The role of α1-antitrypsin deficiency in the pathogenesis of immune disorders

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

35, 363-380 (1985)

REVIEW The Role of cx,-Antitrypsin Deficiency in the Pathogenesis Immune Disorders

of

SAMUEL N. BREIT,~ DENIS WAKEFIELD, J. PAUL ROBINSON, ELIZABETH LUCKHURST, PEGGY CLARK,* AND RONALD PENNY Department of Immunology, St. Vincent’s Hospital University of New South Wales, Sydney, New South Pathology and Medical Research, Westmead,

and the Department of Medicine, Wales, and *The Institute of Clinical New South Wales. Australia

The association between cu,-antitrypsin (al-AT) deficiency and a number of immune mediated diseases including rheumatoid arthritis, anterior uveitis. systemic lupus erythematosus, and asthma suggests that al-AT may be important not only as an antiinflammatory protein but also as an immune regulator. That the relationship between decreased amounts of this inhibitor and these diseases is causal is suggested by both some of its physical properties and evidence indicating it is able to modulate immune function. a,-Antitrypsin has a high plasma concentration, very broad range of inhibitory activity and is an acute phase reactant. Among other things, it is able to modulate lymphocyte proliferation and cytotoxicity, and monocyte and neutrophil function. Additionally, some of these changes are demonstrable in rziro in patients with severe cx,antitrypsin deficiency. This paper reviews the important physicochemical characteristics of this protein, the association of its presence in decreased amounts with immune disorders, and finally the important mechanism that may underlie this disease association. G 1985 Academic Pres\, Inc

INTRODUCTION

Since the original report of Laurel1 and Eriksson (1) in 1963 linking severe (oilAT) deficiency with emphysema, associations of mild degrees of al-AT deficiency with a large number of other disorders such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), uveitis, and asthma have been discovered. A common feature of the pathogenesis of most of these diseases has been immune abnormalities and a strong inflammatory component. This paper reviews the basic characteristics of al-AT then explores its association with immune disorders and the role its deficiency may play in their pathogenesis. GENETICS

al-AT is coded for by a pair of codominant alleles which influence many of its properties including plasma levels, response as an acute phase reactant, amino ’ To whom correspondence should be sent at: Department of Immunology. St. Vincent’s Hospital. Victoria St., Sydney, N.S.W. 2010, Australia. ’ Abbreviations used: al-AT, u,-antitrypsin; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; AS, ankylosing spondylitis; AAU. acute anterior uveitis; CH50, total hemolytic complement activity; PH50, total alternative-pathway-derived hemolytic complement activity. 363 0090- 1229185 $1.50 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

acid composition and electrophoretic mobility. With electrophoretic techniques. more than 26 alleles have been determined for al-AT (2), each being designated by a letter of the alphabet, the commonest being M, S, and Z (3-5). With improved techniques three subtypes of M (designated MI, MZ, and M3) with more subtle differences in electrophoretic mobility have also been detected (6. 7). At a clinical level, the important al-AT variants are those associated with decreased plasma levels, the most common being the S and Z alleles. These are present in about IO’% of the Australian population (see Table I) which is comparable to the distribution seen in many northern European populations. Plasma al-AT concentrations in PiMM individuals were felt to be in the range of I .82.8 g/l (8,9). However, Jeppson et al. have recalibrated the al-AT standard which has resulted in a relative decrease in all values and a new normal mean of 1.3 g/l (10). This broad range is due to its acute phase reactant properties and plasma levels may even rise further in patients with inflammatory diseases. PiMS, PiMZ, and PiZZ are associated with mean plasma levels reduced to 79, 57, and 12% of normal, respectively (9). This leads to considerable overlap between PiMM and PiMS and MZ. Thus, plasma levels will not reliably detect the latter two phenotypes although they may be useful in the detection of severely deficient phenotypes such as PiZZ, and recognition of phenotypes such as PiM-, which are associated with normal electrophoretic patterns but decreased plasma levels. Some studies have suggested an increased number of al-AT-deficient phenotypes in women with large families (I 1) and in twins and parents of twins (12). This suggests that at least one reason for the survival of these alleles relates to increased fertility. There is some experimental support for this view as al-AT is known to inhibit sperm acrosin which is important in ovum penetration. Probably because of this, addition of al-AT to rabbit sperm results in decreased fertility (13). It is also known to be present in cervical mucus and undergoes a dramatic drop in level prior to ovulaton (14) suggesting it may have a role in regulating the viscosity of mucus and therefore sperm penetration. Bagdasarian et (11.( 15) have also demonstrated al-AT in ovarian stromal cells which are known to participate in the maturation of Graflian follicles and formation of the corpus luteum. Regulation of ovum release by (WI-AT could explain the apparent increased incidence of twins in al-AT-deficient families. The structural gene coding for al-AT is linked to the gene for the immunoglobulin heavy chain as demonstrated by the Cm allotype (16. 17). Strong evidence suggests the gene for al-AT is located on chromosome 14 (18, 19). SOME CLINICAL

ASSOCIATIONS

FOR al-AT DEFICIENCY

Lurlg Diseases In 1963, Laurel1 and Eriksson reported inherited al-AT deficiency and noted a remarkable incidence of severe emphysema in these subjects (I). The associations of severe &l-AT deficiency (usually PiZZ) and emphysema remains undisputed and has been extensively reviewed by others (1, 20-24). al-AT deficiency has also been linked to a number of other respiratory disor-

a,-ANTITRYPSIN

DEFICIENCY

365

ders. A number of groups have noted an increased incidence of deficient phenotypes PiMS and PiMZ in asthma (25-30). Studies in infantile asthma have shown an overall incidence in 298 children of 22.5% with as many as 46.2% of the nonatopic group have a deficient phenotype (26). Children with deficient phenotypes PiMS and PiMZ have hyperreactive airways from an early age (3 1, 32). Those children that develop asthma express it at an earlier age (26) and need more bronchodilators and steroids (27, 29) indicating a more aggressive disease. Geddes and co-workers (33) also noted an association of Cal-AT deficiency with interstitial pulmonary disease. They found that 14.7% of patients with fibrosing alveolitis were PiMZ (control frequency 3%). Most remarkable however, was their finding that 50% of those patients with both rheumatoid arthritis and interstitial lung disease were al-AT deficient with increase in both PiMS and PiMZ frequency to about 23%. Karsh and colleagues (34) however found no such increase in patients with pulmonary involvement secondary to Sjogren’s syndrome. Connective

