelial cell may vary from organ to organ the secretory system seems thus far to be confined to those organs having either cuboidal or columnar-type epithelial cells in association with mucous membranes or secreting glands such as the salivary acini. The only fluid examined to date which seems to be related to stratified squamous epithelium is the gingival or crevicular fluid. This does not have the characteristics of other external secretions, and fluorescent studies of the gingival tissue (Brandtzaeg and Kraus, 1!365) do not show the characteristic predominance of yA-containing plasma cells. It will be of some interest, therefore, to study fluids bathing other stratified squamous

24

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCK

epithelia such as the vagina, although here some difficulty niay be encountered in determining the origin of such fluids; whether they are derived from the vaginal wall andlor from the cervical secretions. Pertinent in this context is the recent report, as yet unconfirmed (Page and Remington, 1967), that human sweat has a yG/yA ratio of 3 : l and contains SP determinants, although these were not dekitely shown to be attached to yA. If this interesting observation is confirmed it will suggest a unique circumstance for this secretion, since it is thought to be formed primarily by transudation and the epithelial surface which this fluid bathes consists of stratified squamous epithelium. VI.

Chemical and Immunological Characteristics of Secretory Immunoglobulins

In this section emphasis will be placed primarily on the methods of isolation and the chemical, physical, and immunological properties of secretory yA. Very little work has been reported with the other secretory immunoglobulins, primarily because of the low concentration of yG and yM in external secretions, and the resulting technical problems involved. Studies of these immunoglobulins may, however, be a fruitful area for future investigations. A. ISOLATION The technique necessary to isolate apparently homogeneous preparations of secretory immunoglobulins varies for different secretions. Since the concentration of yA is considerably higher (about 0.5 to 1.0 gm.%) in colostrum, this fluid is preferred when significant amounts of secretory ./A are required. It should be pointed out, however, that the concentration of yA relative to other immunoglobulins varies not only from sample to sample but also depends upon the period in lactation. Early samples taken within the first 48 hours contain the highest relative concentrations of yA, whereas later samples show lower y-globulin levels and increasing amounts of yG. One useful scheme for the isolation of secretory yA from colostrum and saliva is presented in Fig. 5. The yields from colostrum using this method are approximately 25%. In the case of parotid saliva using a two-step procedure [stepwise diethylaminoethyl ( DEAE ) elution followed by Sephadex G-2001, the yields approach 40% but due to the relatively small concentration of yA in this fluid this procedure requires large volumes of starting material. Similar schemes have been employed for obtaining both y G and yA from nasal secretions (Rossen et al., 1966a). Various methods have been suggested for the isolation of

25

SECRETORY Ihlh~UNOGLOBULINS

7 1 Colostrum

Serurn([rcitic

p H 4.6 with acetic acid

Supernale

fluid)

50% ( NH,l

ppt

Neutrolize starch block electrophoresis

t in

&

d

l

SO,

saline

f 0.05M ZnSO, pH 7.0

yA Containing fractions I

V pH 614 0.10 M

t

Sephadex G 200 Eluted with 0.14M soline

I

YA

FIG.5. Scheme for isolation of yA from human serum, ascitic fluid, colostrum, and saliva.

-,A from serum (Heremans et al., 1962; Vaerman, 1966), but none of the techniques is entirely satisfactory and yields are small (of the order of 10 to 25%). Because of this, ascitic fluid, which is available in large amounts, from patients with cirrhosis of the liver has been used as a convenient starting material. Chemical and immunological studies on ascitic %uid yA reveal it to be apparently identical to yA isolated from serum (Tomasi et al., 1965) from which it is undoubtedly derived by transudation. When fresh, normal, human serum is examined by density gradient ultracentrifugation, no more than 515%of the yA is distributed in areas having sedimentation coefficients greater than 7 S (Tomasi et al., 1965). It is not known with certainty whether the 7s yA which is found in colostrum and nasal fluids (about 20%of the total yA in each secretion) represents protein derived directly from serum or from the secretory molecule which has undergone dissociation or failed to complex with the SP, although some evidence has been presented that it is derived from serum (Butler et al., 1967). The available evidence points to the fact that the higher polymers of yA (approximately 18 S ) which are found in many fluids represent polymers of the secretory molecule (Tomasi et al., 1965). Intermediate sizes of yA (9-14 S ) which are found in small amounts in serum are also present in ascitic fluid and these lack the

26

THOMAS €3. TOMASI, JR. AND JOHN BIENENSTOCK

specific secretory determinants. Higher polymers (18 S and above) cannot be detected in ascitic fluid although they are present in small amounts in normal serum. It should be pointed out that in testing for the purity of immunoglobulin preparations, samples which appear to be homogeneous by ultracentrifugation, zone electrophoresis, and by antigenic analysis often show impurities which can be detected. by immunization of rabbits or other animals. For example, there is a particular secretion-specific protein, MMC, with physical characteristics very close to those of secretory yA which is highly antigenic and is often detectable only upon immunization. Likewise lactoferrin often contaminates secretory yA preparations, and antisecretory antisera frequently contain antibodies to both MMC and lactoferrin (Fig. 3 ) . In this regard, tests for purity should always include the use of multiple antisera including those made against whole secretions. B. IMMUNOLOGICAL PROPERTIES Using antisera made against serum yA or isolated yA myeloma proteins, no immunological differences have been reported between the serum and secretory yA. Thus, from this standpoint, there is no evidence that the a chains of secretory yA differ from those of serum. However, using certain antisera made against secretory yA isolated from either saliva, colostrum, or nasal fluids, immunological specificity for the secretory yA can be detected. This is shown in Fig. 6 by the spurring of secretory yA over serum yA, suggesting the presence in the 1 1 s yA of an antigenic determinant not found in serum. It should be clearly emphasized that the demonstration of this type of unilateral or single spur is crucial evidence for the existence of antisecretory yA-specific antibodies in an antiserum. Following absorption of an antisecretory yA antiserum with normal (lyophilized) serum, activity remains only against the secretory molecule. The absorbed antiserum, if it is truly specific, will not react with myeloma sera containing high concentrations of yA polymers ranging from 9 to 1 4 s or with sera from patients with systemic lupus erythematosus ( SLE ), Laennec’s cirrhosis, and Sjogren’s syndrome who have relatively large amounts of polymeric yA but unlike the myeloma polymers have a broad range of electrophoretic mobilities. Also these antisera do not react with preparations containing 11s yA anti-B antibodies isolated from serum by specific precipitation with blood group B substance (Tomasi et al., 1965). The secretory molecules isolated from the saliva, colostrum, as well as those found in urine, nasal fluids, tears, bronchial washings, and GI

SECRETORY IMMUNOGLOBULINS

27

FIG.6. Ouchterlony plate showing antigenic relationships for yA from normal human serum ( N H S ) , colostrum (Col.), urine (yA,,) and saliva (Sal.). The center well contains an antiserum which reacts both with secretory “piece” and yA determinants.

fluids are immunologically identical when examined with antisecretory antisera (Tomasi et al., 1965; Rossen et al., 1966b; Bienenstock and Tomasi, 1968; Hanson and Johansson, 1967). The extra antigenic determinant present in the secretory yA which is lacking in serum yA has been shown to be due to the presence of the SP, also called transport “piece,” T component, and T chain. This can be demonstrated, as shown in Fig. 7, using isolated SP preparations obtained either from agammaglobulinemic urine (or saliva) and SP obtained from dissociation of the intact secretory molecule by reductive cleavage (see Section VI,E ) . In investigations of SP reactive material in a given whole secretion using an SP-specific antiserum ( antisecretory yA antisera absorbed with normal human serum), it is essential that Ouchterlony analysis be performed in such a manner as to demonstrate the immunological identity of the precipitin arc given by the whole secretion and that given by a homogeneous secretory yA preparation. Otherwise it is impossible to determine with certainty whether a given precipitin arc obtained with a whole secretion is due to SP, either free or attached to yA, or to a contaminant. Immunoelectrophoresis is also helpful in identifying the characteristic mobility of 11S secretory yA and free SP and may assist in distinguishing these from each other as well as other contaminating components (Fig. 3 ) . Evidence that the antigenic specificity of the secretory molecule

28

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

FIG. 7. Gel diffusion experiment showing immunological relationships of secrctory yA, secretory “piece” (SP), and serum yA. Well 1-reduced and alkylated colostral yA containing both a chains and SP; well 2-SP from agammaglobulinemic urine; well %SP isolated from secretory yA; well 4-normal human saliva; wells 5 and Gnormal human serum; well 7-anticolostral yA absorbed with yC.

is associated only with higher polymers of yA is suggested by immunological studies on the yA fractions isolated from colostrum. The 7 s colostral yA is identical immunologically to the 7 s serum yA, whereas both the 11 S and 18 S colostral polymers spur over both of the 7 S proteins (Tomasi et d.,1965). Similar results have been obtained with nasal fluid 7 S and 11 S yA proteins (Rossen et al., 1966b). C. CHEMICAL PROPERTIES OF SECRETORY IMMUNOGLOBULINS Some of the differential chemical properties of serum vs. secretory 111. The major component in the various secretions appears to be an 11 S molecule having a molecular weight of 385,000 although as mentioned above, higher polymers ( 16-20 S ) do occur in most secretions. The molecular weight obtained by the equilibrium sedimentation techniques of Yphantis ( 1864) is similar to that obtained by Cebra and Small (1967) on rabbit colostral yA. The higher molecular weight of serum y A (170,000) compared to yG (150,000) has been shown to be due primarily to the large molecular size of the ,a chain (65,OOO) compared with the y chain (50,000)(Cohen and Milstein, 1967). The molecular weight of isolated H chains from human colostral y A has not been determined, but rabbit colostral 01 chains have a molecular weight of 64,000 + 3000 (Cebra and Small, 1967). Since yA are outlined in Table

29

SECRETORY IMMUNOGLOBULINS

TABLE COMPARISON OF PROPERTIES OF A -,

III FROM

Human serum y.4

Property

SERIJMAND SECRETIONS" Human secretory yA

Rabbitb colostral yA

~

Sedimentation coefficient (so?"w) Molecular weight Carbohydrate Hexose (%) Hexosamine (%) Fucose (%) Sialic acid (%) Moles disulfide per 170,000 p. N-terminal amino acids Gm factors InV factors Extinction cvefficiertt Partial specific volume ( V )

6 SS 170,000 5 2 9 0 22 18 15 Asp, (:luc -

+

h'&k,,

11.4 S

~~

xs

6.2

3 2

4

3 2

Id

0.73" 0 65 16 Asp, Glu,' Lys" -

+

13 4 ) I 0 72!P

10

370,000

:385,000

13 9

n

13.5 0 703

~

Data from Tomasi et al. (396.5, IOSS), unless otherwise specified. Cebra and Robbins (1966) aiid Cebra and Small (19137). Heimburger et nl. (1964). Hanson and Johanssoii (1967). Bernier et al. (1965). Axelsson et al. (1966). 0 Havez el al. (1967). * Schultre and Heremans (1966). a

human colostral yA shares H-chain antigenic determinants with serum N chains presumably the H chains of colostral yA are similar or identical to those of serum yA. This is supported by immunological studies (see Section V1,B) which thus far have failed to demonstrate any differences between the CY chain of colostral yA and those of serum. However, urea, starch gel electrophoresis at acid pH suggests differences in mobility between colostral and serum yA H chains (Fig. 8) (Cederblad et al., 1966; Mehta and Tomasi, unpublished) although the significance of this observation has not yet been clarified. The L chains of human colostral yA show a diffuse zone on acid urea gels similar to serum yA and yG. In alkaline urea gels, multiple banding is seen in the L-chain area similar to serum yA and yG but in addition a unique anodally fast band is seen (Fig. 8 ) , which represents SP (Cederblad et cd., 1966; Hanson and Johansson, 1967; Rejnek et al., 1966). A similar fast band representing rabbit T chain is shown by reduced and alkylated, rabbit colostral yA on disc electrophoresis in urea (Cebra and Small, 1967).

30

THOMAS B. TOMASI, JR. AM) JOHN BJENENSTOCK Acid pH

YM

yAcol.

Alkaline p H yAser.

Light chain area Heavy chain

area

Origin-

4

U

FIG.8. Schematic representation of relative mobilities of reduced and alkylated secretory and serum immunoglobulins in acid and alkaline, urea starch gel electrophoresis. At alkaline pH the secretory “piece” moves as a fast anodal band.

Relatively little data are available concerning the carbohydrate content of the secretory molecule. The total hexose content is similar to that of serum yA. The figure for sialic acid content may be somewhat low since determinations were performed on colostral samples that had been exposed to acid pH conditions which are known to remove part of this moiety. As with serum yA, changes in mobility can be demonstrated following treatment of the secretory molecule with neuraminidase. The amino acid composition data on the secretory molecules isolated from saliva and colostrum are compared with serum yA and rabbit colostral yA in Table IV. Secretory yA from colostrum and saliva are very similar and probably identical within the limits of the experimental methods. However, there are significant differences in amino acid content between the human secretory and serum yA proteins. The halfcystine content determined as carboxymethyl cysteine by amino acid analysis is similar for serum and secretory yA, and essentially identical values for these human proteins have been obtained by sulfhydryl titrations using the Ellman technique (Tomasi et at., 1968). The SP is characterized by a high content of glycine and methionine and low proline compared with serum yA. It should be pointed out that the figures reported in the literature for the amino acid content of serum yA vary widely probably, in part, because of the difficulties in obtaining highly purified preparations of serum yA.

L4b11N0 . - h D COMPOSITION

OF

TABLE IV HVM.AN SERUM, SALIVARY A N D COLOSTRAL yA,

AND

RABRIT

Amino acid

IIumair snl. y.4

Humnu col. y.4

Human ser. yA

Col. y-4/ sal. y.4

Ser. ?A/ 881. yA

Human secretory “piece”

Human ser. yA

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

54.7 21.8 50.6 93.4 107.2 132.2 129.0 111.0 96.0 81.3 30.7 8i.7 7.4 25.5 109.4 39.7 35.5

55.9 20.6 49.9 93 . 4 108.5 127.4 119.5 99.0 88.6 81.6 32.5 89.5 7.2 26.7 109.2 37.3 36.6

63.5 22.0 48.5 93.5 117.0 140.0 124.5 104.0 82.0 81.0 33.5 95.0 8.5 23.0 113.5 41.5 42.5

1.02 0.94 0.99 1.00 1.01 0.96 0.93 0.89 0.92 1.00 1.06 1.02 0.97 1.05 1.00 0.94 1.03

1.16 1.01 0.96 1.00 1.09 1.05 0.97 0.94 0.85 1.00 1.09 1.08 1.15 0.90 1.04 1.05 1.20

4.0 1.4 4.0 7.6 8.0 9.2 8.3 6.6 15.2 5.7 2.4 6.9 1.4 2.1 8.3 4.0 4.7

5.1 1.8 3.9 7.6 9.5 11.3 10.1 8.4 6.6 6.6 2.7 7.7 0.7 1.9 9.2 3.4 3.4 ~

Data from Tomasi (1965) and Tomasi et al. (1968). Cebra and Robbins (1966).

yAa

% Moles

Residues per 160,000 gm.