Tissue Disease

A number of groups have defined an association between rheumatoid arthritis and al-AT deficiency (33, 35-39) while two studies have been unable to support such an assertion (34, 40). It is possible that differences in the severity of disease in the populations studied accounts for this difference. As indicated in Table 1 our study (39) noted a frequency of al-AT-deficient phenotypes (MS plus MZ) of 25% compared with 10% in the control population, with a significant increase in the frequency of both MS and MZ. Although detailed assessment of the severity of RA was not undertaken, there did not appear to be a marked difference in severity between deficient and nondeficient subjects with RA suggesting that al-AT deficiency may be a factor in the disease predisposition rather than influencing only severity. However, formal studies addressing this issue need to be carried out. Most of the studies show an increase in the frequency of the Z allele as either PiMZ, PiSZ. or PiZZ. the frequency of PiMS is not significantly increased except in our study (39) and one other (33). In the latter investigation of subjects with RA and interstitial pulmonary involvement, both PiMS and PiMZ were increased dramatically to 22.7% suggesting that these phenotypes may predispose to interstitial pulmonary involvement in RA. In a follow up survey in Sweden which detected 246 PiZZ individuals, a very high frequency of RA of 4.5% was noted (22). This suggests that PiZZ predisposes to RA, quite probably to a greater degree than just a single dose of the Z allele, and the low incidence of PiZZ in surveys of RA is simply a reflection of the rarity of this phenotype. It is particularly interesting, that from this same study of samples that were referred predominantly because of emphysema, 0.8% had SLE. 15% had glomerulonephritis, and 0.4% had ankylosing spondylitis. This suggests that the frequency of immune disorders is very high in subjects with the PiZZ phenotype. Our group was the first to note an association of al-AT-deficient phenotypes

BREIT El. AL.

366

TABLE al-AT

PHENOTYWS

I

IN RHEUMAIIC

DISEASES UI-AT

Controls Rheumatoid arthritis SLE Scleroderma

N

MM

MS

339 80 5x 48

89 75*,fj** 93

7 15** 14” 6

* P < 0.05. ** P < 0.02.

with SLE (39) with an increase in PiMS from 7 to 14% and PiMZ from 3 to 7%, although the latter fell just short of statistical significance (Table 1). Because PiMZ has the lowest frequency of the three common phenotypes it is more difficult to demonstrate statistical significance with relatively small numbers. It therefore may well be that the frequency of PiMZ is also increased. The only other study to subsequently address this question was unable to find such an association (34). This study, however, suffered from several serious deficiencies. First, the number studied, 40, was small and no control population was phenotyped. Additionally, they were unable to detect a single MS subject, thus raising questions as to the adequacy of the phenotyping. However, a survey of 246 patients with PiZZ in Sweden did note an incidence of SLE of 0.8% further suggesting that al-AT deficiency is in fact associated with this disease (22). Although detailed assessment of disease severity was not undertaken as part of this study, there did not apear to be a marked difference in severity between SLE patients with and without al-AT deficiency. If this impression proves to be accurate it would suggest that al-AT deficiency in SLE, as for RA, has some basic influence on the pathogenesis of the disease, rather than simply modifying its severity. The evidence on the incidence of al-AT deficiency in juvenile chronic arthritis is conflicting. Arnaud et al. found an increase of PiMZ to 10.4% in 96 patients (41). Another group however (38) were unable to substantiate this finding in a similar number of subjects. Neither group however has adequately subcharacterised their population. Juvenile chronic arthritis is known to consist of at least five different subgroups with greatly differing disease patterns. The differences between these two studies may well be due to the different composition of their populations. Ankylosing spondylitis (AS) is another inflammatory disease affecting predominantly the axial skeleton and large joints. One group has looked at 175 patients with this disease and found an increase in PiZZ to 3% and PiMF to 9% (36). There are, however, a number of unusual features of the study that render its results suspect. If there was a real increase in the Z allele it is surprising (but not impossible) that they found no increase in PiMZ as well. The increase in PiMF is also rather difficult to explain as it is an uncommon allele that is not a deficient

(Y,-ANTITRYPSIN

367

DEFICIENCY

variant. Brewerton and coworkers (42) found no increase in PiMZ in 30 uncomplicated patients with AS but did find an increase to 30% in AS patients with acute anterior uveitis (AAU). It is not possible to decide from this data if PiMZ predispose just to AAU or also to a lesser degree to AS as well. The only other group to examine this issue (40) found no increase in deficient phenotypes in AS (or psoriatic arthritis) but the number studied is so small as to make a meaningful conclusion impossible. The only other connective tissue disease to have been studied in detail is scleroderma (43, 44). There was no increase in deficient phenotypes in 46 patients with this disease 28 of whom had diffuse involvement and 18 of which had the CREST syndrome (43). This is similar to our observation in this disorder (44) (Table 1). It is however of some interest that in our study all three subjects with PiMS had diffuse disease and no subject with the CREST variant had a deficient phenotype. Eye Diseases Anterior uveitis is a common and important immunologically mediated inflammatory eye disease that is associated with PiMS and PiMZ phenotypes. Brewerton et al. (42) found that 25% of 80 patients with this disorder were PiMZ irrespective of whether they were idiopathic or associated with ankylosing spondylitis. No comment was made about other phenotypes. Three other studies (4547), however, were unable to substantiate these findings. One of these studies, (45) is somewhat suspect in that no patient with PiMZ could be found in 133 subjects. Additionally, none of the four studies gave adequate information on the sorts of anterior uveitis studied. Our results indicate in fact that these differences may well be due to patient selection. Our results show a significantly increased incidence of al-AT deficiency (MS and MZ) in patients with anterior uveitis, being most prevalent in those patients with chronic (40%), bilateral (60%), or recurrent (20%) disease (48) (Table 2). By contrast, none of the 36 patients with a solitary episode of anterior uveitis after a 6-month followup period were found to be al-AT deficient. These results were not influenced by the presence of the HLA B27 antigen, which seemed to function TABLE a,-ANTITRYPSIN

PHENOTYPES

2

IN INFLAMMATORY

EYE

DISEASE

al-AT phenotype (%,)

Controls Anterior uveitis Acute Chronic Bilateral Recurrent Posterior uveitis Retinal vasculitis

No.

MM

MS

MZ

MS + MZ

339

89

7

3

IO

36 10 5 39 16 19

100* 60*t

0

0

0*

0

40*t

80t

40*t 40*t 15*t

17 84

5 II

40t

* P < 0.001-0.05 compared wifh controls. -FP < 0.05 compared with acute anterior uveitis.

20 5 12 3

6o*t 20*t 17 16

368

BKEIT ET .41..

as a separate independent variable predisposing particularly to recurrent disease. Thus while overall there is only a slightly but significantly increased incidence of PiMS in anterior uveitis. when patients are carefully categorized as to the nature and severity of their disease, it is evident that the more severe subtypes of anterior uveitis display a marked increase in the incidence of al-AT-deficient phenotypes. Retinal vasculitis and posterior uveitis are both particularly rare disorders and hence a very long period is required to accumulate sufficient numbers to accurately delineate any association with al-AT deficiency (Table 2). In the case of posterior uveitis however, the frequency of MZ does appear to be markedly (but as yet not significantly) increased to 12% (48) suggesting that further exploration of this question may well be worthwhile. Skin Diseases Although there have been several studies of cxl-AT in urticaria, most are inadequate, as al-AT levels rather than phenotyping were used. Decreased al-AT levels have been found in angioedema (49, 50) and cold urticaria (49) but not in chronic urticaria. Eftekhari et al. (5 1) were able to confirm an association between al-AT deficiency and cold urticaria by phenotyping but were unable to find an association with chronic urticaria or cholinergic urticaria in a small number of patients. Beckman ef al. have also reported an increased prevalence of PiMS and PiMZ in contact dermatitis (52). There have been three interesting reports of the association of PiZZ with systemic Weber Christian disease in three patients (52-54). We have recently reported al-AT deficiency (with PiZZ) and Weber Christian disease in two brothers (54). The occurrence of two such rare disorders in the one family makes it highly unlikely that this association is purely by chance. Why a third member of the family with PiZZ did not develop Weber Christian disease is not clear. Like most genetic risk factors, al-AT deficiency probably has a complex relationship to Weber Christian disease and factors including other genetic markers and an appropriate environmental stimulus are also likely to be required. A study of al-AT deficiency in psoriasis has also noted an increase in PiMZ to 12.5% (55) but no comment was made about clinical differentiation between deficient and nondeficient subjects. Gastrointestinal Diseases As with pulmonary involvement, the association of the 2 allele with liver disease has been appreciated for a long time but its prognosis is controversial. There is no evidence for an association with immune mediated liver disease and hence further discussion is beyond the scope of this paper. Readers are referred to other extensive works on this subject (22, 56-63). Renal Diseases An association between &l-AT deficiency and renal disease was first reported in 1969 in a patient with PiZZ, glomerulonephritis (GN), emphysema, and polyarteritis nodosa (64). Some years later there appeared a report of three children with PiZZ and cirrhosis who had a membranoproliferative GN (65). One of these

a,-ANTITRYPSIN

369

DEFICIENCY

was studied in detail and proved to have IgM, C3, and &l-AT deposited in glomeruli. There have been several other reports of al-AT deficiency and glomerulonephritis (66-68) including a French series of 39 patients (66). Many but not all cases have been of mesangioproliferative GN frequently associated with cirrhosis and with &l-AT deposition in glomeruli. Although there is no definitive study of al-AT phenotypes in GN. these reports suggest there may well be a genuine association. PHYSICOCHEMICAL

PROPERTIES OF al-AT

al-AT is the major serine protease inhibitor of human plasma with a molecular weight of about 5 1,000 (69-72). The three common alleles, A4, Z, and S are known to differ at one of their 394 amino acids (70, 71) resulting in proteins with slightly differing characteristics such as migration on electrophoresis serum concentration and acute phase reactant properties. al-AT is known to be synthesized in hepatocytes (73, 74) and macrophages (75, 76) and has a half-life of about 5-6 days (77). Unlike most of the other protease inhibitors al-AT is an acute phase reactant so that increased amounts are produced at times of extra need. al-AT is an inhibitor of serine proteases, which represent the largest group of mammalian enzymes, and are characterized by serine residues at their active sites. This active site is present within a crevice and the serine residue (78) assisted by adjacent histadine and aspartic acid undertake a nucleophilic attack on the substrate’s peptide bond. (79). The substrate specificity of the enzyme is conferred by its three-dimensional structure which constrains access to its active site. al-AT inhibits serin esterases by acting as a competitive substrate, the enzyme cleaving a 4000- to 8000-Da fragment from &l-AT (80). Once attached to the enzyme, al-AT forms a very stable complex which does not permit the enzyme to interact with other substrates. al-AT has an extraordinarily broad range of enzyme inhibitory activity (Table 3) exceeded in this only by a,-macroglobulin, whose mechanism of action is unique in allowing it to inhibit enzymes irrespective of class. It remains a difficult problem to know which of the interactions of al-AT is physiologically important and which are purely in vitro effects. This situation is made more difficult by the fact that &l-AT is present in such high plasma concentrations that shear quantity TABLE SPECTRUM

OF INHIBITORY

3 ACTIVITY

OF

al-AT

Major importance

Uncertain importance

Minor importance

PMN neutral proteases Elastase Cathepsin G Collagenase Trypsin Chymotrypsin Factor XIa Urokinase

Plasminogen activator Sperm acrosin Renin

Thrombin Plasmin Kallikrein Hageman factor

370

BREIT

ET AL,.

may well make up for somewhat low association constants. Additionally, 55% of al-AT is distributed in the extravascular compartment (81) and in various secretions (82) where the other high-molecular-weight inhibitors are unable to enter. In such areas enzymes normally preferentially inactivated by other inhibitors in plasma, may now be inactivated by al-AT. MECHANISMS

FOR THE ASSOCIATION OF al-AT DEFICIENCY MEDIATED INFLAMMATORY DISEASES

IN IMMUNE

Over recent years a considerable amount of research has been directed at the pathogenesis of autoimmune disorders such as RA and SLE. A discussion of this material is beyond the scope of this article but it has been extensively reviewed previously (83-91). In the case of SLE, one of the predominant etiological factors appears to be abnormalities in T-cell regulation of immune responses with a decrease in suppression or an increase in helper activity and intrinsic B-cell abnormalities with a propensity to hyperreactivity. The situation in RA is considerably less clearcut. Current dogma incriminates abnormal immune reactivity directed at collagen and IgG. Abnormalities in immunoregulation have been demonstrated less consistently than in SLE. However, the two mechanisms described in SLE, are probably also important pathogenic mechanisms in RA as in many other autoimmune diseases (92). Considerably less is known of the pathogenesis of inflammatory eye diseases such as anterior uveitis but immunological factors (probably cell-mediated immunity) are felt to be important in their pathogenesis (93). Two general mechanisms may explain the association of al-AT deficiency and immune mediated inflammatory disease: immunoregulatory abnormalities may directly predispose to these diseases and inflammatory abnormalities may worsen their expression. Immunoregulatory

Abnormalities

Abnormalities in lymphocyte function in al-AT deficiency may well be of relevance in determining its association with immune disorders. In a study of otherwise healthy &l-AT-deficient subjects (PiZZ), we have obtained both in vivo and in vitro evidence to support such an assertion. al-AT-deficient subjects exhibit marked serum-mediated enhancement of lymphocyte responsiveness to PHA especially at suboptimal doses (94). Using highly purified al-AT, this effect can be shown (at least to a large degree) to be due to al-AT itself and not to other secondary factors (95). Addition of increasing amounts of al-AT results in an exponential decrease in the PHA response which reaches a plateau when the alAT in the serum supplement is increased to 2-4 g/l, which is the normal physiological range (95) (Fig. 1). Therefore, the modulatory effect of al-AT is important up to normal serum levels and only reduction in al-AT levels below this range, as occurs in &l-AT-deficient subjects, results in increased lymphocyte activation. al-AT appears to have a similar but somewhat less marked effect on concanavalin A (Con A) responses but no effect whatsoever on pokeweed mitogen (PWM)induced proliferation (95). Detailed assessment of the mechanism of action of alAT indicates a direct effect on adherent cells and no direct action on proliferating T cells (95). It probably acts by the inhibition of a membrane-bound serine es-

q-ANTITRYPSIN

01,

0

I SERUN CO?K.