a

COLOSTRAL

~

Human col. yA 4 1 4 7 9 10 10 8 7 6 2 7 0 2 9 3 3

~~~~

7 7 2 9 2 8 1 4 5 9 7 6 6 2 2 2 1

Ha1hith col. 7.k 3 7 1 4 ‘77 9 6 10 6 9 0 10 R 9 6 b h

6 3 6 0 2

2 2 9 4 4

i i 3 4 3.0

32

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

Salivary yA lacks the Gin genetic determinants which are characteristic only of the YG molecule, but does contain the InV genetic determinants present on the K type of light chain. However, four out of five nasal fluid yA samples lacked the InV allotypes although all possessed K light chains (Rossen et a,?., 1966b). The significance of this apparent difference between parotid and nasal fluid yA remains to be elucidated. Both the K and types of L chains are present in the colostral, salivary, and nasal fluid yA, although the ratios have not been accurately quantitated (Tomasi et al., 1965; Rossen et a,?., 196613). The electrophoretic mobility of human secretory and serum yA are similar in agar, polyacrylamide, and cellulose acetate electrophoresis ( Tomasi, unpublished). Only a few studies have been done on the enzymatic degradation of the secretory molecule. Using the proteolytic enzymes papain, trypsin, and chymotrypsin C, serum 7 S yA and 11S yA myeloma polymers are degraded to 3.8s Fab-like subunits; no Fc or F’c material is apparent (Tomasi and Czenvinski, 1968; Cederblad et al., 1966). The secretory molecule under similar conditions of enzyme concentration and incubation appears to be considerably more resistant to proteolysis with these enzymes and only a small fraction is degraded; the majority of the protein remains as an 11S molecule as determined by analytical ultracentrifugation ( Tomasi and Czerwinski, I968 ) . However, in these studies, proteolysis may actually have occurred; the 11S conformation being maintained by covalent and/ or noncovalent forces. Similar types of experiments involving incubation of purified preparations of immunoglobulins (yG, yM, serum, and secretory yA) with whole GI juice derived from the jejunum indicate that secretory yA is more resistant to proteolysis than the other immunoglobulins (Plaut et al., 1968). However, other studies (Cederblad et al., 1966) report that, although serum and secretory yA both appear to be more resistant to proteolysis than yG, the secretory molecule can be split by proteolysis in vitro by both papain and trypsin. The product is a 3.5s Fab-like fragment, and again no evidence of Fc material was found. The secretory molecule also appears to be quite resistant to reductive cleavage (Tomasi et al., 1965). Following treatment with high concentrations of P-mercaptoethanol (up to 0.5 M ) , no observable dissociation can be detected in the ultracentrifuge. In some cases, partial degradation of secretory yA is found following alkylation of the reduced molecule, but this occcurs irregularly and is dependent on the concentrations of reducing agent used. Following reduction and alkylation and treatment with concentrations of urea as low as 2 M , dissociation regularly occurs suggesting

30

SECRETORY IMMUNOGLOBULINS

that although disulfide bonds have been split the molecule is held together by noncovalent forces. Since serum or myeloma ll S polymers are sensitive to both enzymatic and reductive treatment, it is assumed that the relative resistance of the secretory molecule to dissociation is related to the presence of the SP. The possible biological significance of the resistance to enzymatic and reductive dissociation is discussed in a later section. D. THREE-DIMENSIONAL CONFORMATION Table V shows the optical rotatory dispersion (ORD) data obtained on yA proteins in an aqueous solvent and in 2-chloroethanol (Tomasi, 1965). For comparison, data obtained on 7G are included. The immunoglobulins in certain respects are unusual in their ORD behavior. Although they lack significant amounts of helices as determined by their b,, value in the classic Moffitt equation, they do show a specific rotation at 546 mp suggesting the presence of som2 type of organized structure (for a review, see Urnes and Doty, 1961). Both TABLE L' OPTICALROTATORYDISPERSION DATAOF Protein

rc

Serum 7 S 7.A Salivary 11 S y A Reduced yG 1:edured 11 S -,Ll

Solver1t

[fflalf,

Water Water Water 2-Chloroet h a i d 2-Chloroet haiiol

-14 -46 - 40 - 17 -2.5

ya"fh

1)"

% Helix

0

0

0

0 0 41

1)

-264 -269

41

a Optical rotatory dispersion data on yC; and serum and salivary ?A in aqueous arid organic ("-chloroetli:ttiol) solvelits. Proleiris reduced wilh 0.2 M BME for 6 hours at 20°C. * Data from Tomasi (1965).

serum and secretory yA are very similar to yG in their rotatory behavior. Certain organic solvents such as 2-chloroethanol allow more optima1 conditions for helix formation and many proteins in this solvent have close to 100%of their residues invoIved in heIix formation. Both yG and yA form a maximum of only 40%helix in this solvent. Cleavage of disulfide bonds, in an attempt to remove any possible restrictive influence of this linkage, does not significantly affect the final helical content in 2-chloroethanol. Whether the low helical content of the immunoglobulins is related to the presence in these molecules of a high content ( al?out 40%) of those amino acids (swine, threonine, proline, valine, and

34

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

isoleucine) that, as sugggested by Blout (1962), do not readily participate in helix formation is a matter of speculation. The type of threedimensional conformation which gives rise to the optical properties presented above is a matter of debate. However, the ORD data as well as studies using polarized infrared light are consistent with the presence of significant amounts of cross-p structure in these proteins. Essentially no information is available concerning either the tertiary or quaternary structure of the secretory yA molecule.

E. ISOLATION AND CHARACTERISTICS OF T H E SECRETORY ‘‘PE~” One technique used for the isolation of SP is outlined in Fig. 9. Following reduction, ablation, and chromatography in 1N propionic acid, it is important to take cuts from the Sephadex G-100 chromatograph (the shaded area in Fig. 9 ) which exclude L-chain determinants ( measured immunologically). These fractions are contaminated with H-chain determinants, but subsequent chromatography on

U

Homogeneous y A (colostral) .

0.2M BME 25OC, 12 hours. 0 . 3 M iodoacetomide

I H choin

Sephadex G 100 in 0.5N propionic acid

II

Sephodex 6200 in 0.14M NoC I

/Piece”

J\

FIG. 9. Schematic outline of the isolation of secretory “piece” from colostral yA (see text for details ).

Sephadex G-200 yields SP preparations which are homogeneous as tested by ultracentrifugation, disc electrophoresis, and immunological methods. Free SP is also present in secretions of patients with various types of dysproteinemias characterized by absence of serum yA. The SP isolated from agammaglobulinemic fluids is immunologically identical with that isolated by the above technique from the intact molecule (Fig. 7). Some of the properties of the SP isolated from colostral yA are outlined in Tahle VI.

35

SECRETORY IMMUNOGLOBULINS

The mode of linkage of the SP to the yA portion of the molecule is a matter of debate. Hong et al. (1966) have reported that SP can be dis; sociated using 6 M guanidine suggesting a noncovalent linkage. This is consistent with the reports of Cebra and Small (1967) on rabbit colostral yA. However, other studies (Tomasi and Czerwinski, 1968) suggest that only a fraction (approximately 20%) of the SP is bound noncovalently, the majority being linked by disulfide bonds. The evidence for this is based on the following studies. Chromatography of secretory yA on Biogel PlOO in 1N propionic acid or 6 M guanidine (containing 0.02 M iodoacetamide ) separates a small second peak containing unattached 1ABLE VI PROPERTIES OF SECRETORY PIECE"^ Sedimentation coefficient Molecular weight Carbohydrate (%) Moles of disulfide per mole Partial specific volume (P)

4.2 8 58,000

9.5 5 0,726

Data from Tomrtsi et (11. (1968).

SP. However, examination of the major peak eluted in the void volume indicates that SP determinants are still attached to yA. Rechromatography in 6 M guanidine results in a single peak containing intact secretory yA, which on treatment with small concentrations of reducing agents, releases the SP. In this regard recent studies of the binding of albumin to certain yA myeloma proteins suggest that this bonding is also through disulfide bonds ( Mannik, 1967). Noncovalent interactions undoubtedly also occur as suggested by the radiolabeling experiments discussed below, and in this respect the binding of SP resembles that of H and L chains in which both types of force are involved. The mobility of SP (isolated from secretory y A ) on immunoelectrophoresis is shown in Fig. 10. Free “piece” present in the secretions of agammaglobulinemic subjects has a similar mobility although minor variations in mobility are noted. As shown in Fig. 10, no reaction occurs between SP and an anti-L-chain antiserum. Immunodiffusion studies in our laboratory have likewise failed to demonstrate any antigenic relationship between SP and L chains; findings which are in accord with those of Hanson and Johansson (1967). The results of Hong et al. (1966) suggesting the existence of common antigenic determinants between SP and L chains may have been due to contamination of SP preparations

36

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

FIG.10. Immunoelectrophoresis of colostral yA (upper well) and isolated secretory “piece” (lower well). Upper trough-antiserum yA antiserum; center trough-anticolostral yA antiserum; lower trough-anti-L-chain antiserum. Anode on right.

with L chains or the formation of mixed dimers between L chains and SP. The molecular weight of isolated SP of 58,000 is not entirely compatible with the chromatographic position on Sephadex of the free SP present in agammaglobulinemic secretions or normal urine. Free SP has an elution volume less than that of albumin which is more consistent with a molecular weight of approximately 80 to 100,OOO by this technique. This suggests that the chromatographic behavior may be related to asymmetry although further work is necessary to clarify this point. It was also suspected that in agammaglobulinemia, the free “piece” might be a dimer, particularly in view of the findings with rabbit colostral yA suggesting that SP, with a molecular weight of 50,000, is composed of two 25,000 monomeric subunits linked by disulfide bonds (Cebra and Small, 1967). However, no evidence could be obtained with human SP of either covalent ( disulfide) or noncovalent associations between monomeric subunits, on treatment of free SP, isolated from agammaglobulinemic saliva, with reducing agents and 6 M guaindine ( Tomasi and Czenvinski, unpublished). Experiments with radiolabeled SP indicate that the in vitm complexing of SP is relatively specific for yA (Tomasi and Bienenstock, 1968). Addition of lS1I-1abeled SP to whole serum or isolated preparations of immunoglobulins shows that complexing occurs only with serum yA and not with yG or y M (see Fig. 11).The finding that reduced and alkylated SP obtained from dissociation of colostral yA will bind to serum yA suggests that noncovalent interactions are involved. Similar experiments with labeled SP obtained from agammaglobulinemic urine also demonstrate a specific interaction with yA. The site of linkage of the SP to the yA portion is unknown. However, since SP appears to have specific affinity for yA, it might be expected

37

SECRETORY IMMUNOGLOBULINS

FIG. 11. Radiolabeling experiments showing specific coniplexing of I3’Ilabeled secretory “piece” with serum yA. Complexing does not occur with yG, yM, transferrin, or other senmi proteins. Stained immunoelectrophoretic patterns on left, corresponding radioautographs on right. ( NHS-normal human serum. )

:hat the covalent and noncovalent associations occur with the chain. Likewise the number of SP molecules per mole of secretory yA has not been accurately determined. From the molecular weights one would expect a dimer of 7 S yA plus one SP molecule or one 7 S yA plus three or four SP molecules. The former seems more likely on the basis of approximate yields of SP, L, and H chains following reduction, but until the relative concentrations of SP and L chains are accurately determined, this question cannot be definitely answered. A tentative schematic model based on one SP per secretory yA molecule is shown in Fig. 12. The biological functions of SP are not fully understood. It appears from the data presented above that the secretory molecule is quite resistant to proteolysis and reductive cleavage. This is probably a result of the presence of SP in the 11 S secretory molecule, since myeloma and serum ./A polymers of 11 S size do not show a comparable degree of resistance under similar conditions. Teleologically, such stability would be of considerable biological advantage to an antibody molecule of which the activity is confined to complex fluids containing proteolytic enzymes such as external secretions. The available evidence indicates that the SP is synthesized in epi(Y

38

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

FIG. 12. Schematic model of secretory yA. Disulfide bonds represented by solid bars, and secretory “piece” by the triangular area.

thelial cells, and it has been suggested that SP is added to the yA protein in the process of transport across the mucous membrane. However, it is possible that complexing of SP and yA occurs in the secretion following transport and there is, therefore, no good evidence that SP is in any way involved in facilitating transport of yA. Since yA has a tendency to complex with a variety of proteins (Heremans, 1960; Mannik, 1967; Levitt and Cooperband, 1967; Ganrot, 1967), it is conceivable that its combination with SP is nonspecific and has little biological significance. This thesis has certainly not been excluded, but the specificity of the reaction of SP for yA in the radiolabeling studies outlined above, the occurrence of SP and yA together in all external fluids thus far examined, and the stability conferred on the secretory molecule by SP, suggest that it may have important and specific functions. The question of an enzymatic activity for SP has not been investigated except that recent work in our laboratory using antihuman amylase antisera have excluded amylase activity in SP (Bienenstock and Tomasi, unpublished). Further work in this area seems warranted particularly in view of the possibility suggested by the work of Adinolfi et al. (1966b) that secretory yA, together with lysozyme and complement, may have a lytic action on Escherichia coli.

SECRETORY IMMUNOGLOBULINS

VII.

39

Sites of Synthesis of Secretory Immunoglobulins

A. IMMUNOLOGICAL STUDIES

.

From quantitative studies of immunoglobulin levels in parotid saliva and serum in health and in a variety of disease states, it is apparent that there is often little correlation between yA levels found in saliva and serum (Claman et al., 1967; South et al., 1966). In patients with hypergammaglobulinemia of varied origin which was associated with elevated levels of serum yA, there is little correlation between salivary concentrations of this immunoglobulin and those found in serum (Claman et al., 1967; see also Table VII). No increase in salivary yA concentration was noted (Tomasi et al., 1965) in 8 patients with yA myeloma, 2 of whom had a high concentration of 11 S yA polymers. In 1 myeloma patient, density gradient ultracentrifugation was performed and it was shown that the salivary yA was predominantly 1 1 s in type and possessed secretory antigenic determinants, whereas the serum myeloma was predominately 7 S. In the parotid saliva of neonates, salivary yA can be found in the absence of measurable serum yA using a quantitative complement fixation technique which will detect as little as 0.25 pg. of yA per milliliter of serum ( Sullivan and Tomasi, unpublished). Similar results have been reported by South et al. (1967) and by Selner et al. (1967), the latter using a sensitive electroimmunodiffusion technique. However, apart from neonatal secretions, secretory yA has not been found in the absence of serum yA provided sensitive Ouchterlony or complement fixation techniques are used to detect serum yA. There is one discordant report from McFarlin et al. (1965) that in patients with ataxia telangiectasia and absent serum yA, plasma cells containing yA could be demonstrated in the parotid gland and bone marrow and that, in addition, yA could be detected in concentrated parotid saliva. These findings have so far not been confirmed by other investigators. However, Swanson et al. (1968) have reported one patient with a malabsorption syndrome and absent serum yA (less than 5.0 mg3) in whom fluorescent studies of the GI tract revealed a normal number of yA-staining plasma cells. However, as shown in Table VII, certain patients may have markedly depressed serum levels of yA (one-tenth of normal) and yet show essentially normal levels of salivary y A. As seen in Table 11, free or unattached secretory “piece” can be found in the concentrated saliva of all neonates and most children (South et at., 1967), and it is occasionally found in salivary samples from adults. Free SP is also found in most colostral samples and in all normal

40

THOMAS B. TOMASI, JH. AND JOHN BIENENSTOCK

urine ( Sullivan and Tomasi, unpublished; Bienenstock and Tomasi, 1968) . In addition, in patients with agammaglobulinemia, ataxia telangiectasia, and in healthy subjects who lack yA, free SP has been demonstrated in saliva, colostrum, bronchial washings, and urine (South et d., 1966; Bienenstock and Tomasi, 1968; Tomasi and Czenvinski, 1968; Hanson and Johansson, 1967). One possibility to account for the relative predominance of yA in external secretions is that a preferential secretory mechanism exists in the organ systems involved for transporting yA from serum to secretion TABLE V I I COMPARISON OF ?A LEVELSIN SERUM A N D RALIVA'~ Patients ~~

18 Normal N. C. L. s.

s.

M.

A. v. N. T.

M. H. M. M. R. I. M. F. J. V.

rA h4yelonia (7 8 ) ?A Myeloma (7 S) rA Myeloma (11 S) ?A Myeloma (11S) SLE SLE SLE Ragweed allergy Ataxia telang. Ataxia telang.

160

> 1000 > 1000 > 1000 > 1000

570 550 620 21 0 0

6.3 3.6 5.6 3.9 4.3 2.3 2.3 4.3 4.6 0 0

Patielits selected to illustrate the dissociation between serum and Ralivary levels of Data from Tomasi et al. (1965) and unpublished. b SLE-systemic lupus eiythematosus; te1ang.-telangiectasia. 11 S ?A standard.

7.4.

perhaps similar to that responsible for the selective transport of yG across the placental barrier (see Brambell, 1966). A specific recognition site might then be expected to be present on the yA molecule to account for facilitated transport. In this regard, South et al. (19f36), as a result of studies involving infusions of whole normal plasma into agammaglobulinemics, demonstrated transport of yA into the saliva of 1 and possibly 2 out of the 5 patients studied. The transport mechanism appeared to be specific for the yA molecule since yG and yM were absent from the saliva despite the high levels of these immunoglobulins in the serum. The quantitative aspects of transport were not determined, nor was it demonstrated whether the yA which was found in the saliva following the infusions was secretory (11S ) in nature. However, two

SECRETORY IILIMUKOGLOBULINS

41

recent studies (Selner et al., 1967; Haworth and Uilling, 1966) following excliangc transfusions in newborns with cqdiroblastosis have failed to demonstrate any transport of yA from scmiin to saliva. This was not related to the levels of serum yA achieved, since higher concentrations of yA were found following some of the exchange transfusions than in the infusion experiments of South et al. (1966). The findings following exchange transfusions are consistent with the in vivo 1311-labeledyA studies in adults (see Section VI1,C) which also failed to demonstrate transport of yA into saliva. Some 7 s yA occurs in normal colostrum, nasal secretions, and 1965; Bienenstock and Tomasi, urine (Rossen et ul., 1966a; Tomasi et d., 1968). The 7 S yA found in secretions is immunologically identical to serum yA. Whether this represents yA transported from serum as previously suggested (Butler et nl., 1967) or degradation of the 11S yA to “monomeric” 7 S form remains to be proven.

B. IMMUNOFLUORESCENT STUDIES Immunofluorescent techniques, although useful in investigations of sites of synthesis, can, however, give only indirect evidence that the synthetic source of a spec& immunoglobulin in a secretion is, indeed, the cells stained by the corresponding antiserum. Other difficulties, particularly relating to the secretory system, are encountered. For example, it has been found exceptionally difficult both in our laboratory and that of other investigators (Hanson and Johansson, 1967) to make monospecific antisera which will react only with SP, since when the antisera are tested against whole secretions, reactions are found in the majority of cases with nonimmunoglobulin secretion-specific components (see Section IV). Despite these problems, immunoffuorescent studies have shed considerable light on the probable source of immunoglobulins in several organ systems and such studies are summarized in Table I. From these results it can be seen that there is good accord between the quantitative predominance of a particular immunoglobulin class in a secretion and the distribution of plasma cells staining with specific antisera in that tissue. Thus, the yh-containing cells predominate in the lamina propria along the whole of the GI tract, in the parotid and submaxillary glands, nasal and bronchial mucosa, as well as the lacrimal glands. In gingival tissue, however, yG cells appear to predominate and in the lamina propria of the appendix the numbers of yA- and yGcontaining plasma cells are approximately equal. In the latter organ, lymphoid follicles stain approximately equally with all antisera used,

42

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

and a similar finding has been demonstrated with nasopharyngeal tonsils. There are no reports as yet of a systematic investigation into the Peyer’s patches and similar lymphoid collections of the GI tract. The morphological type of cells stained with immunofluorescent antisera in these studies are most often described as members of the plasma cell series, However, most investigators stress that these c d s have wide variations in morphological character, though many of them are identifiable as typical plasma cells with eccentric nuclei. Crabbe et al. (1965; Crabbb 1967) assumed a globular shape for nucleus and cell alike and calculated an average nucleocytoplasmic ratio of 0.7 for cells containing immunoglobulins in the lamina propria of the intestinal tract. Little difference was found for this ratio between cells stained with different antisera. The studies of Tomasi et d.(1965) demonstrated staining of plasmalike cells and cells of uncertain morphological type in the interstitium of the parotid gland with antisera directed against the secretory yA molecule and also with antisera specific for 7 S serum yA. No staining of the epithelial cells was found with a specific anti-7S yA antiserum. However, staining of the epithelial cells occurred with an antisecretory yA antiserum and with an antisecretory yA antiserum that was absorbed and, thus, reacted only with the secretory molecule and was presumably directed against SP alone. As a result of this work, as well as other evidence discussed later in this section, it was suggested that local production of yA might be occurring in interstitial plasma cells and that SP might be added to the yA molecule at the epithelial cell level in transit to the lumen. Rossen et al. ( 1 9 6 7 ~ ) have reported that both SP and 7 S yA staining occurred in serous acinar cells of the submaxillary salivary gland and in the epithelial cells of the bronchial mucosa. These authors have also described the existence of SP and yA determinants in cells beneath the epithelial layer and suggested that SP and yA were produced by the same cell, probably a plasmalike cell. However, other studies also performed on bronchial tissues ( Martinez-Tello and Blanc, 1968; and see Fig. 4 ) have not confirmed these findings. Moreover, it is now well established that SP and yA can be independently synthesized since in the newborn and agammaglobulinemic, SP is found in the absence of immunoglobulins and in the apparent absence of plasma cells. If Rosqen’s observations are valid, one would also have to hypothesize that there were two completely different types of +A-producing cells, since y A-containing plasma cells in peripheral and splenic lymphoid tissue do not stain for SP. Eidelman (personal communication) has

SECRETORY IMMUNOGLOBULINS

43

shown in a study of rectal biopsies of neonates the almost complete absence of plasma cells in the lamina propria until about 7 to 10 days of life when this structure rather suddenly receives a full complement of plasma cells similar in numbers to that found in the adult. Immunofluorescent staining of these tissues shows no staining in the lamina propria in the absence of plasma cells and an appropriate predominance of yA-containing plasma cells without SP folIowing colonization with plasma cells. Gelzayd et al. (1967a), in immunofluorescent studies of the rectum and colon, found fluorescence with anti-yA antisera in the apical portion of the epithelial cells and also in the mucus on the luminal side. Epithelial yA fluorescence might represent transported yA from the lamina propria, and several investigators (summarized in Table I ) have shown faint or occasional staining of the epithelial layer with antiserum yA antisera. However, as mentioned above and as shown in Fig. 4, little or no yA epithelial fluorescence was found in the parotid or bronchial mucosal glands with an antiserum yA antiserum although striking luminal fluorescence was observed. Moreover, Crabbe (1967) in his investigations on the GI tract did not find epithelial staining with antiserum yA antisera anywhere along the GI tract except for occasional staining of the epithelial layer of the nasopharyngeal tonsil, These discordant results need further clarification.

C . I n Vivo RADIOACTIVE TRACER STUDIES The metabolic behavior of serum yA labeled with l3II and yG labeled with I Z a I was studied (Tomasi et al., 1965) in 1 normal and 4 subjects with various diseases. No evidence of transport of intact yG or yA from serum to saliva was found. McFarlin et al. (1965) detected transfer of only trace amounts of labeled 7 s yA in 2 out of 3 cases studied and none in a third. Butler et al. (1967) found that labeled 11S yA isolated from nasal secretions was not transported into nasal secretions when administered intravenously. However, homologous and autologous 7s yA did appear in the nasal secretions apparently unchanged. These findings suggested that serum 7 S yA might be a source of the 7 s yA found in nasal secretions, but that the major portion of the ./A which was of the 11S secretory type was synthesized de no00 locally in nasal tissues. In the turnover studies of Tomasi et al. (1965) with labeled 7 S yA, no protein-bound radioactivity was found in the urine. This is of interest in regard to the derivation of urinary secretory yA (Bienenstock and

44

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCK

Tomasi, 1968) since, if the urinary yA were derived from serum by glomerular filtration or selective secretion, protein-bound radioactivity would have been expected.

D. TISSUECULTURE Incorporation of ’“-labeled amino acids into yA has been demonstrated by radioimmunoelectrophoresis with mammary gland tissue obtained from humans, rabbits, and monkeys (Hochwald et aE., 1964). Labeling of the other immunoglobulin classes was not found. Similar studies with human parotid tissue (Tomasi et al., 1965) demonstrated labeling of 11 S secretory yA in tissue culture and only trace amounts of yG were found to be labeled and, then, only after prolonged exposure of the radioactive slides to the sensitive film, Whether the small amount of y G synthesized was produced by white cells present in blood contaminating the tissue fragments or by plasma cells indigenous to the gland could not be determined. Analogous findings were reported by Hochwald et al. (1964) in tissue culture studies of monkey submandibular glands. In the two studies mentioned above, no attempt was made to determine whether incorporation of label occurred into yA, SPYor both. Thus, the only conclusion which is justified on the basis of this work is that at least one of the components of the secretory molecule was being synthesized by the tissue fragments. The possibility that only the SP is synthesized locally and that the yA portion is transported from serum cannot be excluded from these studies. Asofsky and Small (1967) used a similar technique and demonstrated incorporation of 14C-labeled amino acids into rabbit colostral yA. However, fractionation of the harvested culture fluid showed that the majority (about 75%)of the radioactive label was incorporated into the T chain and little into the yA heavy-chain or light-chain components. The results suggested to these investigators that the major source of the yA portion of the secretory molecule in rabbit colostrum originates from serum yA rather than from local synthesis by yA-producing plasma cells in the gland. Alternative explanations for these results considered unlikely by the authors, including a sizable pool of 7 s serum yA in the mammary tissue at the start of the study and an unusual distribution of amino acids in the SP. It is also possible that there is a disproportionate local rate of synthesis of yA and SP although in this case one might expect more incorporation of label into the yA portioii tlraii demonstrated in these studies, The work of Askonas et a2. (1954) would support the conclusion of Asofsky and Small. These workers, on the basis of the kinetics of incorporation into serum and colostral proteins of labeled amino acids given parenter-

SECRETORY IMMUNOGLOBULINS

45

ally, concfndcd that thv colostrd ~-gloltuliiisin goats and Inbl>its were derived primarily from serum. Similar ronclusions regarding the origin of milk y-globulins in the cow were reported by Dixori et a / . (1961) and Pierce and Feinstein (1965). It seems likely, therefore, that the mammary gland at least in certain species is able to transport immunoglobulins from serum. The transport mechanisms must be highly selective since it apparently involves predominantly yA in the rabbit and a fast yGglobulin in the sheep and cow (see Section X). Moreover, the mechanism whereby serum yA ( 7 and 9 S forms in the rabbit) becomes converted to 11 S secretory yA in the gland is still obscure but probably involves complexing with SP as in the human. Asofsky and Thorbecke (1961), by using the technique of organ culture, demonstrated labeling of yG and yA in cultures of human ileum. Labeling of yM occurred less frequently and cultures of one sample of appendical tissue was found to label only yG. In labeling experiments with monkey tissues, similar results were found with monkey ileum which incorporated labeled amino acids into both yG and yM. No antisera were available specifically directed against monkey yA. Surprisingly, monkey kidney was shown to incorporate label into yG and not yM. Antibody synthesis of unknown immunoglobulin classes has also been shown to occur with in ~ i t mtissue culture explants of rabbit vagina by Bell and Wolf (1967). Only rabbits immunized with diphtheria toxoid by the local vaginal route were shown to produce specific vaginal antibody. Immunization of the uterus and intravenous or footpad immunizations with Freunds adjuvant, produced high serum titers of antibody but no antibody production could be detected in vaginal tissue cultures. Neither the immunoglobulin class nor the type of cells responsible for immunoglobulin production were investigated.

E.

POSSIBLE

MECHANISMS OF SECRETION OF yA

The above observations are most consistent with the following interpretation. In the human salivary glands and probably in the GI and respiratory tracts, a significant fraction of the immunoglobulins present in the secretions are synthesized locally in tissue plasmalike cells which are found in close anatomical relationship to the glandular and mucous membrane epithelia. The possibility of some transport from serum cannot be excluded particularly if inflammation of the mucous membrane occurs for any reason. In agammaglobulinemia, if high enough serum levels of yA are produced by infusion, some yA may reach critical transport sites and be secreted. However, this is an inconstant

46

-

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

llSyA

External secretions

(7syA), +“piece”

Glandu lor plasma cell

E pi t he1 ia I cel Is

FIG. 13. Schematic representation of a postulated mechanism for the formation of secretory yA.

finding and does not normally occur to a significant extent, perhaps in part because of failure of yA to permeate capillaries or a “diffusion advantage” of locally produced yA, or for some other more complex reason. The locally produced yA which is assumed (but not proven) to be identical to serum yA, then traverses the mucous membrane or glandular epithelium by some as yet unknown mechanism. The SP, a nonimmunoglobulin glycoprotein with apparent specificity for the yA molecule, is synthesized by the epithelial cells. It complexes firmly with the yA portion, probably by both disuIfide as well as noncovalent forces, to form the intact 11 S secretory molecule. Where the SP and yA portions unite, whether inside the epithelial cell, on one of its surfaces, or in the lumen is not known. This hypothesis is summarized schematically in Fig. 13. It has been suggested (Tomasi et al., 1965; South et al., 1966) that SP might in some way facilitate the transport of yA and for this reason it has been referred to as transport “piece.” However, although an attractive possibility, no good evidence is presently available to support this thesis. In the case of mammary tissue of certain animals the available evidence suggests that the major portion of the colostral immunoglobulins are derived by transport from serum. Transport is, however, highly selective and only certain serum immunoglobulins are secreted (yA in the rabbit). The SP in the rabbit is synthesized locally in the mammary gland but its function, if any in facilitating transport, is unknown. Whether the human mammary gland is similar in these respects to those of animals and selectively transports immunoglobulins from serum or whether it synthesizes yA and perhaps other immunoglobulins locally as seems likely for other external secretions remains to be determined. Although the interpretations outlined above seem most likely on the basis of currently available information, other possibilities should be

SECRETORY IMMUNOGLOBULINS

47

entertained. The possibility of cellular transport by migrating lymphocytes through the epithelial surface to the lumen must be considered. In both man and animals the presence of lymphocytes within the epithelial cells of intestine, trachea, and urinary tract are well established (Trowell, 1958; Andrew, 1965; Andrew and Collings, 1946; see also Darlington and Rogers, 1966). These epithelial lymphocytes of man show considerable morphological similarity to the globule leukocytes described in animals in response to nematode infestation (see Dobson, 1966a,b,c; Whur, 1967). It is possible that both the epithelial lymphocytes and globular leukocytes do not actually lie inside epithelial cells but rather in the natural intercellular channels described by Tormey and Diamond (1967), which are apparently common to most epithelial structures involved in absorptive processes. These channels are firmly closed by a tight junction on the apical side of the cell but are open with gaps in the basement membrane on the basal side. Thus, the various studies (outlined in Table I ) showing fluorescence of epithelial cells might represent yA being transported in lymphocytes. In this regard, some fluorescence has been detected in intercellular positions by Brandtzaeg et al. (1967). However, Gelzayd et al. (1967a) detected yA fluorescence primarily in the apical portion of the epithelial cells, whereas epithelial lymphocytes are seldom found in this region since usually they lie in the basal portions beneath the cell nucleus. Furthermore, the bulk of evidencz (Darlington and Rogers, 1966) suggests that actual migration of lymphocytes into the lumen rarely occurs. The observation that yA staining is primarily apical would also be difficult to explain if yA transport occurred primarily by a mechanism involving reverse pinocytosis by epithelial cells as is often assumed for the transport of other large molecules. Another hypothesis which has been considered (Tomasi, 1965) involves the transudation or secretion of serum proteins with selective degradation by the proteolytic enzymes present in these fluids, the yA being more resistant to proteolysis than the other proteins. In this regard some evidence for an increased resistance to proteolysis of both serum and particularly secretory yA, compared with y G and yM, has been presented (Plaut et al., 1968; Tomasi and Czerwinski, 1968). However, this mechanism can be excluded in the case of parotid saliva. Previous work has shown that parotid fluid lacks proteolytic activity (Chauncey, 1961). Addition of yG to parotid fluid and subsequent incubation did not reveal significant degradation of the y G molecule ( Tomasi, 1965). Moreover, this thesis would leave unexplained the vast predominance of YA-type plasma cells in the organs of the externaI secretory system.

48

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

Biological Properties of Secretory yA

VIII.

A. “NATURAL” SECRETORY ANTIBODIES 1. Isohemugglutinins Serum isohemagglutinins have been described with intermediate sedimentation coefficients (approximately 11S ) (Rockey and Kunkel, 1962; Rawson and Abelson, 1964; Ishizaka et al., 1965), and these for the most part appear to be due to polymeric serum yA. Serum yA polymers can be differentiated from the 11s secretory yA by the absence of the specific SP determinants and also by the fact that serum 11s yA dissociates on treatment with reducing agents with loss of agglutinating activity, whereas secretory yA is unaffected. Tomasi et d. (1965) demonstrated that salivary and colostral secretory yA in both 11s and 18 S polymer ranges contained isoagglutinin activity, and similar results have been obtained by Adinolfi et aZ. (1966a). The polymeric form of serum yA is approximately 7 times more efficient in hemagglutination than the monomeric form on a weight basis (Ishizaka et al., 1965). However, no information is available on the number of antibodycombining sites and the relative efficiency of the 11s secretory yA molecule. Adinolfi et al. (1966a) in a study of colostral isohemagglutinins demonstrated antibody activity both in the yM and yA classes but not in the yG. In addition, they were able to show in several cases that the titer of colostral yA antibody exceeded that found in serum. Although isosgglutinins have been described in several other external secretions, the immunoglobulin class of these antibodies has in most cases not been investigated, with the exception of urine. Isoagglutinins could not be found in normal concentrated urine (Hanson and Tan, 1965; Prager and Bearden, 1965) However, on immunization of such individuals with blood group substances, isoagglutinins of the y G class have been described by both Prager and Bearden (1965) and Hanson and Tan (1965). Cross-reactivity between blood group substances and microorganisms have been well described, and it seems likely that the isohemagglutinins found in serum are a result of exogenous stimulation by cross-reacting antigens present in bacteria and viruses (Springer, 1967). For example, a single oral feeding of blood group-active Escherichia coli 086 to germfree animals (having no anti-B antibodies) produces high serum anti-B titers. Feeding experiments in man have given similar results. Likewise, myxoviruses such as influenza and fowl-plague virus contain significant blood group A and Forssman antigen specificity as components of their a