2 ALFtlA

371

DEFICIENCY

3 I ANTITRYPSIN

4

1 5

CGA,

FIG. I. The effect of (WI-AT on the PHA response of lymphocytes cultured in the presence of alAT sera. The results are expressed as means 2 1 standard error of 23 experiments and are scaled so that the value obtained without added al-AT is considered 100%.

terase which is already active prior to the addition of phytohemagglutinin (PHA). The fact that al-AT does not completely inhibit PHA responsiveness but reduces it by 40-60% indicates that it inhibits the production of a factor or factors from adherent cells that enhance but are not absolutely essential for T-cell proliferation. This would be consistent with an effect on interleukin 1 production. That these in vitro findings are of clinical relevance is further supported by in vivo studies in which &l-AT deficient subjects exhibited exaggerated cell-mediated immunity, as manifest by marked acceleration of delayed hypersensitivity responses (94). Macrophages secrete a number of factors regulating the growth and differentiation of many other ceil types. It seems possible that al-AT may also inhibit the production of at least some of these factors, which could lead to alteration in growth and regulation of cells other than lymphocytes. The hepatocyte and the macrophage are the only cells known to synthesize alAT (73-76) although one group also noted de now synthesis by the lymphocyte fraction from partially fractionated mononuclear cells (96). The stimulus and control of its synthesis and secretion are not known, but as for many macrophage factors, may well be selective. It would seem self defeating for a cell to simultaneously secrete a protease and its inhibitor so it is likely that some differential secretion of these substances occurs with time. On the other hand, if its function were purely to prevent autolysis of proteases leaking from lysosomes it is difficult to understand why it is secreted into the culture medium in the absence of phagocytic stimuli. If nothing else this provides strong presumptive evidence for a role for &I-AT in macrophage mediated functions, but at this stage, its role and relative importance are uncertain. Few previous studies concerning the effect of al-AT on human lymphocyte proliferation have been published. One group could demonstrate no inhibition of the mixed lymphocyte response (97), whilst another showed inhibition of the

372

BREIT

E-I- AI..

PHA-induced proliferation (98). The latter group have been able to demonstrate surface membrane serine esterase on unstimulated human T-cell-depleted tonsillar cells which was inhibited by CYI-AT, a,-macroglobulin and soya bean trypsin inhibitor (99, 100). They felt this enzyme was on the surface of B cells, however. sufficient characterization of the cell was not undertaken and it therefore may well have been a monocyte. Other groups have also demonstrated finding of culAT to Con A-stimulated peripheral blood mononuclear cells (100) and to both stimulated and unstimulated lymphocytes and monocytes (102). In the latter publication, surface expression of cul-AT was greatly increased by cell activation with mitogens. There is also direct evidence from another source that macrophages have a surface membrane elastase like enzyme (103) and perhaps these studies are detecting binding to this substance. Therefore, the (ul-AT and adherent cellmediated inhibition of the PHA response may be due to the inhibition of this enzyme. The results of our functional studies suggest it is less likely that al-AT has any direct inhibitory effects on the cell surface enzymes of T cells or B cells although this is certainly still possible in view of the CYI-AT binding. However, there are also a number of other aspects of lymphocyte function that might be influenced by al-AT. Enzymes such as human neutrophil elastase and cathepsin G (104). human skin proteases (105), plasminogen activator (PA) and urokinase (106) are mitogenic and can induce and enhance antibody synthesis probably by acting directly on B cells. These enzymes may also influence lymphocyte function indirectly. For example, they can cleave IgG, liberating its Fc fragment that is known to augment lymphocyte responses (107). All these findings suggest that serine esterases are likely to play an important role in nonspecific enhancement of B-cell immune responses, especially at sites of inflammation. As al-AT is the major protease inhibitor acting on these enzymes it is reasonable to suppose that al-AT would be able to modulate their function. In (YI-AT deficiency decreased inhibition of these enzymes could result in further immune enhancement. al-AT is able to inhibit the cytotoxic reactions of lymphocytes including antibody-dependent cell-mediated cytotoxicity (108, 109), T-cell-mediated cytotoxicity (108). and natural killer activity, the latter possibly due to interaction with an elastase-like enzyme on the cell surface (109, 110). The evidence for inhibition of T-cell cytotoxicity is quite weak largely because of the crudity of the al-AT preparation used (108). It is entirely unknown if the inhibition of these cytotoxic functions is due to an effect on the activation of the cytotoxic cell or due to interaction with substances mediating target cell destruction. Hypothetical

Model jbr the Ejfect of al-AT

on Lymphocyte

Function

A hypothetical scheme for action of &l-AT is presented in Fig. 2. The data outlined suggest strongly that al-AT is able to modulate T-cell proliferation due to its effect on monocyte function. al-AT may also inhibit the serine esteraseinduced polyclonal activation and enhancement of B-cell function. An attractive but still unproven hypothesis would be that deficiency of oLI -AT leads to increased T-helper activity and polyclonal B-cell activation leading to abnormalities in immunoregulation which may predispose al-AT-deficient subjects to autoimmune disease.

a,-ANTITRYPSIN

IL2

EFFECTOR

TH

YONDCYTE

373

DEFIC’IENC’Y

FUNCTION

ANTIBODY

FIG. 2. Possible sites of action of al-AT on T- and B-cell function.