49

SECRETORY IMhlUNOGLOBULINS

virus coat. The isoagglutinins in external secretions are probably also formcd as a result of cross-reactions nit11 microorganisms but this has not been directly demonstratcd. 2. Viral and Bacterial Antibodios “Natural” antibodies apparently formed in the absence of overt infection or immunization, against a variety of bacteria and viruses, have been found in nasal secretions, saliva, small intestinal secretions (Fig.

FIG. 14. Immunofluorescent stuclies of Escherichh culi incubated with normal serum ffluorescent anti-7 S small intestinal secretions and serum. ( A ) E . c d i yA; ( B ) E. culi f intestinal secretions -/- fluorescent anti-7 S yA; ( C ) E . coli f intestinal secretions serum fluorescent antisecretory “piece” (SP); ( D ) E . coli ffluorescent anti-SP. [Data from Tourville et al. ( 1968a).]

+

+

+

CHARACTERIZATION

TABLE VIII SECRETORY A4NTIBODIES

OF “NATURAL”

Antibody class. Secretion Saliva

antigen

Nasal Small intestinal Colostm1

Blood group subs. Escheriehiu coli Lactobacillus, Streptococcus salivarius, Streptococcus mitis Salmonella typhosa, Shigella dysenteriae Polio I, Coxsackie A9, ECHO 28, Parainfluenza 3 Eseherichia coli Blood group subs.

Urine

Eseheriehia coli Blood group subs. Eseheriehia coli

yG

yh

y M

Reference

+*

Tomasi et al. (1965) Tourville etal. (1968b) +I Green (1966) +t Evans and Mergenhagen (1965) Artenstein et al. (1964) Tourville et al. (1968a) Tomasi et al. (1965) +t Adinolfi et al. (1966a) +t Adinolfi et a!. (1966b) - Prager and Bearden (1965) Hanson and Tan (1965) - Tourville et al. (1968b)

+ +* +

+ +

+- +* + +* + -

+ +*

a Key to symbols: (+) Evidence of strong activity in given immunoglobulin class; (-) evidence that activity not, in given immunoglobulin class; (*) good evidence for secretory ?A activity; (t) suggestive evidence for secretory ?A activity.

SECRETORY IhlMUNOGLOBULINS

51

14 ) , colostrum, and urine. Investigations concerning the characterization of these antibodies are summarized in Table VIII where appropriate references for more detailed information are given. In Section VI, there is a critical discussion of the methods (direct and indirect) of determining antibody activity in a given immunoglobulin class. B. SECRETORY ANTIBODIESFOLLOWING IMMUNIZATION 1 . Active Zininunization

,

Parenteral immunization in many instances can endow protection upon the individual providing a significant humoral antibody response is mounted. However, it is well established in the case of polio (Salk) vaccine that although protection can be demonstrated on successful immunization, prevention of the carrier state is not necessarily accomplished. This suggests that, although the stage of viremia can be prevented by circulating antibody, colonization of the GI tract and replication of the virus locally is not necessarily inhibited by serum antibodies. Since the portals of entry of many pathogenic viruses and bacteria are the GI and respiratory tracts, study of the role of secretory antibody and the procedures by which local immune mechanisms can be more effectively stimulated, could be of considerable importance in developing more effective immunization programs. a. Gastrointestinal Antibodies. Besredka ( 1927) appears to have first postulated a mechanism of local intestinal immunity against cholera. Burrows and Havens in 1948 demonstrated the effectiveness of oral live vaccination against cholera in both man and guinea pig and showed that protection could be correlated with the presence of coproantibody and not with the level of serum antibody. Neither urinary nor fecal antibody appeared to be derived from serum, at least by direct transudation. Oral immunization in man by dietary proteins routinely ingested may also stimulate production of serum antibody in apparently healthy people (Gunther et al., 1960; Peterson and Good, 1963; Saperstein et al., 1963; Rothberg and Farr, 1965). Rabbits immunized orally with low concentrations of bovine serum albumin will eventually develop serum antibody levels indistinguishable from those of animals immunized by intravenous or subcutaneous routes ( Rothberg et al., 1967). After oral immunization in man with attenuated poliovirus, or living or dead bacteria, specific antibodies can also be detected in the serum (Sabin et al., 1961; Buser and Schar, 1961; Thomson et al., 1948; Freter, 1962). [For a review of similar work relating to coproantibody, see Freter ( 1%2), Pierce (1959), and Freter and Gangarosa (1963).] Koshland and Burrows (1950) and

52

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

Koshland (1953) suggested that the mode of immunization may well affect the eventual production of coproantibody in animal experiments. The use of Freund's adjuvant in conjunction with intraperitoneal instillation of vaccine did not result in any coproantibody formation despite high levels in the serum, However, these investigators were detecting antibody activity by complement fixation techniques, and noncomplement-fixing antibodies (such as secretory y A ) would have been missed by the techniques employed. Since it is recognized that in the guinea pig the production of yl and y. antibodies can be predetermined by the mode of immunization, these results are perhaps not surprising. However, these experiments point out the possibility that adjuvants such as alum often used in immunization procedures in man might modify the type of antibody eventually produced at a local site. In a comparative study of live oral vaccination (Sabin) and killed parenteral (Salk) vaccination with poliovirus in children, Ogra et d. (1968) showed that the serum antibody responses and their distribution in the three major immunoglobulin classes were approximately the same with either route of immunization using the sensitive radioimmunodiffusion technique employing "P-labeled poliovirus ( Fig. 15). However, after parenteral immunization with killed poliovirus, no secretory antibody could be detected in nasal washings or duodenal juice. Oral vaccination with live poliovirus did produce predominantly a y A immunoglobulin response in the secretions studied (no yG or yM antibody could be detected), whereas serum antibody measured simultaneously could be found in all three classes but was present in highest concentrations in yG. Investigations were not carried out on the antibody response t0,oral killed poliovirus in order to determine whether the use of live vs. killed vaccine is of importance in the local response. Hodes et aZ. ( 1964) have demonstrated that colostrum contains predominantly yA polio antibody and the same group of investigators (Berger et al., 1967) demonstrated only yA antibody to poliovirus in saliva and duodenal juice, whereas, in urine, yG antibody was found in addition to yA. The mode of immunization was not recorded in these studies. Oral killed cholera vaccine can produce local or coproantibody production ( Freter, 1962; Freter and Gangarosa, 1963) with minimum serum antibody levels. However, daily doses of vaccine had to be given for 4 weeks to produce satisfactory titers and subsequent weekly doses of oral vaccine were necessary to maintain coproantibody levels. Recruitment of local immune competent cells either in the wall of the gut or mesenteric lymph nodes might account for the local production of antibody. This suggestion is supported by the cell transfer experiments

53

SECRETORY IMMUNOGLOBULINS

of Thind (196f3). Cells obtained from the mesenteric lymph nodes or peripheral lymphoid tissues ( axillary or popliteal nodes) of rabbits, immunized either intravenously with cholera vaccine or fed the same vaccine intragastrically, were transferred to normal recipients. Mesenteric lymph node cells when obtained from animals immunized by either

&

128-

0

m t2

a 32v) 3

g > P

B -1 90

=

0 u

Y

8-

2-

Lu 0:


NASAL

I WCCINNION!

16

t

32

48

DAYS

t

64

80

a DUODENALr~ 96

FIG. 15. Relation of poliovirus antibodies in serum and secretions following live oral vs. killed p a r e n t e d vaccination. Only live oral vaccine resulted in significant titers in secretions. No yC or yh4 antibodies were detected in secretions. Antibody class measured by binding of 3'P-labeled poliovirus in radioimmunodifLive vaccine per 0s; ) inactivated vaccine intramuscularly. fusion. (-) [After Ogra et al. (1968).] (-0-0-

route were capable of transferring an immune response. Cells obtained from the axillary and popliteal nodes of animals receiving intragastric inoculation were incapable of producing a response in the recipients. However, when ceIIs from the same peripheral Iymphoid tissue were obtained from animals receiving intravenous immunization, they were able to transfer antibody production to the recipients. The presence of preformed antibody in the transferred cells was effectively exchded. Felsenfeld et al. (1967) have also obtained similar evidence of local

54

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

antibody production in the mesenteric lymph nodes of monkeys immunized parenterally with cholera toxin. These experiments point out quite clearly that parenteral immunization can give rise to antibody formation in local ( G I ) sites. Experiments in human volunteers given conventional cholera vaccine parenterally also show that the parenteral vaccine induces both circulating and coproantibody ( Freter, 1962). Thus, the relatively recent demonstration in field trials ( Phillips, 1966) that parenteral cholera vaccines are effective in reducing the case rate during epidemics may be due to the protection afforded by coproantibody; the latter being formed as a result of systemically administered antigen reaching local GI antibody-producing sites. The short-term immunity induced by the cholera vaccine might be explained by the relatively rapid disappearance. of coproantibody ( compared with serum antibody) as has been demonstrated in experimental animals. If this were the case, then perhaps continued administration of appropriately spaced oral vaccines to maintain coproantibody after its induction by the parenteral route would be more effective in prolonging immunity. The role of killed vs. live attenuated vaccines given orally (Mukerjee, 1965) is also worthy of further investigation since conceivable local replication of the live vibrio in the GI tract, as occurs with Sabin polio vaccine, could result in a more prolonged coproantibody response. The role of oral vaccines in cholera is well discussed by Freter (1965), but it should be emphasized that despite the highly suggestive evidence available, the functional role of coproantibody requires further clarification by experimental investigations on the site of production, the mechanism by which antigen reaches these sites, and by clinical studies on the biological consequences of local antibody production. b. Respiratory Antibodies. Results quite similar to those described for the GI tract have been obtained by a number of investigators interested in the immunity of the respiratory tract. Smith et al. (1966) demonstrated that resistance to infection with Type I parainfluenza virus was correlated with the presence of nasal antibody which was a better index of resistance to infection than serum antibody. However, in contradistinction to infection with the live virus, parenteral immunization with inactivated virus although resulting in significant serum titers failed to produce either nasal antibody or resistance. Smorodintsev and Chalkina (1955) had also suggested on the basis of clinical trials that live influenza virus was best as an immunizing agent when introduced intranasally and was more effective than parenteral immunization. The antibody in nasal secretions responsible for resistance to reinfection was found to be predominantly secretory yA (Smith et al., 1967). Antiviral

SECRETORY IMMUNOGLOBULINS

55

activity against a variety of different viral agents (including influenza, polio, adenovirus, ECHO, rhinoviruses, and coxsackie) have been described in normal nasal secretions (Artenstein et al., 1964). Investigations utilizing both direct and indirect techniques including sedimentation and chromatographic characteristics and absorption with specific antisera have clearly shown that the antiviral activity of nasal secretions is associated primarily with intermediate sedimenting yA (Table IX) . In some normals, and during respiratory infections, nasal secretions also show YG antiviral activity. What proportion of the yG activity is derived by transudation from serum vs. local production is unknown. However, during respiratory virus infections there is an increase in the concentration of several plasma proteins including yG in nasal secretions (Rossen et al., 1965), and in view of the predominance of viral antibodies of the yG class in most sera it seems likely that at least a portion of the yG activity in nasal fluids is derived from serum. It is of considerable interest that following either parenteral injection of the inactivated vaccine or infection with the live virus, serum antibody is in both cases primarily yG although y M and occasionally yA antibody is found (Smith et al., 1967; Lehrich et al., 1966). Following rhinovirus infection, neutralizing antibody activity is detected first in serum followed by its appearance simultaneously in three external secretions: parotid saliva, tears, and nasal fluids (Douglas et d., 1967). In order to explain the occurrence of antibodies in secretions distant from the site of virus replication it was suggested that antigen is disseminated from the original site of implantation (nasal mucosa) . The antigen must be disseminated early in infection (to explain the rapid rise in serum antibody) and is probably noninfectious since no evidence could be found of active virus in serum, lacrimal, or parotid fluids. In any case such a thesis would explain the integrated behavior of the antibody responses in serum and several secretions which were noted in this and other studies (Cate et al., 1966). Both serum and nasal antibodies to Francisella tularensis could be stimulated in respiratory tract secretions by either parenteral or aerosol immunization using a live attenuated vaccine ( Buescher and Bellanti, 1966). There was no clear correlation between the titers developed in the serum with those in nasal secretions following parenteral immunization. Moreover, absorption studies with specific antisera demonstrated that the antibody in the serum was primarily yM, whereas nasal antibody was entireIy yA (Buescher and Bellanti, 1966; Bellanti et al., 1967). Resistance to subsequent aerosol challenge with the same vaccine was found to be related to the dosage of bacillus administered and if a high

TABLE IX

CHARACTERIZATION OF SECRETORY ANTIBODIES FOLLOWING INFECTION OB IMMUNIZATION Antibody classa Secretion Nasal

Antigen

YA

Tr

+ +t +

Parainfluenza Type I Influenza A2 Polio I, 11, I11

+ Tr

Francisella tularensis

-

Salmonella typhosa endotofin Rhinovirus

Tr

Polio Rhinovirus Rhinovirus Tears Colostrum Polio, Coxsackie B5, Escherichia coli, Staphylococcus Duodenal Polio Saliva

Urine

rG

Salmonella typhi “H” Clostridium leiuni Blood group subs. Polio Escherichia coli “0”

-

Tr Tr -

-

+ + + + + +

+t

+t +t +t +t

+ +t + +t + + +

+t Tr -

+-

YM

Reference Smith et al. (1967) Artenstein et al. (1964) Bellanti et al. (1965) Ogra et d. (1968) Buescher and Bellanti (1966) Bellanti et al. (1967) Rossen et al. (1967b) Douglas et al. (1967) Rassen et al. (1966~) Cate et al. (1966) Berger et al. (1967) Douglas et al. (1967) Douglas et al. (1967) Hodes et al. (1964) Berger et al. (1967) Ogra et al. (1968) Turner and Rowe (1964) Turner and Rowe (1967) Hanmn and Tan (1965) Prager and Beerden (1965) Berger et al. (1967) Vosti and Remington (1968)

Key to symbols: (+) Evidence of strong activity in given immunoglobulin class; (-) evidence that activity not in given immunoglobulin class; (Tr) trace activity; (t) suggestive evidence for secretory yA activity.

SECRETORY IMMUNOGLOBULINS

57

enough dose of live F . tularensis were given, resistance was overcome even in the presence of significant titers of nasal antibody. Hornick and Eigelsbach (1966) have noted that the serum antibody response following aerosol immunization with live F . tularensis is more rapid than the response to parenteral vaccination and that the aerosol route appeared to produce a more effective immunity, although, as the authors point out, it remains to be determined whether this route is more advantageous for large-scale immunizations than the conventional dermal procedure. Intravenous injection of Salmonella typhosa endotoxin is followed by the appearance of antibody in nasal washings coincident with antibody in serum (Rossen et al., 1967b). In both secretions and serum the antibody response appeared to be heterogeneous although the methods used were indirect and, therefore, did not clearly establish the class of antibodies. However, from the sedimentation and other studies it appeared that there was little correlation between the types of antibodies in serum and nasal washings of the same individual and the predominant antibody in the nasal secretions was intermediate in sedimentation. It appears, therefore, that at least with certain antigens, both parenteral and aerosol (local) immunization results in the production of serum as well as secretory (nasal) antibodies. It is clear, however, that antibodies of different classes may predominate in the two fluids in the same individual. Whether, after aerosol immunization, antibody formed locdly in the respiratory tract contributes to serum antibody or whether the antigen is absorbed and reaches peripheral lymphoid tissue has not been determined. The latter possibility is suggested by the fact that serum antibody of different classes ( yG and yM in addition to y A ) are formed. It also seems likely that some of the parenteraIIy administered antigen reaches local respiratory sites of antibody production. However, the work with polio vaccine (Ogra et al., 1968) suggests that this is not always the case and whether mucoantibodies are formed following parenteral immunization may depend on a number of factors including the character of the antigen (type of virus, live vs. killed, etc.), the route and dose administered as well as the external secretion under investigation. c. Genitourinary Antibodies. Naylor and Caldwell ( 1953), following the earlier reports by Burrows and Havens (1948), demonstrated in studies on urinary enteric carriers the presence of antibodies in unconcentrated urine specific for the infecting organism. On immunization with an unrelated Salmonella, high titers were found in the serum but urinary titers were considerably lower than those against the carrier suggesting that pathotopic potentiation was not a significant factor.

58

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

T u n e r and Rowe (1964) investigated the response of human adults to Salmonella typhi immunization with a killed vaccine injected subcutaneously. Antibodies to the flagellar antigen were subsequently found both in the yA and yG classes in urine, whereas the serum response appeared to be principally yM and yA. No yM antibody activity was found in urine (also see Vosti and Remington, 1968). A similar distribution of antibody in the yA and yG classes in urine was found after immunization with tetanus toxoid (Turner and Rowe, 1967). Immunoglobulin fragments are known to occur in several external secretions such as urine and the secretions of the GI tract. Normal urine contains light chains and fragments of the heavy chains of yG such as Fc and F’c (Berggard, 1961; Turner and Rowe, 1966; Berggard and Bennich, 1967). It is unclear at present what percentage of these fragments results from degradation of parent molecules (in tissues, serum, or urine) and what proportion represents a by-product along the metabolic pathways of immunoglobulin production ( Bienenstock, 1968). It has been suggested that a specific concentrating or excretion mechanism exists in the kidney to account for the apparent preferential excretion of L chains (Solomon et al., 1964; Berggard, 1961). Although there have been reports of antibody activity of free L chains, the binding affinity is at best weak and their functional activity is questionable. The Fab fragment containing one antibody-combining site has not, thus far, been conclusively identified in normal urine or other secretions. Low-molecular-weight ( 10-20,000), biologically active molecules or fragments have been described in the urine which possess bivalent agglutinating and weak, complement-fixing, antibody activity ( Merler et al., 1963; Merler, 1966). These fragments when isolated contained only X light-chain determinants, and no K or InV determinants were detected ( Merler, 1966). Low-molecular-weight antibody activities have also been described in urine by Hanson and Tan ( 1965), and similar size molecules capable of agglutination have recently been described in the sera of man and several animal species by Rossen et al. (1967a) and in saliva and tears by Douglas et al. (1967). It is difficult to conceive how such fragments are constructed in the light of current knowledge of immunoglobulin structure. Turner and Rowe (1964, 1967) have not been able to confirm the existence of low-molecular-weight antibodies in urine. If antibodies with such low molecular weights do, in fact, exist in serum, they might by virtue of their small size and easy diffusibility play a significant functional role in secretions and in extravascular compartments throughout the body. Straus (1961) was able to demonstrate apparent local antibody pro-

SECRETORY IMMUNOGLOBULINS

59

duction in the cervical mucus of female volunteers following parented typhoid immunization. Antibody was detected earliest in the cervical mucus followed by serum antibody. When immunization was performed with soluble typhoid vaccine locally in the vagina the antibody response was higher in the cervical mucus than in the serum and persisted for a longer period of time. 2. Passive Inmunization There are numerous reports (Visek and Thomson, 1961; Thomson and Visek, 1963; Batty and Bullen, 1961; Schubert, 1938; Burrows and Havens, 1948) indicating that active parented immunization with certain antigens can give rise to antibodies both in serum and external secretions such as saliva and intestinal fluids. Such experiments cannot, however, be interpreted as evidence of transfer of antibody from serum to secretion since it is likely that antigen reaches local antibody-forming sites (Campbell and Garvey, 1965). Several studies have appeared on the transport of antibodies into external secretions following passive immunization in normal animals. Visek and Thomson (1961) reported that, following passive administration of rabbit antiurease antisera to rabbits, antibody activity appeared in the intestinal lumen. However, the quantitative aspects of transport were not determined. Batty and Bullen (1961) injected Clostridiurn welclrii antitoxin intravenously into rabbits and sheep and found antitoxin activity in the intestinal contents ranging up to a maximum of 1.3% of the serum concentration. The transfer of heterologous antitoxin was only slightly greater than homologous antitoxin, and there were essentially no differences between the pepsin-treated and the unaltered antibody. In these experiments it was interesting that the titers in the duodenum were definitely lower than those in the ileum and were quite constant with time despite a significant fall in the serum titers. Burrows and Havens ( 1948) administered homologous and heterologous cholera antitoxin intraperitoneally to guinea pigs and found significant titers (24% of serum titers) of antitoxin in urine and feces. However, there was a more rapid disappearance of antibody from the feces and after 2 weeks fecal antibody could not be detected in spite of persisting high serum levels of antitoxin. These authors concluded that although antibody is definitely excreted in feces and urine following passive administration, the kinetics were not consistent with a simple diffusion of semm antibody into the bowel. In these experiments antitoxin was also administered passively into the lumen of the bowel of adult guinea pigs. Surprisingly, significant serum titers were obtained so that the authors

80

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

were able to conclude that the passage of immune globulins across the intestinal tract occurred readily in either direction. In many of the experiments involving passive transfer, relatively large volumes of serum were administered and it is questionable whether these animals should be considered normal. This is particularly pertinent when heterologous antisera are used. In addition, in some of the studies the long persistence of serum titers suggested the possibility of the presence of antigen as well as antibody in the antisera administered. Since in cholera the infection is confined to the intestinal lumen (Burrows et a]., 1947) and the intesinal mucosa appears to retain its gross integrity except for some submucosal edema (Greenough, 1966; Fresh et al., 1964), this disease has lent itself to the study of immunoglobulin transfer across the intestinal wall. However, studies in cholera are not strictly applicable to the question of transfer in normal subjects. Freter (1965) found that adult guinea pigs were protected from otherwise fatal cholera infection by the oral administration of rabbit antisera to Vibrio cholerae. No protection was observed when the antiserum was given intraperitoneally even in the presence of high titers of circulating antibody. Similar experiments in adult rabbits showed little protection by intraperitoneal administration of antiserum, although some delay occurred in onset in symptoms ( McIntyre and Feeley, 1964). Analogous findings have been reported by Finkelstein et al. (1964). Finkelstein ( 1965) has shown that parenterally administered antisera, which have potent neutralizing activity against the choleragenic principle elaborated by vibrios in culture, did not protect the recipients against oral challenge with the choleragen. It was concluded in this study that since the choleragen is confined to the intestinal lumen in cholera and exerts its effect locally, no antibody had crossed the intestinal wall. Panse et al. (1964) investigated the effect of antisera, raised in adult rabbits, on the resistance of suckling rabbits to cholera. These investigators concluded that only antisera originally made against live vibrios afforded protection to the infant rabbits when given parenterally. Antisera made against somatic or flagellar antigens, or a lysate of vibrios did not protect the recipients against oral challenge with live organisms. Successful protection against oral challenge with vibrio by the use of parented passive administration of antisera against V. cholera was also found by Feeley (1965). This investigator showed little correlation between protective effect and levels of agglutinating or vibriocidal antibody and suggested that the immunoglobulin class of antibody in the donor antiserum might give a better correlation with protection. Further experiments (Panse and Dutta, 1964) on infant rabbits born of immunized

SECRETORY IMMUNOGLOBULINS

61

mothers demonstrated complete protection only in those animals whose mothers had received live vibrio vaccine. No protection was observed in infant rabbits whose mothers had received formolized or inactivated vaccine despite good serum antibody responses in the mothers. The results of these passive transfer experiments are confusing; some apparently show passage of antibody into the bowel lumen while others fail to demonstrate significant protection from parenterally administered antibody. These inconsistencies may result from a number of experimental factors. The volume of antisera administered and the dose and virulence of the organism in relation to the amount and types of antibody in the antisera may be important factors. As pointed out in several of these reports, antibodies against a variety of vibrio antigens are produced on immunization with this organism. It is probable that the main choleragenic principle is an exotoxin, and only certain antisera contain antitoxin antibodies. In addition, the immunoglobulin class of antibody could be important in detennining whether antibody is transferred to the intestinal lumen. Many of these experiments have been performed in the infant rabbit or the adult guinea pig and far-reaching analogies in the human and other species would at present be unjustified. In addition, though the morphology of the intestine may be relatively maintained the integrity of the capillaries and lymphatics may be affected in cholera (Sprinz, 1966) and the possibility exists that some leakage of serum antibody into the intestinal lumen occurs.

FIXATION C. COMPLEMENT Unlike yG and yM serum antibodies, serum yA does not have the ability to activate the first complement component in the sequence of complement fixation (Ishizaka et d,1966a). South et al. (1966) were unable to demonstrate complement fixation by secretory yA after attempting to heat-aggregate the molecule, and colostral yA isohemagglutinins do not fix complement (Tomasi and Duda, unpublished; Adinolti et al., 1966a). Adinolfi et al. (1966b) have suggested on the basis of experiments with natural colostral yA antibodies to Escherichia coli that lysis by secretory yA occurs only in the presence of lysozyme and a source of complement. Removal of any one component from the system (antibody, lysozyme, complement) prevented lysis of the bacteria. Lysozyme is found in most of the external secretions discussed in this review in relatively large quantities. The complement components ,8,c and PIEhave also been demonstrated in most secretions. Component C’1 has been shown to be synthesized by guinea pig small intestine (Colten et al., 1966). Surprisingly PICand PIE were not found in six

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

samples of concentrated parotid saliva (Tomasi and Bienenstock, unpublished) using specific antisera in gel diffusion. The observations concerning a possible interrelationship of complement components, lysozyme, and secretory yA are of considerable interest and deserve further study. IX.

Secretory Immunoglobulins in Disease

One of the technical problems which must be emphasized in investigations of secretory immunoglobulins in disease is that disturbances of the normal physiological integrity of organ structures may occur and lead to the passage of serum proteins into secretions. These considerations make it difficult fully to evaluate the question of local synthesis of antibodies except when experiments are performed analogous to those devised by Oakley et al. (1955) in which the relative titers of antibodies against two unrelated antigens are measured in serum and secretions (see Section IV).

A. ANTIBODYDEFICIENCY STATES Patients with both the congenital and acquired forms of agammaglobulinemia manifest as a primary clinical characteristic recurrent infections. These patients show particular susceptibility to pyogenic infections and, although a variety of organ systems may be involved, the respiratory tract is a particularly frequent site of infection. It is well established that patients with agammaglobulinemia, in addition to manifesting deficiencies of circulating immunoglobulins of varying degree, also show deficiencies of the immunoglobulins in their secretions (South et at., 1966). Examination of various tissues by light microscopy has shown a marked deficiency in plasma cells and similarly fluorescent antibody studies have shown a paucity of 7-globulin-containing cells both in peripheral lymphoid tissues and at local sites such as the respiratory tract ( Martinez-Tello and Blanc, 1968) and GI tract (Eidelman et al., 1966). Since in most cases of agammaglobulinemia both serum and secretory antibodies are lacking or severely deficient, it is difficult to determine what role systemic vs. local antibody mediated immunity plays in the susceptibility of these patients to infection. In this regard it is known that resistance to infection with certain agents may be controlled either by circulating antibody or at the local level presumably, at least in part, by secretory antibody. For example, in the case of poliovirus, current evidence suggests that central nervous system (CNS) involvement can be prevented either by inhibiting the virus at the site of entry presumably by local antibody or by preventing the

SECRETORY IMMUNOGLOBULINS

63

stage of viremia which can be effectively accomplished by circulating antibody. In contrast, resistance to those diseases that are primarily restricted to mucous membranes and are not characterized by systemic involvement (e.g., cholera) might be expected to be related more closely to local immune mechanisms. It is reasonable to hypothesize in our present state of ignorance that resistance to and recovery from infections which are restricted to mucous membranes might result from the cooperative effects of both circulating and locally produced antibody. For example, colonization of a virus in the respiratory tract could be prevented, depending upon the dose and virulence of the virus, primarily by local immune mechanisms and evidence is available to support this thesis (Ogra et nl., 1968; Buescher and Bellanti, 1966). Should, however, local defense mechanisms be inadequate then the resulting inflammatory reaction would result in transudation of serum antibody and thus allow its contribution, along with many other factors, to the local defense reaction. These considerations have some bearing on the efficacy of treatment of dysproteinemias with 7-globulin. It is known that periodic parenteral administration of therapeutic doses of commercial preparations of y-globulin may result in significant decreases in the incidence of infections in patients with agammaglobulinemia. One might suppose that, in cases where administration of 7-globulin has a beneficial effect, the circulating antibodies prevent viremia or septicemia, or they may reach the local site subsequent to colonizatio~by the microorganism and the induction of an inflammatory reaction. However, in some cases administration of 7-globulin is only partially or not at all effective in reducing infectious complications. The reasons for therapeutic failures are, of course, complex and include inadequate dosage, lack of antibody activity in the y-globulin preparation, etc. Pertinent to this discussion, is the possibility that systemically administered antibody does not reach local sites in sufficient amounts. A number of studies supporting this hypothesis have been presented in this review. There is little evidence in the human that the yG antibodies which are the predominant class present in commercial preparations are able to reach mucous membranes and their secretions. It has been suggested that, perhaps, because of the predominance of yA in most secretions, therapy with preparations rich in this immunoglobulin would be more efficacious. However, to our knowledge preparations of human yA suitable for therapeutic use are not commercially available. Moreover, as mentioned above ( see Section VII ), there is evidence suggesting that yA administered parenterally is not excreted onto mucous surfaces to a significant extent. It seems un-

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THOMAS B. TOMASI, JR. AND JOHN BENENSTOCK

likely, therefore, that preparations containing high concentrations of y A, which would be extremely difficult and costly to prepare in the amounts necessary for clinical use, would be any more effective than the y-globulin preparations now available. Agammaglobulinemia also occurs in hereditary thymic aplasia (Swisstype agammaglobulinemia). In this disorder there are defects in both circulating antibody and cellular ( delayed-type ) mechanisms. The characteristic clinical syndrome is so severe and complex that it is difficult to determine what role deficiencies in the secretory system play in the disease syndrome. Patients with acquired agammaglobulinemia show a high incidence (20-5048) of diarrhea and malabsorption syndromes. The most common histological features on examination of the small bowel include blunted villi, a lymphocytic infiltration, and the absence of plasma cells in the lamina propria. Specific organisms are rarely cultured and antibiotic therapy is only occasionally effective. The administration of y-globulin has been reported in some patients to be beneficial, but in the majority it has little effect on the GI symptoms. In Type I1 dysgammaglobulinemia, characterized by absent serum yA and yM with normal or slightly decreased yG, diarrhea and malabsorption are sometimes associated with infestation with Giardia lamblia. These patients commonly have a striking nodular lymphoid hyperplasia (Hermans et al., 1966) which can often be visualized on roentgenographic examination of the small intestine. Failure to culture specific organisms, the lack of response to administered antibiotics, together with the fact that patients with congenital agammaglobulinemia rarely manifest GI disease make infection an unlikely etiological agent for the malabsorption in these dysproteinemias. The role of the giardia has not been clearly defined but it seems unlikely that it causes the GI manifestations. The relationship of the y-globulin deficiency in these syndromes both in the serum and secretions to the GI disease is as yet obscure. Little definitive information is available concerning the secretory antibody system in the GI tract in these disorders (Bull and Tomasi, 1968). Crabbk and Heremans (1966a, 196%) have recently pointed out an association between isolated -yA deficiency and a malabsorption syndrome closely resembling nontropical sprue. These patients, in addition to absence of serum yA, show deficiencies of yA in several exocrine secretions (Cattan et al., 1966; Crabbe and Heremans, 196%). Fluorescent antibody studies of the GI tract showed markedly decreased numbers of yA-containing cells in the lamina propria of biopsy specimens. The authors considered these patients to represent a new syn-

SECRETORY IMMUNOGLOBULINS

65

drome differing from nontropical sprue primarily in its association with yA deficiency. The clinical picture resembles quite closely nontropical sprue and the steatorrhea and histological (spruelike) changes in the intestinal tract frequently improved on a glutenfree diet. The yA levels in these patients are unaffected by treatment. The relationship of the yA deficiency to the intestinal maIabsorption syndrome is unknown. It should be noted that in these patients there is an apparent replacement of yA- by yM-containing cells in the intestinal tract and whether an absolute or relative antibody deficiency syndrome, in fact, exists in the GI tract and other exocrine secretions remains to be established by quantitative studies of immunoglobulin levels and specific antibody activities. As appears to be the case with the patients with hereditary telangiectasia discussed below, it may be that total immunoglobulin levels in the secretions of these patients are nearly normal, consisting primarily of yG and particularly yM. In this regard, the two healthy individuals described by Rockey at al. (1964) with absent yA also appeared to replace yA in their secretions with both yG and yM (Tomasi et al., 1965), as did the patient reported by South et al. (1966). Little can be offered except speculation to explain the pathophysiology of the relationship between the yA deficiency and the intestinal disease. One could postulate that the immune deficit and the intestinal disease are directly related as cause and effect. A relative antibody deficiency at the mucosal surface could allow infection with fastidious and as yet undefined infectious agents. Mucosal damage could also be produced by products of bacteria, wheat, or milk which would otherwise be inactivated by immune mechanisms. Also, the immune deficit and the gastroenteropathy could be a manifestation of the same underlying disease process, but the antibody deficiency might have no direct pathogenic relationship to the GI disease. In this respect it should be noted that there are a variety of diseases in which isolated yA deficiencies have been described in higher than normal frequency. These include hereditary telangiectasia, lupus erythematosus, cirrhosis of the liver, and Still's disease (for review, see Bull and Tomasi, 1968). In these disorders there is no apparent relationship between the basic disease process and the immune deficit. Finally, it may be that gastroenteropathy and the immune deficit are totally unrelated. In this regard it has been found that isolated yA deficiency may occur in a significant number (1in 400 to 700) of normal individuals (Bachmann, 1965). A recent interesting study (Swanson et al., 1968) reports that malabsorption may occur with absent yA in serum but with the presence of YA-containing cells in the intestinal tract. This patient had steatorrhea