Injlammator?, Abnormalities

While immunoregulatory abnormalities may predispose to immune disorders, inflammatory abnormalities may worsen their expression. Irrespective of the etiology of the disease, no tissue damage results without an inflammatory response in which phagocytic cells and complement plays a major role. There is some evidence of excessive inflammatory responses in al-AT-deficient subjects. Studies of the activation of monocytes and neutrophils using zymosan-induced chemiluminescence (CL) have demonstrated enhanced activation of both of these cells in the presence of al-AT-deficient sera compared with control sera (94). However, using highly purified al-AT, no suppression of the CL response was demonstrable, indicating that the enhancement noted in al-AT-deficient sera was due to other secondary factors (Ill). As the CL assay is dependent on alternative pathway-derived complement and these sera are known to have elevation of the important alternative pathway and attack sequence proteins C3, CS and factor B (94), it would appear quite possible that the enhancement was due to this factor. This study therefore does not support the hypothesis that UI-AT acts on the cell surface serine esterases that are known to be involved in the activation of phagocytic cells. However, as a number of different receptors and mechanisms exist for activation of these cells, the results do not completely exclude a direct effect of al-AT or enzymes involved in their activation. Two studies have investigated the effect of al-AT on phagocyte motility. In one, al-AT was found to have a very significant effect on chemokinesis, reducing it by about 35% for monocytes and neutrophils (110). A small (10%) but significant inhibition of casein-induced chemotaxis was probably due to the fact that casein had some chemokinetic as well as chemotactic properties. In 1975, Goetzl (112) reported a rather complex effect of al-AT on C3a- and CSa-induced neutrophil chemotaxis. He found that cells pulsed with or I-AT for a very short time exhibited

374

BKElT

E’I /\I..

enhanced random migration and decreased chemotaxis. More prolonged incubation resulted in abrogation of this effect. However. the purity and function activity of his al-AT was not specified, and this is important considering the difficulties involved in its isolation. Additionally, the dose of al-AT used (0. I g/l) is very small considering its normal physiological serum level of 2-4 g/l. Several authors have previously demonstrated that an active and an activatable serine esterase are involved in the chemotactic response ( I I?- 115). al-AT presumably inhibits a cell membrane serine esterase which is activated by a chemokinetic signal. The reported finding of accelerated delayed hypersensitivity responses in al-AT-deficient subjects could conceivably be an irr I+W corollary of this reaction, representing accelerated locomotion of macrophages. Neutral proteases secreted from both monocyte and neutrophil especially elastase, cathepsin G, collagenase, and possibly plasminogen activator are potently inhibited by (ul-AT not only in the circulation, but most importantly in extra vascular fluids where its low molecular weight allows it to diffuse. These enzymes are directly responsible for the majority of the tissue destruction in inflammatory responses due to their effects on substances such as elastin ( 116- Il8), cartilage and structural collagen (119, 120). basement membrane (121, 122), fibrin (123). and tibronectin (124). Additionally, they generate chemotactic factors from fibrin (123) and complement (125, 126). produce an enhancing factor of IgG (107). and activate mediators such as complement ( 125, 126), kininogen (127), and angiotensinogen (128) and activate other cells such as lymphocytes and monocytes. It would therefore appear very likely that in al-AT deficiency, decreased inhibition of these enzymes would result in increased direct tissue injury and indirect injury through activation of other cells and mediator systems (Fig. 3). This tissue destruction, through the production of increased amounts of altered self antigens, may perhaps also provide extra impetus for the development of autoimmune diseases. Complement is a major initiator and amplifier of both immune and nonimmune NEIJTRAL

PROTEASES

ACTIVATION

INFLAMMATION

FIG. 3. Possible sites of action of al-AT on neutral protease-induced

tissue injury.

a,-ANTITRYPSIN

DEFICIENCY

375

mediated tissue injury. The role of al-AT in regulating the complement system is probably largely restricted to inhibiting its activation by neutral proteases. While of uncertain significance, we and one other group have demonstrated elevation of alternative pathway and attack sequence proteins, C3, factor B, and CS in al-AT-deficient patients, parallelled by an increase in the total alternativepathway-derived hemolytic activity (PH50) (94). No difference was noted in classical pathway components Clq, C4, or in the total hemolytic complement activity (CH50). We were also not able to demonstrate the interaction of al-AT with complement components in immune complexes or any direct inhibition by purified al-AT of functional complement activity using CHSO and PH50 assays (unpublished observation). al-AT, however, has been shown by one group to bind C3 and inhibit C3-mediated phagocytosis (129, 130). Two other studies have investigated complement profiles in al-AT deficiency. In a mixed group of @l-ATdeficient subjects Arnaud et al. have observed elevated levels of Clq, C3, and C5 and in some groups factor B but normal C4 levels (131). Another group, using functional assays found no variation from normal except for depressed C4 levels (132). These patients however all had severe liver disease known to greatly influence the metabolism of many proteins. cxl-AT also has a role in the inhibition of several mediator pathways. It is the major inhibitor of factor XIa of the coagulation pathway (133), can inhibit factor Xa (134) an has lesser inhibitory activity against thrombin (135). Its activity against factors XIIIa, Xa, and VIIa has not been determined. It also inhibits plasma renin (136), the neutrophil cathepsin G activating angiotensinogen (127), and a skin-derived protease mediating neutrophil infiltration and increased vascular permeability (137). It has no significant effect on activated Hageman factor, kallikrein, or plasmin. It does however inhibit the neutral protease-mediated activation of Hageman factor (138, 139), kininogen (140, 141), and neutrophil-mediated fibrinolysis (142). The increased severity of some inflammatory diseases in al-AT-deficient subjects may therefore in part be explained by increased motility and activation of monocytes and neutrophils the former due to the absence of an inhibitor of neutrophil and monocyte locomotion and the latter due to secondary changes in serum resulting in enhanced chemiluminescence. Its inhibition of enzymes of the major mediator pathways is probably not of great significance. However, the capacity to inhibit the many neutral proteases that both cause direct tissue damage and activate many mediator pathways is of great importance. CONCLUSION

The sum of this evidence suggest that al-AT deficiency is an important genetic factor in inflammatory and immune diseases. This is mediated through the effect of al-AT on specific immune responses on the one hand and inflammatory response on the other (Fig. 4). Its deficiency is likely to lead to increased activity of a number of inflammatory pathways leading to excessive inflammatory responses. Additionally, these responses might occur to minimal insults which under normal circumstances would initiate minimal inflammation. Thus, not only would tissue damage occur but a predisposition to autoimmune disease might

BKEIT

376

ET Al..