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

and, on intestinal biopsy, showed blunted villi and infiltration of the lamina propria with lymphocytes and plasma-cytoid cells. Fluorescent studies of the lamina propria of the appendix and colon showed normal numbers of yA- and y M-containing cells. The patient's serum contained less than 0.05 mg./ml. of yA as detected by gel diffusion. The authors considered two explanations to account for the discrepancy between the tissue and serum yA: ( I ) the release of yA by the cells of the GI tract (and elsewhere) into the serum is abnormal, and ( 2 ) the cells responsible for the synthesis of serum yA and those in the lamina propria are independent and controlled by different mechanisms. This study is similar in certain aspects to the cases of hereditary telangiectasia reported by McFarlin et al. (1965). In these patients, serum yA was absent but yA-staining cells were found in bone marrow and parotid tissue and yA was also demonstrated in the saliva. The apparent absence of serum yA in the presence of secretory yA is, however, difficult to reconcile with other studies. For example, Eidelman et al. (1966), in a study of fluorescent cells in 21 normal controls, 6 patients with sprue,' and 5 hypogammaglobulinemic subjects reported a direct relationship between numbers of yA-containing intestinal plasma cells and the serum level of yA. This suggested to them the possibility that the GI tract may be a major site of synthesis of serum yA. Also, in patients with hereditary telangiectasia, rectal biopsies have shown a complete absence of ykcontaining cells in the lamina propria (Eidelman and Davis, 1967). Investigations in our laboratory of a large number of patients with both isolated and multiple immunoglobulin deficiencies have invariably shown that when yA is absent from the serum when estimated by sensitive techniques such as gel diffusion and complement fixation, it is also undetectable in salivary secretions. However, as pointed out (Table VII), some patients with low levels of serum yA have normal secretory yA concentrations. One wonders, therefore, in the situations discussed above showing dissociation between serum and secretory yA whether careful examination of serum by sensitive techniques may not have revealed the presence of yA, although from the data presented this does not seem to be the case in the report by Swanson et al. (1968). In hereditary telangiectasia, approximately 80%of the patients have absent serum yA. Stobo and Tomasi (1967) and Bellanti et al. (1966) have reported that the secretory yA is also absent in these patients and that there is a replacement of yA with yG and particularly yM; the total level of immunoglobulins in parotid saliva being essentially normal. Moreover, antibody activity in the form of isohemagglutinins has been found in saliva of patients with hereditary telangiectasia (Stobo and

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67

Toiixisi, 1967). Bellanti ct al. (1966) have shown that such patients on immunization with srvcral viruses develop nasal antibody activity attributable to and yM. In addition the levels of immunoglobulins in the serum or saliva could not be correlated with susceptibility to infection ( Stobo and Tomasi, 1967). The available evidence suggests that selective serum or secretory deficiencies of yA per se do not account for the increased susceptibility of these patients to infections. It may be that the susceptibility to infection displayed by patients with ataxia telangiectasia is more attributable to defects in delayed hypersensitivity than to the immunoglobulin abnormalities. However, further studies, particularly of the secretory antibody response to various types of antigenic challenge, are necessary before definite conclusions can be reached. From the above discussion it is obvious that at the present time it is not possible to assess the significance of the association of specific disease syndromes with serum or secretory immunoglobulin deficiencies. Not all patients with a yA deficiency have recurrent infections or malabsorption syndrome since this immunoglobulin deficiency can occur in apparently healthy individuals. Various diseases exist in association with a yA deficiency in which the primary pathological features are markedly different and have no apparent relationship to one another; e.g., hereditary telangiectasia with yA deficiency and Still's disease with yA deficiency. Moreover, patients with ataxia telangiectasia do not have arthritis as a cardinal manifestation of their disease nor do patients with Still's disease and yA deficiency have CNS involvement. In addition, in many of the diseases which have been associated with immunological deficiencies, apparently identical clinical syndromes exist in which the immunoglobulin levels are perfectly normal.

B. GASTROINTESTINAL DISEASES 1 . Caries and Periodontal Disease

A large number of reports have appeared concerning the possible relationship of the antibodies present in saliva to dental caries and periodontal disease. [For reviews, see Green ( 1966), Afonsky ( 196l), and Ellison and Mandel (1963).] This problem is, however, extremely complex both because of the paucity of information available concerning the character and function of salivary constituents, and our ignorance of the etiological factors involved in the production of caries and periodontal disease. There is reasonable information available suggesting that microorganisms, particularly streptococci may be involved in production of caries. This is perhaps best illustrated by experiments in

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

which certain strains of streptococci werc found to produce caries upon introduction into germfree animals (Gibbons et nl., 1966). There are many reports describing the occurrence of antibodies directed against a variety of oral bacteria including streptococci in human saliva (see Kraus and Konno, 1963; Evans and Mergenhagen, 1965). The difficulty has been in defining the role of these antibodies in resistance to the development of caries. Bacteriolytic factors have been found in saliva which are effective against oral lactobacilli and various strains of streptococci. It has been reported that the lytic factor( s ) has properties similar to secretory yA in its behavior on DEAE and Sephadex chromatography and is found in yA-rich fractions isolated from parotid saliva ( Green, 1966). Some evidence has been presented that higher concentrations of both bacteriolytic factor (Green, 1966) and the yA class of immunoglobulins (Lehner et al., 1967) are found in caries-immune individuals compared to those who are susceptible to caries. This is also supported by the findings of Geller and Rovelstad (1959) that there is a relative deficiency of yglobulin, determined by electrophoresis, in the parotid saliva of the caries-prone group as compared with the low-caries group. Although these findings are of considerable interest and worth pursuing, it has certainly not been clearly established that the bacteriolytic factor referred to above is, indeed, y A-globulin. Moreover, the differences in salivary levels of antibodies as well as other substances between groups of caries-prone and caries-resistant individuals are not totally convincing. With periodontal disease, as with caries, the situation is extremely complex, and it seems likely that multiple factors may be involved in its development. Several workers have reported that the severity of the disease is a linear function of the accumulation of dental plaques which are composed almost entirely of bacteria (Socransky et al., 1963). Although the disease may be primarily bacterial, a number of other host factors may enter into susceptibility and individual differences in the seventy of the disease. Little definitive information is available concerning the role of salivary antibodies in determining susceptibility to periodontal disease. In an extensive study by Brandtzaeg and Kraus (1965), no evidence could be found that autoimmune phenomena were involved as had been suggested by others. However, the histological observation of immunoglobulin-producing cells primarily yG in infiamed gingiva suggested to these authors the possibility of a hypersensitivity reaction occurring locally in the inflamed tissue possibly due to antigenic products of oral bacteria diffusing into the gingival connective tissue. It is of interest in this regard that the gingival pocket fluid which

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69

is in intimate contact with the tooth is similar in immunoglobulin content to that of serum and, thus, is not typical of other external secretions ( Brandtzaeg, 1965).

2. Pernicious Anemia and Gastritis Antibodies to gastric parietal cell constituents and intrinsic factorB,? complex have been described in serum and gastric juice of patients with pernicious anemia (PA) and atrophic gastritis (Table X). Parietal cell antibodies occur in the serum of approximately 75%of patients with PA and appear to be mainly YG in type although yA antibodies are also found. The yG class of antibody seems to predominate in gastric juice (Jeffries and Sleisenger, 1965; Fisher et al., 1965). In 5 patients with circulating antibodies to both gastric parietal and thyroid acinar cells, Fisher et al. (1965) found that the gastric juice of all 5 patients lacked antibodies against the thyroid acinar antigen, whereas 4 out 5 contained the antiparietal cell antibodies, This suggested to the authors that the parietal cell antibody was either synthesized locally in the stomach or that selective transport from serum occurred only with the parietal cell antibody. It seems likely that the parietal cell antibodies demonstrated by immunofluorescence are in large part responsible for the fixation of complement with gastric mucosal extracts. However, other noncomplement-fixing antibodies to parietal cell constituents (such as antibody to intrinsic factor) may also be responsible for positive fluorescence. The presence of parietal cell antibodies seems to be related to gastric inflammation and have been described in chronic gastritis (Taylor et al., 1961) and iron-deficiency anemia (Dagg et al., 1964), as well as in a significant number of apparently healthy individuals (Taylor et al., 1962). A high incidence of these antibodies also occur in patients with various types of thyroid disease and their relatives (Doniach et al., 1965). Thus this antibody is relatively “nonspecific” and of little value in the diagnosis of pernicious anemia. In contrast to parietal cell antibody the antibody to intrinsic factor (IF) is much more specific for PA. It is seldom present in the serum of normal individuals of any age and is not found in gastritis without PA ( Fisher and Taylor, 1965), although it occasionally occurs in thyroid disease (Schiller et al., 1965). In juvenile PA, significant gastritis and achlorhydria are usually absent and I F antibodies are not founcl (Doniach et ul., 1965; McIntyre et al., 1965). Serum antibodies to I F are found in approximately 60%of patients with PA, and like the parietal cell antibody are predominately of the yG class (Carmel and Herbert, 1967). Antibodies to IF have also been

TABLE X

SECRETORYANTIBODIES IN DISEASE Secretion Saliva

Disease

Type of antibody

systemic lupus Rheumatoid arthritis

Antinuclear factors Rheumatoid factor

Gastric juice

Pernicious anemia Pernicious anemia and atrophic gast,ritis

Intrinsic factor-Blz antibody Gastric parietal cell antibody

Urine

Pernicious anemia systemic lupus

Intrinsic factor-Blz antibody Antinuclear factors

Rheumatoid arthrit,is

Rheumatoid factor

a

Shown to be of secretory type.

Antibody class

-YA rA

-4"

?A YG rG r G (rA) YG Low mol. wt., -fG -YAe

Reference Tomasi et al. (1965) Heimer and Levin (1966) Tomasi et al. (1965) Camel and Herbert (1967) Fisher and Taylor (1965) Jeff ries and Sleisenger (1965) Fisher et al. (1965) Camel and Herbert (1967) Hanson and Tan (1965) Bienenstock and Tomasi (1967)

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71

found in the gastric juicc of approximately 1 out of 3 patients with PA (Fisher et al., 1966) hiit the immunoglobulin class involved has not been determined. In some patients circulating antibody is present without gastric juice antibody and, in 1 case reported (Fisher et d., 1966), IF inhibitor (presumably antibody) was found in the gastric juice but not in serum. It appears likely from this and other data (see Taylor, 1966) that serum antibody does not always reflect the occurrence of inhibitors of IF in the GI tract. In 1 patient, antibody was demonstrated in the serum, gastric juice, and jejunal juice in the absence of a similar antibody in the saliva (Schade et al., 1966). Carmel and Herbert (1967) have described yA antibodies to IF in the saliva of 1 patient with PA while the gastric and serum antibodies were predominantly yG. Dissociation of antibody between serum, gastric juice, and saliva suggests the possibility of local anti-IF antibody synthesis. It should be pointed out that, with the gastritis that so often accompanies PA, significant serum leakage undoubtedly occurs and could account, at least in part, for the finding of yG antibodies both to parietal cell and IF in gastric juice. The pathogenic significance of these antibodies in external secretions is uncertain. It seems unlikely that the parietaI cell antibody is directly involved in the pathogenesis of PA. In some cases it may be a nonspecific manifestation of inflammatory gastric disease or alternatively an expression of an underlying aberration in immune mechanisms. It is conceivable that in some cases it contributes, perhaps in a secondary fashion, to perpetuation of the local gastric inflammation and thus serves to stimulate more specific antibodies such as those directed against IF. The I F antibodies, on the other hand, could be specifically related to the pathogenesis of PA. Such antibodies present in saliva and GI secretions could inhibit the action of IF in promoting vitamin B,, absorption particularly if IF production was already impaired by gastric disease. In addition it is conceivable that antibody might inhibit synthesis of IF in some as yet unknown manner, as has been reported in other systems (Dray, 1962). The improvement in B,, absorption which follows steroid therapy in patients with PA is associated with a fall in the level of IF antibody in the serum (Jeffries et al., 1962, 1966), and this may be another indication of the role of IF antibodies in the pathogenesis of PA. 3. Celiac Disease

It has been suggested mainly on the basis of clinical observations that celiac disease might be due to local hypersensitivity to wheat gluten

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THOMAS B. TOMASI, JR. AND JOHN BENENSTOCK

(Taylor et nl., 1964). Such observations include the beneficial responses to a glutenfree diet and to steroid therapy, and the occasional occurrence of a dramatic reaction to small doses of gluten, so-called “gliadin shock.” This suggestion is strengthened by the finding of an increased frequency of antibodies to wheat gluten in the sera of both adults and children with this disorder. Antibodies to milk proteins have also been found in high titers and incidence in celiac sera (Sewell et al., 1963). However the relationship of the serum titers of these antibodies to the GI disease is not clear, and it is conceivable that the diseased bowel allows permeation of macromolecules or sizable fragments of molecules and that the absorbed antigenic material reaches peripheral lymphoid tissues. Considerable evidence reviewed above suggests that coproantibodies can be formed locally in the GI tract. This is supported by experiments in rabbits in which bovine serum albumin given orally induced an effective serum antibody response (Rothberg et al., 1967) and by the ability of intestinal lymphocytes to transfer antibody-producing capacity to other lymphoid tissue (Farr et al., 1960; Farr and Dickinson, 1961). Experiments with orally administered egg albumin have demonstrated antibody production in GI lymphoid cells, using the immunofluorescent technique (Crabbe and Heremans, 1966d), In ceIiac disease it has been postulated that a local hypersensitivity reaction occurs as a result of the reaction of coproantibody (locally produced) and fractions of wheat gliadin. However, careful immunofluorescent studies ( Rubin et al., 1965) showed no evidence of gliadin-binding antibodies in the intestinal biopsies from 9 patients with adult celiac disease, and complement-fixing antigen-antibody aggregates could not be demonstrated in the intestinal mucosa. Autoantibodies against bowel epithelial cells which had been suggested by other workers (Malik et al., 1964) were not found in these studies.

4. Ulcerative Colitis Suggestions that disturbances of immunity may underlie the pathogenesis of ulcerative colitis have been repeatedly voiced and have been recently reviewed by Broberger (1964) and by Kraft and Kirsner ( 1966). Such investigations have mainly emphasized serological aspects and have not had recourse to local secretions. It has been suggested that local immediate-type hypersensitivity reactions to exogenous antigens such as milk proteins may be a causative or exaggerating factor in ulcerative colitis. Evidence for this thesis comes primarily from the clinical response to elimination diets and more recently from direct testing by injections of suspected allergens into the rectal mucosa (Rider and

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73