ANTIGEN ::

.**. f MACROPHAGE

PROTEASES

t* Tu-T,

CELL MEDIATED IMMUNITY

ANTIBODY ‘MEDIATED IMMUNITY

PHAGOCYTE MIGRATION

ENZYME RELEASE-

~‘KI!iS AND

L

COAGUL8. PIBRINOLYSIS

PHYSICAL

INJURY

PHAGOCYTE MIGRATION

‘d -

I

RAG.,! FACTOR

I

FIG. 4. Overview of possible effects of al-AT on inflammatory and immune responses.

result through increased degradation and damage to self antigens. In al-AT-deficient subjects, the accelerated delayed hypersensitivity responses in viva and the in vitro macrophage-mediated enhancement of T-ceil proliferation suggest that al-AT has an effect on immunoregulation. An attractive but as yet unproven hypothesis is that al-AT modulates T-helper function and that enhanced T-helper activity might then well predispose to immune disorders, while exaggerated inflammatory response may worsen its severity. The thrust of all available evidence therefore suggests that this generalized hyperresponsiveness in al-AT deficiency may explain its many associated diseases. ACKNOWLEDGMENTS Dr. Breit, Dr. Wakefield, and Dr. Robinson were supported by Fellowships from the National Health and Medical Research Council of Australia.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Laurell, C. B., and Eriksson, S.. Scund. J. C/in. Lab. fm~esr. 15, 132. 1963. COX, D. W., Johnson, A. M., and Fagerhol. M. K., H~tm. Genet. 53, 429. 1980. Laurell C. B.. and Persson, Ll.. Biochirrr. Biophvs. Acra 310, 500, 1973. Fagerhol, M. K., and Laurell, C. B.. Clip. Chinz. Acw 16, 199, 1967. Arnaud, P., Chapuis-Cellier, C., and Creyssel, R., Protides Biol. Fluids 22, 515, 1974. Frants, R. R., and Eriksson, A. W., Hum. Hered. 26, 435, 1976. Frants, R. R., Noordhoek, G. T., and Eriksson. A. W.. Stand. J. C/in. Lab. Invest. 38, 457, 1978. 8. Heimberger, N., Haupt. H., and Schwick. H. G., In “Proceedings, International Research Con-

a,-ANTITRYPSIN

DEFICIENCY

377

ference on Protease Inhibitors” (H. Fritz, and H. Tscheche. Eds.), pp. i-22, De Gruyter, New York, 1971. 9. Kueppers, F., Humangenetik 15, 1. 1972. IO. Jeppsson, J. 0.. Laurell, C. B., and Fagerhol. M., Eur. J. Biochem. 83, 143, 1978. Il. Fagerhol, M. K., and Gedde-Dahl. T., Hum. Hered. 19, 354. 1969. 12. Lieberman, J., Borhani, N. 0.. and Feinleib, M., C/in. Getter. 15, 29. 1979. 13. Yang, S. L., Zaneveld, L. J.. and Schumacher, G. E B.. Fertil. Sterii. 27, 577. 1976. 14. Schumacher, G. F. B., and Pearl M. J.. Fertil. Steril. 19, 91, 1968. 15. Bagdasarian, A., Wheeler, J.. Stewart. G. J., Ahmed, S. S.. and Colman. R. W., J. C/in. Invest. 67, 281. 1981. 16. Gedde-Dahl, T.. Fagerhol. M. K.. Cook, P. J. L.. and Noades. J.. Ann. Hum. Genet. 35, 393. 1973. 17. Noades, J. E., and Cook, P. J. L.. Cytogenet. Cell Genet. 16, 341, 1976. 18. Croce. C. M.. Shander, M., Martinis. J., Cicurel, L.. et a/., Proc,. Nat/. Acad. Sci. USA 76, 3416. 1979. 19. Cox, D. W., Markovic. V. D.. and Teshima, I. E., Nature (London) 297, 428, 1982. 20. Mittman, C., Barbela. T., and Lieberman, J., Arc/z. Environ. Health 27, 201, 1973. 21. Norum, R. A., Beam, A. G., Briscoe, W. A., and Briscoe. A., Mt. SinaiJ. Med. 44, 821. 1977. 22. Larsson, C., Acta Med. Stand. 204, 345, 1978. 23. Morse, J. 0.. N. Engl. J. Med. 299, 1045, 1978. 24. Morse, J. O., N. Engl. J. Med. 299, 1099. 1978. 25. Fagerhol, M. K., and Hauge, H. E., Acta A//ergo/. 23, 107, 1%9. 26. Arnaud. P.. Chapuis-Cellier. C.. Souillet. G., Carron. R., et al., Trans. Assoc. Amer. P&s. 89, 205. 1976. 27. Katz, R. M., Lieberman. J., and Siegel, S. C.. J. Allergy C/in. fmmuno/. 57, 41, 1976. 28. Rosenfeld, G. B., and Murphy, W. H., .I. Allergy Clin. Immunol. 57, 218, 1976. 29. Hyde, J. S., Werner, P.. Kumar, C. M., and Moore. B. S., Ann. Allergy 43, 8, 1979. 30. Hoffmann, J. J. M. L., Kramps. J. A., and Dijkman, J. H., Clin. AiIergy 11, 555, 1981. 3 I. Hyde. J. S., Koeh. D. F., Isenberg, P. D., and Werner, F?. J. Amer. Med. Assoc. 235, 1125. 1976. 32. Werner, P., Hyde, J. S., Lourenco, R. V.. and Talamo, R. C.. Chest 65, 603, 1974. 33. Geddes, D. M., Webley, M., Brewerton, D. A., Turton, C. W.. et al., Lancet 2, 1049. 1977. 34. Karsh. J.. Vergalla. J., and Jones. E. A., Arthritis Rheum. 22, 1I I. 1979. 35. Arnaud, P., Galbraith, R. M.. Faulk, W. P., Black, C., and Hughes, G. V, Lancer 1, 1236, 1979. 36. Buisseret, P. D., Pembrey, M. E.. and Lessof, M. H., Ltrncet 2, 1358. 1977. 37. Cox. D. W., and Huber, 0.. Lancer 1, 1216, 1976. 38. Cox. D. W., and Huber, 0.. Clin. Genet. 17, 153. 1980. 39. Breit S. N., Clark, P., and Penny, R., Aust. N.Z. J. Med. 10, 271, 1980. 40. Sjoblom, K. G., and Wollheim, F. A., Lancer 2, 41, 1977. 41. Arnaud, P., Galbraith, R. M., Faulk, W. P., and Ansell, B. M., J. C/in. Invest, 60, 1442, 1977. 42. Brewerton. D. A., Webley, M.. Murphy, A. H., and Milford Ward, A., L.ancet 1, 1103, 1978. 43. Seibold. J. R., lammarino, R. M.. and Rodnan. G. I?. Arthritis Rheum. 23, 367, 1980. 44. Zilko, P. J., Penny, R., Breit, S. N.. McCluskey, J., and Dawkins, R.. In “Immunogenetics in Rheumatology” (R. L. Dawkins. E T. Christiansen. and P Zilko. Eds.). pp. 248-250, Excerpta Medica, Amsterdam, 1982. 45. Brown, W. T., Mamelok, A. E.. and Bearn, A. G., Lancet 2, 646. 1979. 46. Saari, K. M., Solja, J., Sirpela, M.. Frants, R. R.. and Eriksson A. W., Albrecht von Graefes Arch. Klin. Exp. Ophthalmol. 216, 205, 1981. 47. Grabner, G.. Pausch. V.. and Mayr, W. R.. Ophthulmic, Res. 14, I. 1982. 48. Wakefield. D.. Breit, S. N., Clark, P., and Penny. R., Arfhritis Rheum. 25, 1431, 1982. 49. Doeglas. H. N. G., and Bleumink, E.. Arch. Dermurol. 111, 979, 1975. SO. Crovato, F., and Rebora. A., Arch. Dermatol. 113, 236, 1977. 51. Eftekhari. N.. Milford-Ward, A.. Allen, R.. and Greaves, M. W.. Brit. J. Dermutol. 103, 33. IWO.

378

BREIT ET AL.

52. Beckman, G., Beckman, L., Cedergren, B.. Goransson. K., and Liden. S. Hrrrn. Hered. 31, 5~. 1981.

52a. Warter, J., Storck, I).. Grosshans, E.. Metais, P., rt (II.. .4rrn. Med. /nrerrrc~ 123. 877. 1972. 53. Rubinstein. H. M.. Jaffer, A. M.. Kudma, J. C., Lertratanaku], y.. cr (I/., Anrr. /rr/er-n. ,w~ed. 86, 742, 1917.

54. Breit. S. N., Clark, P., Robinson, J. P.. Luckhurst. E., Dawkins, R. L.. and Penny. R.. Ar(,/i. Dermatol. 119, 198, 1983. 55. Beckman, G., Beckman, L., and Liden, S., Acta Derm.-Venereal. 60, 163. 1980. 56. Sharp, H. L., Bridges, R. A., Krivit, W., and Freier. E. F.. J. Lab. C/in. Med. 73, 934, 1969. 57. Berg, N. O., and Eriksson, S.. N. Engl. J. Med. 287, 1264. 1972. 58. Sharp, H. L., Hosp. Pratt. 6, 83, 1971. 59. Bell, 0. E, and Carrell, R. W., Nature (London) 243, 410. 1973. 60. Sveger, T.. N. Engl. 1. Med. 294, 1316. 1976. 61. Hirschberger, M., and Stickler, Cl. B., Mayo C/in. Proc. 52, 241. 1977. 62. Hodges. J. R., Millward-Sadler, G. H., Barbatis, C., and Wright, R., N. Engl. 1. Med. 304, 557, 1981. 63. Cutz, E., and COX. D. W., Perspect. Pediatr. Pathol. 5, 1, 1979. 64. Miller, E, and Kuschner. M., Amer. J. Med. 46, 615, 1969. 65. Moroz, S. P. Cutz, E., Balfe. J. W.. and Sass-Kortsak, A.. Pediatrics 57, 232. 1976. 66. Mazodier. P.. Pediatre 13, 205. 1977. 67. Rodriguez-Soriano, J.. Fidalgo, I., Camarero, C.. Vallo. A., and Oliveros, R., Acta Paediatr. Stand.

67, 793, 1978.

68. Cutz, E., and Cox. D. W.. Perspect. Pediatr. Pathol. 5, 1, 1979. 69. Miller, R. R.. Kuhlenschmidt, M. S., Coffee, C. J., Kuo, I., and Glew, R. H., J. Biol. 251, 4751,

Chem.

1976.

Jeppsson, J. O., Laurel], C. B., and Fagerhol, M., Eur. J. Biochem. 83, 143, 1978. 71. Carrol, R. W., Jeppsson, J. 0.. Laurel], C. B., Brennan, S. 0.. Owen, M. C.. Vaughan, L., and Boswell, D. R., Nature (London) 298, 329, 1982. 72. Travis, J., and Salvesen, G. S., Annu. Rev. Biochem. 52, 655, 1983. 73. Gitlin, D., and Biasucci, A., J. C/in. Invest. 48, 1433, 1969. 74. Alper. C. A., Raum, D., Awdeh, Z. L., Petersen, B. H., et a/., Clin. Immunol. Immunopathol. 16, 84, 1980. 75. Isaacson, P., Jones, D. B.. and Judd, M. A., Lancet 2, 964, 1979. 76. Wilson, G. B., Walker, J. H., Watkins, J. H., and Wolgroch, D., Proc. Sot. Exp. Biol. Med. 164, 105, 1980. 77. Laurell. C. B.. Nosslin, B., and Jeppsson, J. O., Clin. Sci. Mol. Med. 52, 457. 1977. 78. Matthews, B. W., Sigler, P. B., Henderson, R., and Blow, 1). M., Nature (London) 214, 652, 1967. 79. Blow, D. M., Birktoft, J. J., and Hartley, B. S., Nature (London) 221, 337. ]%9. 80. Johnson, D. A., and Travis, J.. Biochem. Biophys. Res. Commun. 72, 33, 1976. 81. Jeppsson, J. O., Laurell, C. B., Nosslin, B., and Cox, D. W., Clin. Sci. Mol. Med. 55, 103. 1978. 82. Gadek, J. E., Fells. G. A.. Zimmerman, R. L.. Rennard, S. I., and Crystal, R. G., J. Clin. Invest. 68, 889, 1981. 83. Decker, J. L.. Steinberg, A. D., Reinertsen, J. L., Plotz. I? H., ef al., Ann. Intern. Med. 91, 587, 1979. 84. Miller, K. B., and Schwartz. R. S.. N. Engl. J. Med. 301, 803. 1979. 85. Williams, R. C., Hosp. Pratt. 13, 53, 1978. 86. Sakane. T., Steinberg, A. D., and Green, I., Proc. Nat/. Acad. Sci. USA 75, 3464, 1978. 87. Sagawa, A., and Abdou, N. I., J. Clin. Invest. 62, 789, 1978. 88. Spitler, L. E., In “Manual of Clinical Immunology” (N. R. Rose. and H. Friedman, Eds.), pp. 200-212, Amer. Sot. Microbial., Washington, D.C.. 1980. 89. Winchester, R. J.. (Ed.), “Proceedings of the ARA Conference on New Directions from Research in Systemic Lupus Erythematosus.” Arthritis Foundation, Georgia, 1977. 70.