Moeller, 1962). However, in general, the evidence is far from convincing that a reaginic mucosal allergy plays a significant role in the majority of cases of ulcerative colitis. Circulating antibodies against colonic extracts have been reported in both children and adults with ulcerative colitis (Broberger and Perlmann, 1959). These antibodies react with colon antigens from germfree rats (Perlmann et ul., 1965) and in vitro with human fetal colonic cells in tissue culture ( Broberger and Perlmann, 1963). They are, therefore, not directed against contaminating bacteria. Recently evidence has been provided for an immunological cross-reaction between a colon antigen from germfree animals and a lipopolysaccharide extractable from Escherichiu coli O,, (Perlmann et al., 1965). Using the fluorescent antibody technique these antibodies react with cytoplasmic antigens in colonic epithelial cells ( Broberger and Perlmann, 1962). This observation has led to the hypothesis that antibodies formed against intestinal bacteria may cross-react with colonic mucosaI antigens and produce disease. This mechanism is similar to that postulated for the development of carditis in rheumatic fever secondary to the reaction of antibodies formed against streptococci with chemically related cardiac muscle antigens. Colon-reactive antibodies have been found in the regional nodes of the colon, but not in the small intestinal lymph glands of the same individual ( Perlmann and Broberger, 1960), suggesting their local production in the large bowel. It is conceivable, therefore, that if the postulated immunological phenomenon occurs, it may be a result of local immune reactions. In this regard it is known that there is little correlation between the circulating antibody and clinical course ( Harrison, 1965), and anticolon antibodies may occur in the absence of ulcerative lesions in the colon. However, little definitive information except that mentioned above, is available to support a local immune reaction. Histologically the colon in ulcerative colitis shows a cellular infiltrate with increased numbers of lymphocytes and plasma cells together with other cell types. Fluorescent studies have shown decreased numbers of yA cells but with areas of amorphous extracellular material which stains with anti yA antisera (Gelzayd et al., 1967b). This may represent yA released from cells. Fluorescent studies have failed to visualize aggregates of y-globulin and complement at the mucosal epithelial cell layer presumably the location of antigen. One report has appeared in which inmunofluorescent studies in a patient with ulcerative colitis demonstrated the presence of an unusually large number of plasma cells staining with anti-yD antisera in

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THOMAS B. TOMASI, JR. AND JOHN BlENENSTOCK

several rectal biopsies ( Crabb6 and Heremans, 1966b). These cells were distributed in the mucosa and granulomatous lesions. This finding is unusual since yD-staining cells are rarely found in normal intestinal and lymphoid tissues except in the nasopharyngeal adenoids. The significance of this observation awaits clarification.

5. Gastrointestinal Allergy Gastrointestinal allergic reactions presumably mediated by local antibody directed against a specific antigen have been frequently suggested but rarely well documented. A thoroughly studied case has been reported of malabsorption secondary to hypersensitivity to p-lactoglobulin ( Davidson et ab., 1965). Waldmann et al. (1967) have suggested that a true allergic GI enteropathy can occur with GI edema, protein loss, and eosinophilia together with more usual systemic manifestations of allergic disease such as eczema, asthma, and rhinitis. Evidence was presented that allergy to milk proteins was related to this type of GI disease in 3 of 6 patients who showed characteristic small bowel biopsies with marked eosinophilia. Local allergy to milk apparently giving rise to occult GI hemorrhage in infants has also been described by Wilson ( 1965). Although these infants have high titers of antibodies against milk proteins in the serum, the immunological nature of these disorders remains to be established. Coproantibodies to cows’ milk proteins have been found in children with GI bleeding and protein-losing enteropathy (Katz et ab., 1967). Milk allergy was suspected on the basis of these antibodies and a beneficial response to elimination of milk from the diet. It is of interest that several patients demonstrated coproantibodies in the absence of detectable precipitins in their sera. In addition, among those infants who die mysteriously of sudden cot death, allergy to cows’ milk has been suggested by some investigators to play a major role (Parish et al., 1964). Again, the role of antibodies to milk proteins which occur in high titers in the serum of these infants (as well as a significant percentage of normals) is uncertain. In this regard, using the fluorescent technique, specific antibody production in peripheral lymph nodes has been shown following oral administration of egg albumin in humans (Crabb6 and Heremans, 1966d) and rabbits. Rothberg et al. (1967) have found that small repeated oral doses of bovine serum albumin eventually produce serum antibody titers similar to those obtained by other routes of irnmunizat‘ion.

6. Parasitic Infestation The veterinary literature in recent years is replete with suggestions that reaginic antibody appears to be related to the self-cure phe-

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75

noinenon found in parasitic nematode infestations in several animal species (for review, see Bloch, 1967; Ogilvic, 1967; Dobson, 1966a,b,c). The production of reaginic-type antibody in animals in response to helminth infestation has been possible in animals only with the introduction of living worms into the GI tract (Soulsby, 1962), and worm extracts given parenterally do not stimulate active immunity (Ogilvie, 1967). However, it is possible to transfer immunity to normal animals by passive parented administration of the serum of naturally infected animals (Mulligan et al., 1965; Ogilvie, 1964; Ogilvie et al., 1960). Such experiments would suggest the ability of reaginic antibody to fix locally to the mucosa of the GI tract. Barth et al. (1966) demonstrated that nonspecific anaphylactic reactions in the rat gut, induced by an unrelated antigen-antibody system, have no direct detrimental effect on the worms but will enhance their elimination from the gut by passively administered immune serum. This has been interpreted by the authors as evidence that protective antibodies do not cross the intact gut but that the anaphylactic reaction and, in the natural infection, the worms themselves affect the integrity of the gut wall in such a way as to allow passage of serum antibodies. The nature of the protective antibodies has not been clearly established but they appear to resemble human reagins in that they are intermediate sedimenting, fast migrating antibodies which are skin sensitizing in the homologous species ( Ogilvie, 1967). Blocking antibodies of the 7s and 19 S types are also found in the sera of infected animals. The exact mechanism whereby protection occurs is unknown, although it seems likely that the site of the protective action is in the intestinal tract where the worms are localized. A direct effect on the worms themselves is unlikely since the immune serum causes no apparent effect on the worms in uitro nor does it interfere with their oxygen uptake (Mulligan et al., 1965). It should be emphasized that although reaginic antibodies could have a protective function in man, as apparently they do in animals against parasitic infestation, no good evidence is available that this is, indeed, the case. In fact, thus far the allergic reactions which have been recorded in man in association with parasitic infestations have been demonstrably detrimental to the host ( Bloch, 1967).

7. Cholera The work in this area has, for the most part, been discussed in other sections (see Sections I1 and VII1,B) and will be only briefly reviewed here. From the work of Besredka in 1927 to the present day, the concept of local immunity of the GI tract against cholera has been upheld by

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THOMAS B. TOMASI,

JR. AND JOHN BIENENSTOCK

many investigators. Burrows and co-workers ( 1947; Burrows and Havens, 1948) clearly demonstrated in their extensive studies that protection was related to copro- and not serum antibody. In the rice water stool of most patients suffering from cholera, antibody can be found on about the fourth day of disease (Freter et at., 1965). Since in this disease the intestinal mucosa by and large retains its integrity and the violent diarrhea and passage of rice water stool appears to be due to the elaboration of a toxin by the vibrio, antibodies to cholera toxin itself are currently being investigated. In human cholera stool, secretory yA and undegraded yM can be detected (Northrup, Bienenstock, and Tomasi, unpublished) but they have not been shown to be directed against either the toxin or the intact vibrio. In the experiments of Felsenfeld et al. (1967), monkeys were given the vibrio toxin of Burrows via duodenal tube. The numbers of cholera toxin, antibody-producing cells in the regional mesenteric nodes were somewhat greater than those in the spleen of the same animal, All three classes of immunoglobulins were formed, but yG-containing cells predominated by about 3 to 1over yA cells. The toxin-neutralizing capacity of jejunal yG per milligram of globulin was greater than that of serum yG. Very high levels of yAneutralizing activity were found in the jejunal contents at a time when saliva and serum yA antibody titers were considerably lower. These experiments suggested that local production of both yG and yA antitoxin antibody occur in the regional nodes (and, perhaps, intestinal wall) but they do not exclude some participation by antibody derived from serum. As the authors rightly point out it is quite possible that antibodies are both produced locally and excreted from serum into the lumen.

C. RHEUMATOIDAND ANTINUCLEAR FACTORS Evidence has been presented for the occurrence of rheumatoid factors ( R F ) in external secretions, including saliva (Tomasi et al., 1965; Heimer and Levin, 1966) and urine (Bienenstock and Tomasi, 1967) of patients with rheumatoid arthritis. These patients had no apparent cIinical evidence of invoIvement of the salivary gland or urinary system and the urinary protein excretion was less than 100 mg. per 24 hours. Both the salivary and urinary RF were found primarily in the yA class and, in the urine, it was shown to be of the 11S secretory type. In urine, yG and yM R F were not found, and there was little correlation between serum and urine titers although urinary R F was demonstrated only in those patients with positive serum titers. In the patients reported with yA RF activity in external secretions, simultaneous

SECRETORY IMMUNOGLOBULINS

77

determination of the character of the serum RF was not mentioned although it is known from other studies (Heimer and Levin, 1966; Torrigiani and Roitt, 1967) that serum yA R F probably exist. No experiments were designed to determine if the salivary gland and renal tract can independently participate in the production of RF or whqther it is derived from serum. Antinuclear factors with intermediate sedimentation characteristics (presumed but not proven to be yA) have been described in saliva of some patients with systemic lupus (Tomasi et al., 1965). Hanson and Tan (1965) have reported urinary antinuclear factors in both yG and low-molecular-weight fractions.

D. SECRETORY IMMUNOGLOBULINS IN RESPIRATORY ALLERGIES Many of the clinical manifestations of common allergies such as hay fever and asthma occur at the mucous membranes of the respiratory tract. It is presumed that the release of pharmacologically active agents which are responsible for clinical symptoms are triggered by immediatetype hypersensitivity reactions involving antigen-antibody interactions at or near the mucous membrane surface. Since the mucous membranes of the respiratory tract are bathed by secretions that contain predominantly secretory yA antibodies, considerable recent interest has centered around the possible participation of local immunoglobulins in respiratory allergies. In man the anaphylactic (reaginic or skin-sensitizing) antibody in serum, previously thought to be yA, has been recently shown by Ishizaka et al. (1966b,c) to belong to a new class of immunoglobulins termed YE. The YE is a frequent contaminant of preparations of serum yA, and anti-yA antisera may contain antibodies directed against the spec& H chain of the yE molecule. Serum y E appears to be responsible for the Prausnitz-Kiistner (PK) reaction in most allergic patients and is probably the same antibody which sensitizes monkey ileum for the release of histamine in the classic Schultz-Dale reaction ( Arbesman et al., 1968). Recently, a myeloma protein (yND) has been found which is capable of inhibiting the PK reactions in high dilution and is presumably identical with y E (Stanworth et al., 1967). Increased levels of this immunoglobulin have been described in the serum of patients with allergies (Johansson, 1967; Johansson and Bennich, 1967). Although it appears that in most cases reaginic antibodies are YE, in certain allergic individuals reagins may be found in other immunoglobulin classes, A report by Reid et aE. (1966) suggests the existence of y G reagins, and we have recently examined a patient with absent serum yA (and pre-

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

sumably Y E ) in whom ragweed reagins appeared to be present in both yG and yM. It will be of some interest in these rare cases to determine which of the subgroups of yG are involved. Investigations of nasal secretions have demonstrated PK activity in ragweed-sensitive patients (Samter and Becker, 1947; Remington et al., 1964) and in the nasal edema fluid from patients with nasal polyps { Berdal, 1952), Remington and co-workers found positive PX reactions in six out of seven nasal washings from patients with ragweed allergy. Also, PK reactivity has been described in tears (Settipane et al., 1965) in 2 out of 7 atopic individuals with high reagin titers in their serum. One of these patients’ serum contained high titers of blocking antibody which could not be demonstrated in lacrimal fluid. Arbesman et al. (1968) investigated the parotid secretions of ragweed allergic individuals. Secretions from 17 allergic patients were all negative when used in passive transfer tests to human skin. Even highly concentrated parotid secretions obtained from patients with serum PK titers greater than 1:800 were negative. Failure of the parotid fluid to sensitize human skin was not related to short periods of fixation of antibody to skin sites since challenge with antigen within 4 hours after sensitization gave no reaction. Inhibition of skin sensitization by some factor present in the saliva was also excluded. These findings are discordant with those reported by Ishizaka et al. (1964) who demonstrated PK activity in the saliva of 2 patients with ragweed allergies. It is possible that in these latter studies the saliva was contaminated with serum components (YE), and it seems likely that parotid fluids differ from nasal fluids in which positive PK titers can be regularly demonstrated. An interesting observation in the patients studied by Arbesman et al. (1968) is that despite the negative salivary PK reactions, the saliva of these same patients were all capable of sensitizing the monkey ileum in the Schultz-Dale reaction. The Schultz-Dale antibody was intermediate in sedimentation (approximately 11 S ) on density gradient ultracentrifugation and was heat labile at 56°C. for 4 hours. Absorption experiments with antisera directed against secretory yA, SP, and serum yA showed that all of these antisera were able to remove monkey ileum-fixing activity from the saliva, whereas similar absorptions did not remove activity from the sera of the same subjects. Absorption with anti-yG antiserum had no effect on either serum or salivary titers. In one typical experiment (Table XI), sera and parotid saliva from the same individual were concentrated so that titers in both were 1:3000 in the monkey ileum Schultz-Dale test. Absorptions with an anti-yA or an anti-SP antiserum were able to remove the salivary but not serum ac-

79

S E W T O R Y IMMUNOGLOBULINS

tivity. This patient’s serum also had a PK titer of 1: 1500 and yet the concentrated salivary sample was negative. Thiis, there appears to be a clear difference between the reaginic-type antibody components in the serum and parotid secretions of allergic subjects. The evidence available suggests that the 11s heat-labile component in parotid secretions which sensitizes the monkey ileum in vitro is secretory yA, while the analogous antibody in serum is probably YE. In addition, the parotid TABLE XI EFFECTOF ABSORPTION WITH VARIOUS ANTISERA ON IIAGWEED-SENSITIVE SERUM .4ND SALIVA“.‘

Specimen

PK titer

Serum Parotid saliva

1500 0

Titers after absorption with Schultzragweed Dale Antisecrebinding. activityd Anti-yG Anti-YA tory yA Anti-SP

YE

+ 0

3000 3000

3000 3000

3000 0

3000 0

3000 0

a Serum but not saliva contained reagin (PK) and YE-binding activity. Both serum and saliva contained Schultz-Dale activity but t,hat in saliva was removed by prior absorption with anti yA or antisecretory “piece” (SP), whereas serum activity was not. Data from Dolovich et al. f 1968). Determined by radioimmunodiffusion with pJo1 c antigen. Using monkey ileum.

saliva does not appear to contain YE antiragweed antibodies. This suggestion is strengthened by the recent observation of Dolovich et al. ( 1968) who, using a sensitive radioimmunodiffusion technique, were unable to demonstrate YE-type ragweed antibodies in saliva despite their presence in serum and nasal secretions from the same individuals. Whether the reagin activity and yE ragweed-binding antibodies found in nasal fluid are synthesized locally or derived from serum has not been investigated. Immunofluorescent studies of tissue from normal vs. allergic individuals using YE-specific antisera will be of some interest in this regard. X.

Secretory Immunoglobulins in Animals

No attempt will be made in this section to review the now rather extensive literature concerning the characterization of serum immunoglobulins of various animal species. Suffice it to say that there are significant differences in the numbers and types of immunoglobulins which

80

THOMAS B. TOMASI, JR. AM) JOHN BIENENSTOCK

have been described in various species. Considerable difficulty has been encountered in defining which immunoglobulin in a given animal species is analogous to a particular immunoglobulin class in the human. Most species have a well-defined and easily recognizable yG which is the predominant immunoglobulin class and is low in carbohydrate and 7s in sedimentation, In some species, such as the guinea pig, 7s yglobulins seem to be divided into a slow ( y 2 ) and fast ( yl ) component which differ in their antigenic and biological properties. In other species, a clear-cut division has not been observed. In all species thus far examined there appears to be a yM system with a sedimentation of 19s quite analogous to the human macroglobulin. However, considerable difficulties have been encountered in defining which of the immunoglobulins, if any, in a particular animal serum is analogous to human yA. Comparisons with human yA are usually made on the basis of fast electrophoretic mobility, high carbohydrate content, and, frequently but not always, an intermediate sedimentation coefficient (9-11 S). However, these properties provide only indirect evidence, and many of these are, for example, also shared by human yE. Whether the intermediate-sedimenting immunoglobulins with reagin activity which have been described in other species (see Bloch, 1967) are analogous to human yE or yA has not been clearly shown. Other criteria have been on the basis of antigenic cross-reactions and the mobility of H chains on urea starch gel electrophoresis, although these criteria have not been extensively investigated in a large number of species. It has been found that cross-reactions utilizing antisera against human immunoglobulins are quantitatively greater between the analogous immunoglobulin classes ( Stobo, Mehta, and Tomasi, unpublished). For example, using certain antisera against human yM the cross-reactions of macroglobulins from several animal species are significantly greater than between the yG proteins from the same species. The mobility of H chains in acid urea starch gels is also relatively characteristic of the immunoglobulin class and, for example, has helped to identify the dogfish immunoglobulin system as being most analogous to human y M, observations which were later supported by peptide mapping and other studies ( Marchalonis and Edelman, 1965). Another important criterion in establishing an animal immunoglobulin as analogous to yA has been its occurrence in external secretions such as saliva and colostrum. This has been well demonstrated by the work in rabbits (Cebra and Robbins, 1966) and in the dog (Johnson and Vaughan, 1967). However, this criterion does not always apply, as discussed below for cow and sheep. ( See Section X,B. )

SECR13TORY IMMUNOGLOBULINS

A.

81

RABBIT

Feinstein ( 1963) first isolated an immunoglobulin from rabbit colostrum analogous to human yA. Subsequent examination and isolation of rabbit yA from colostrum was performed by Cebra and Robbins (1966) and by Sell (1967). In rabbit colostrum, the yA class appears to be the predominant immunoglobulin although small amounts of yG are also found. Unlike human colostrum, yM appears to be present in very small amount (Sell, 1967). Both Sell and Feinstein reported detecting allotypic A locus antigenic determinants (also found on rabbit yG H chains) in rabbit colostral yA, whereas Cebra and Robbins (1966) were unable to confirm these results. A detailed study of the structure of rabbit colostral yA performed by Cebra and Small (1967) points out the marked similarity in the characteristics of this immunoglobulin to those of human secretory yA including molecular size, carbohydrate content, and presence of a polypeptide chain ( T chain or T component) which appears to correspond to SP. Comparison of some properties of these molecules is shown in Tables I11 and IV. Unique antigenicity of the rabbit colostral yA similar to the human secretory yA was not reported but appears to be present (Bienenstock and Tomasi, unpublished). Immunofluorescent studies with specific antisera demonstrated a predominance of yA-containing cells in the lamina propria of the small intestine in normal rabbits and animals infested with Trichinella (Crandall et al., 1967). The yA cells made up 2 1 0 % of the immunoglobulin-containing cells in the spleen as compared with 80-90a: in the lamina propria of the rabbit intestinal tract. Ouchterlony analysis of immunoglobulins in intestinal contents and in gut extracts also showed a high relative concentration of yA although quantitative studies were not performed. The low numbers of yA cells (relative to yG cells) in rabbit spleen compared with the human may be correlated with the lower concentration of yA in rabbit serum (approximately one-eighth that of the human) (Onoue et al., 1966; Cebra and Robbins, 1966). I n vitro tissue culture of rabbit lactating mammary tissue demonstrated incorporation of labeled lysine and isoleucine only into the SP and not into yA heavy or light chains. This evidence was taken to represent independent synthesis of SP by the rabbit mammary gland and selective transport of yA from serum (Asofsky and Small, 1967). A similar suggestion had been made in 1954 b y Askonas et al. who reported that much of the immunoglobulin found in rabbit (and also goat) colostrum was apparently derived from serum ( see Section VII) . Biswas ( 1961 )

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

also suggested a selective transport mechanism to account for the secretion of circulating immunoglobulins into the milk of rats. This thesis was based on differences in serum vs. colostral titers of antibodies during the primary as opposed to secondary responses. However, local production could not be excluded by these studies.

B. COW AND SHEEP Smith and Holm (1948) showed that the immunoglobulin fractions of cow colostrum and milk are identical and that both are similar to the T globulin of cow plasma. Extensive studies by Dixon et al. (1961) have clearly shown a concentrating mechanism present in the alveolar cells of the mammary gland in the cow responsible for the transfer of large amounts of immunoglobulins from serum to colostrum and milk. Immunofluorescent studies in the normal lactating udder demonstrated few plasma cells staining with specific anti-immunoglobulin antiserum. Pierce and Feinstein ( 1965) demonstrated the predominance in bovine colostrum of fast yl-immunoglobulin and showed that there was an almost complete exclusion in this secretion of the YG-globulin with slowest electrophoretic mobility ( yz ) which normally predominates in serum. Sullivan and Tomasi (1964) have also shown that the predominant immune globulin found in both bovine and ovine colostrum and saliva is a fast y,-globulin, the majority of which has a sedimentation coefficient of 7s although 10s polymers occur in small amounts. It appears to be antigenically identical to the fast 7 S yG immunoglobulin in cow and sheep serum and is antigenically distinct from serum y z . No antigenic specificity similar to that due to SP in the human could be shown for the secretory immunoglobulins of these species. Thus, there is evidence in these species that the major immunoglobulin in saliva and colostrum is present in significantly lower concentrations in serum and is actively and selectively transported from serum to secretion. This is further supported by studies in agammaglobulinemic newborn calves showing selective transport into saliva of 7,-immunoglobdins (Sullivan and Tomasi, 1964). C. DOG The apparent differences in the secretory immunoglobulins of the various species is further highlighted by the work of Johnson and Vaughan (1967). These workers have demonstrated that in a single sample of dog colostrum two immunoglobulins are present in higher concentrations than that found in serum. The iinmunoglobulin which appears to predominate is a 7 S y, and is present in approximately 4

SECRETORY IMMUNOGLOBULINS

83

times greater concentration than in serum and was not demonstrated in saliva or bronchial secretions. The second immunoglobulin detected in canine colostrum was an intermediate-sedimenting yl protein (int. S yl) and was present in a concentration 80 times that found in the serum. The int. S yl did not cross-react with the yl-immunoglobulin and can be distinguished immunologically from it. In addition, the int. S yl appears to have unique antigenic determinants which may be analogous to human SP. This immunoglobulin is apparently the predominant immunogIobulin of canine saliva. D. MOUSE Examination of mouse colostrum has revealed the presence of three of the immunoglobulins found in normal mouse serum (Fahey and Barth, 1965) including 7 S y,, 7 S y2, and yA. From the evidence presented it is possible that the yA predominates in the mouse colostrum, although this was not clearly established by quantitative techniques. The existence of a mouse secretory system analogous to that of the human and rabbit is further suggested by recent observations (Mandel and Asofsky, 1967) that the mesenteric and intestinal tissue of the mouse contain immunocytes that produce primarily yA. Moreover, it has been known for some time that the most common type of plasmacytoma resulting from the intraperitoneal injection of irritants in mice produces a yA myeloma protein. This suggests that a large proportion of the immunocytes in the peritoneum of the mouse are of the yA-producing type (Potter and Lieberman, 1967). The occurrence in mice of a polypeptide chain analogous to SP or T component has not been described.

E. CONCLUSIONS Local production of antibody following direct immunization into the mammary gland has been shown in the rabbit (Batty and Warrack, 1955), cow (Mitchell et nl., 1954), and goat (Mitchell et al., 1967) although the characterization of the antibody formed in these studies has not been adequately delineated. Despite the local response mentioned above for the immune udder and mammary gland, it appears that these structures in certain species may be capable of concentrating and selectively secreting immune globulins from the serum without necessarily invoking local synthetic mechanisms. In this respect thc secretory systems of the sheep and cow appear to be different from those secretions elaborated by some of the other structures, such i s the human salivary gland, which are involved It is conceivable that this differcnce in the sccrction of i~n~nuno~lol~uliiis.

84

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

is primarily quantitative and that the relative contributions of immuno-

globulins transported from serum vs. that formed locally varies not only with the species but in different organs of the same species. The predominance of 7 S yG rather than 11 S yA and the apparent absence of a polypeptide chain analogous to SP in the human in some species suggests the possibility of a qualitative difference in the secretory system between species. However, further work, particularly using different species for immunization, is necessary to exclude the presence of SP on sheep and cow secretory immunoglobulins. Very little has been reported on the secretory immunoglobulins in inore primitive species. It is interesting, however, that the lamprey, which is more primitive than the earliest vertebrates, has lymphoidlike cells beneath its intestinal mucosa and very little well-defined peripheral lymphoid tissue (Ashback et al., 1964). The predominant antibody formed by this species appears to be intermediate-sedimenting but whether it is analogous to human yA or yM has not been determined. The serum immunoglobulin system of the lemon shark and dogfish (Marchalonis and Edelman, 1965; Clem and Small, 1967), early vertebrate forms, appears to be most analogous to human yM. No information has been reported regarding their secretory systems. XI.

Summary

A number of clinical and experimental observations have served to emphasize that in certain situations neither susceptibility to infection nor resistance following immunization appear to be directly related to serum antibodies. This is particularly well demonstrated by the work of Burrows et al. on cholera and that of Fazekas de St. Groth and his colleagues on respiratory infections. These and other investigators demonstrated the importance of antibodies in the secretions in resistance to infection. Thus the concept of local immunity, originally advanced in the early part of this century, was revived, and furthered by the suggestion that regional immunity might be mediated by locally formed antibody, apparently not derived from serum by simple transudation. In recent years, the characterization and description of the immunoglobulin classes, and the demonstration that the yA-immunoglobulin predominates in most external secretions, has helped to clarify some of these older findings. As a result, nonvascular fluids have been divided into two groups: internal secretions, in which the yG/yA ratios approach those in serum, and external secretions which are characterized by the predominance of yA. It has been found that the secretory yA molecule

SECRETORY IMMUNOGLOBULINS

85

has unique physical, chemical, and antigenic properties, and that these characteristics are conferred on the molecule by the presence of a nonimmunoglobulin glycoprotein termed secretory “piece.” The significance of SP has not been clearly established although it appears to stabilize the yA molecule and renders it relatively resistant to proteolysis. Incomplete information is available regarding the possible role of SP in facilitating the transport of yA from serum to secretions, and it has not yet been completely excluded that SP binds nonspecifically to yA and has little or no biological significance. The origin of the secretory molecule is still a matter of dispute although there is now a sizable body of evidence that the secretory immunoglobulins are not derived from serum by simple transudation. The evidence presently available regarding the site(s) of synthesis of the secretory immunoglobulins can be summarized as follows : 1. There is little correlation between salivary and serum levels of yA during development following birth and in certain diseases associated either with increased or decreased levels of yA in the serum. 2. There is little transport into saliva of radiolabeled serum yA or yG given intravenously to normal adults. Experiments with exchange transfusions in newborns have also failed to show significant transport of immunoglobulins. However, one report has suggested selective transport of yA in 2 patients with agammaglobulinemia when high serum levels were obtained by infusion of plasma. 3. In tissue cultures of human parotid and mammary gland, I4Clabeled amino acids are incorporated specifically into secretory yA. It is not known from these studies whether the label is incorporated into the yA portion, SP, or both. 4. Tissue culture of rabbit mammary gland has demonstrated incorporation of label into colostral yA, but dissociation of the secretory molecule revealed the majority of the label in the SP. 5. Studies on the origin of milk immunoglobulins in sheep, goats, COWS, and rabbits strongly suggest a selective type of transport of y-globulins from serum to colostrum and milk. 6. Fluorescent antibody studies in humans show accumulations of immunoglobulin-containing plasma cells in the lamina propria of the GI and respiratory tracts and, interstitially, between the salivary gland acini. The yG and yM cells are present in varying proportions in different organs but the most consistent and striking finding in all of these tissues is the predominance of yA-containing cells. 7. Fluorescent studies using SP-specific antisera have for the most part shown staining only of the glandular epithelial cells. Likewise, in

86

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

agammaglobulinemic tissues, only epithelial cells stain. One discordant study has appeared suggesting that both SP and yA are made in the same plasmalike cell. 8. In patients with agammaglobulinemia or dysgammaglobulinemia involving a deficiency of serum yA, SP is synthesized in approximately normal amounts. From the above observations it seems likely that in the human the majority of the yA in most normal secretions is synthesized locally. There may, however, be individual variations between secretions, and in the case of the GI tract and nasal fluids, for example, a small but significant fraction of yA may be transported from serum. In the mammary gland of certain animal species such as the cow, sheep, and perhaps the rabbit, there is good evidence that the secretory immunoglobulins are derived from serum and that the transport of immunoglobulins is highly selective. Whether the human mammary gland also shows selective transport rather than local synthesis remains to be determined. Thus there appear to be species variations and also differences between organs of the same species in regard to sites of synthesis and mechanisms of secretion of immunoglobulins. However, the common characteristic in all species examined so far is the presence of a specific immunoglobulin class in proportions quite different to those found in serum. This together with the apparent independent regulation of serum and secretory antibodies in certain situations appears to justify the separation of the secretory immunoglobulin system from that responsible for the production of circulating antibody. Whether the yA synthesized locally in the secretory system contributes to the serum pool of yA is unknown. Although considerable emphasis has been placed in this review on yA both for historical reasons and because of its high concentration in these fluids, it should be pointed out that the role of the other immunoglobulins and immunoglobulin fragments may be extremely important and deserve more attention in future studies. The available information suggests that the secretory immunoglobulins may play an important role in host immune defense against potentially pathogenic organisms. In this regard secretory yA antibody activity has been demonstrated against several microorganisms and viruses. Secretory immunoglobulins may also play a major part in the regulation of normal bacterial and viral flora of mucous membranes. Little definitive information is available regarding the role of deficiencies of secretory immunoglobulins in various diseases. In clinical

SECRETORY IMMUNOGLOBULINS

87

syndromes characterized by deficiencies of serum yA, secretory yA is also absent but is usually replaced by other immunoglobulins. It is unknown whether immune deficiency states exist characterized by primary defects in the secretory immunoglobulin system. The potential role of secretory immunoglobulins in allergic reactions both in the respiratory and GI tracts is of considerable current interest. Since allergic manifestations are initiated locally by antigen-antibody reactions, it is reasonable to postulate that secretory immunoglobulins may be involved in allergic reactions. The finding of secretory antibodies against ragweed and milk proteins in allergic individuals have further strengthened this view, although there has been no direct demonstration that these antibodies, indeed, mediate the allergic reactions. Current interest has also been aroused in the possible function of secretory antibodies in relation to prophylactic immunization. Several studies have suggested that local administration of antigen via the respiratory or GI tracts may be more efficient in stimulating local antibody formation and resistance to infection than the classic parenteral routes. This has been particularly well demonstrated by the oral polio vaccines, which, as opposed to the inactivated parenteral vaccines, are effective in preventing colonization by the virus and the subsequent carrier state. However, whether in other situations, local prophylactic immunization will be a preferable method of vaccination must depend upon further studies involving appropriate clinical trials.

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Ashbach, N. E., Finstad, J., Sarnecki, J., and Pollara, B. (1964). Federation Proc. 23, 346. Askonas, B. A., Campbell, P. N., Humphrey, J. H., and Work, T. S. (1954). Biochem. J. 56, 597. Asofsky, R., and Small, P. A. (1967). Science 158, 932. Asofsky, R., and Thorbecke, G . J. ( 1961). J. Exptl. Med. 114,471. Axelsson, H., Johansson, B. G., and Rymo, L. (1966). Acta Chem. Scand. 20, 2339. Bachmann, R. (1965). Scand. J. Clin. Lab. Invest. 17, 316. Barth, E. E., Jarrett, W. F., and Urquhart, G. M. (1966). Immunology 10, 459. Batty, I., and Bullen, J. J. (1961). J. Pathol. Bacteriol. 81, 447. Batty, I., and Warrack, G. H. (1955). J. Pathol. Bactel.io2.70, 355. Bell, E. B., and Wolf, B. (1967). Nature 214, 423. Bellanti, J. A., Artenstein, M. S., and Buescher, E. L. (1965). J. Immunol. 94, 344. Bellanti, J. A., Artenstein, M. S., and Buescher, E. L. (1966). Pediatl.ics 37, 924. Bellanti, J. A., Buescher, E. L., Brandt, W. E., Dangerfield, H. G., and Crozier, D. (1967). J. Immunol. 98, 171. Berdal, P. (1952). J. AZZergy 23, 11. Berger, R., Ainbender, E., Hodes, H. L., Zepp, H. D., and Hevizy, M. M. (1967). Nature 214, 420. Berggard, I. (1961). Clin. Chim. Acta 6, 545. Berggard, I., and Bennich, H. (1967). Nature 214, 697. Bernier, G. M., Tominaga, K., Easley, C. W., and Putnam, F. W. (1965). Biochemistry 4, 2072. Besredka, A. (1919). Ann. Inst. Pusteur 33, 882, Besredka, A. ( 1927). “Local Immunization,” Williams & Wilkins, Baltimore, Maryland. Best, C. H., and Taylor, N. B., eds. (1966). “The Physiological Basis of Medical Practice” 8th Ed. Williams & Wilkins, Baltimore, Maryland. Bienenstock, J. (1968). J. Immunol. 100, 280. Bienenstock, J., and Tomasi, T. B. (1987). PTOC. 4th Panam. Congr. Rheumat., Mexico City. Bienenstock, J., and Tomasi, T. B. (1968). J . Clin. Invest. 47, 1162. Biswas, E. R. I. ( 1961) . Nature 192, 883. Bloch, K. J. (1967). Progr. Allergy 10, 84. Blout, E. R. (1962). I n “The Dependence of the Conformation of Polypeptides and Proteins Upon Amino-acid Composition in Polyamino-acids, Polypeptides and Proteins” (M. A. Stohman, ed.), p. 275. Univ. of Wisconsin Press, Madison, Wisconsin. Brambell, F. W. R. (1966). Lancet 2, 1087. Brandtzaeg, P. (1965). Arch. Oral Biol. 10, 795. Brandtzaeg, P., and Kraus, F. W. (1965). Odontol. Tidskr. 73, 281. Brandtzaeg, P., Fjellanger, I., and Gjeruldsen, S. T. (1967). Immunochembty 4, 57. Broberger, 0. (1964). Gastroenterobgy 47, 229. Broberger, O., and Perlmann, P. (1959). J. Exptl. Med. 110, 657. Broberger, O., and Perlmann, P. (1962). J. Exptl. Mcd. 115, 13. Broberger, O., and Perlmann, P. (1963). J. Exptl. Med. 117, 705. Buescher, E. L., and Bellanti, J. A. (1966). Bacteriol. Reu. 30, 539. Bull, C. G., and McKee, C. M. (1929). Am. J . Hyg. 9, 490.

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