(r,-ANTITRYPSIN

DEFICIENCY

379

90. Zvaifler, N. J., Adv. Immunoi. 6, 265, 1973. 91. Veys, E. M., Hermanns, P., Goldstein, G., Kung, P., et al.. lnr. J. Immunopharmacol. 3, 313, 1981. 92. Rose, N. R., Sci. Amer. 244, 70, 1981. 93. Wakefield, D., Schrieber, L., and Penny, R., Med. J. AUG. 1, 229, 1982. 94. Breit, S. N., Robinson, J. P., Luckhurst. E., Clark, P., and Penny, R., J. Clin. Lab. Immunol. 7, 127, 1982. 95. Breit, S. N., Luckhurst, E., and Penny, R., J. Immunol. 130, 681, 1983. 96. Ikuta, T., Okubo, H., Kudo, J., Ishibashi, H., and Inove, T., Biochem. Biophys. Res. Commun. 104, 1509, 1982. 97. Hubbard, W. J., Hess, A. D., Hsia, S., and Amos, D. B., J. Immunol. 126, 292, 1981. 98. Bata, J., Deviller, P., Colobert, L., and Lepine, M. P., C.R. Acad. Sci. (Paris) 285, 1499, 1977. 99. Bata, J., Martin, J. P., and Revillard, J. P., Experientia 37, 518, 1981. 100. Bata, J.., Devilier, I?, and Revillard, J. P., Biochem. Biophys. Res. Commun. 98, 209, 1981. 101. Lipsky, J. J., Beminger, R. W., Hyman, L. R., and Talamo, R. C., J. Immunol. 122, 24, 1979. 102. Boldt. D. H., Chan, S. K., and Keaton, K., J. Immunol. 129, 1830, 1982. 103. Lavie. G., Zucker-Franklin, D., and Franklin, E. C., J. fmmunol. 125, 175, 1980. 104. Vischer, T. L., Bretz, V., and Baggiolini, P., J. Exp. Med. 144, 863, 1976. 105. Eskola, J., and Fraki, J. E., Arch. Dermatol. Res. 263, 223, 1978. 106. Cohen, S. D., Israel, E., Spies+Meier, B., and Wainberg, M. A., J. Zmmunol. 126, 1415, 1981. 107. Morgan, E. L., and Weigle, W. O., J. Exp. Med. 151, 1, 1980. 108. Redelman, D., and Hudig, D., J. Immunol. 124, 870, 1980. 109. Ades, E. W., Hinson, A., Chapuis-Celher, C.. and Amaud. P., &and. J. Immunol. 15, 109, 1982. 110. Hudig, D., Haverty, T., Fulcher. C., Redelman, D.. and Mendelsohn. J., J. Immunol. 126, 1569, 1981. 111. Breit, S. N., Robinson, J. I?, and Penny, R., J. C/in. Lab. Immunol. 10, 147, 1983. 112. Goetzl. E. J., Immunology 29, 163, 1975. 113. Ward. P. A., and Becker, E. L., J. Exp. Med. 127, 693, 1966. 114. Ward, P. A., and Becker, E. L., J. Exp. Med. 125, 1001. 1967. 115. Damerau, B.. Grunefeld, E., and Vogt, W., Int. Arch. Allergy Appl. Immunol. 63, 159, 1980. 116. Kueppers, F., and Beam, A. G.. Proc. Sot. Exp. Biol. Med. 121, 1207, 1966. 117. Janoff, A.. and Scherer, J., J. Exp. Med. 128, 1137, 1968. 118. Baumstark, J. S., Arch. Biochem. Biophys. 118, 619, 1967. 119. Malemiud. C. F.. and Janoff, A., Ann. N. Y. Acad. Sci. 256, 254, 1975. 120. Laskowski, M.. and Kato, I., Annu. Rev. Biochem. 49, 593, 1980. 121. Davies, M., Barrett. A. J., Travis. J., Sanders, E., and Coles. G. A., C/in. Sci. Mol. Bio/. 54, 233, 1978. 122. Sanders, E., Cole% G. A., and Davies, M., Dial. Transplant. Nephrol. 13, 541, 1976. 123. Plow, E. F., Biochim. Biophys. Acta 630, 47, 1980. 124. McDonald, J. A., Baum, B. J., Rosenberg, D. M., Kelman, J. A., et al. Lab. Invest. 40, 350, 1979. 125. Hugh, T. E., Taylor, J. C., and Crawford, I. P. J. Immune/. 116, 1737, 1976. 126. Taubman, S. B., Goldschmidt, P. R., and Lepow, I. H.. In “Proceedings, 4th Int. Congress Immunology,” p. 434, 1980. 127. Wasi, S., Movat, H. Z., Pass, E., and Chan J. Y. C. In “Neutral Proteases of Human Polymorphonuclear Leukocytes; Biochemistry. Physiology and Clinical Significance” (K. Havemann, and A. Janoff, Eds.), pp. 245-260, Urban & Schwartzenberg, Baltimore, 1978. 128. Wintroub. B. U., Goetzl, E., and Austen, K. F., J. Exp. Med. 14, 812, 1974. 129. Landen, B., Schmitt, M., and Dierich, M. P., Fed. Proc. 38, 1467. 1979. 130. Mod, A., Fust, G., Gergely, J., Hollan, S., and Dierich, M. P., lmmunobiology 158, 338, 1981. 13 1. Arnaud, P., Creyssel, R.. Bertoux. F. C., Chapuis-Cellier, C.. and Freyria, A. M., Prorides Bioj. F1uid.r

23, 387, 1975.

132. Le PrevoQ, C., Frommel, D., and Dupuy J.-M., J. Pediatr.

87, 571. 1975.

BKEIT

380

ET AL.

133. Heck. L. W., and Kaplan, A. P., Fed. Prxx. 33, 642. 1974. 134. Gitel. S. N.. Medina. V. M.. and Wessler. S.. J. Biul. C~YI?Z.259, 6890. 1984. 135. Downing, M. R., Bloom. J. W.. and Mann, K. G.. 1~ “Chemistry and Biology of Thrombin” tR. L. Lundblad P! c/f.. Eds.). pp. 441-450. Ann Arbor Science Pub. Ann Arbor, Mich.. 1977. 136. Sharpe. S. E. M.. Correman, W., and Lauwers, A.. Bir~c,/rcrn. J. 153, 505. 1976. 137. Fraki. J. E.. Acttr Derm.-Venercol. 57, 393, 1977. 138. Revak. S. D.. Cochrane. C. G.. Johnston. A.. and Hugh. T.. J. Clin. lhr~st. 54, 619. 1974. 139. Kaplan, A. P.. and Austen, K. F.. J. E.up. Med. 133, 672. 1971. 140. Movat. H. Z.. Steinberg, S. G., Habal, E M.. Ranadive. N. S.. and Macmorine. D. R. L.. I/n. tnmol.

Commffm.

2. 247.

1973.

141. Movat, H. Z., C~rr. 7op. furho/. 68, 1 I I, 1979. 142. Franz. R. C.. Coetzee, W. J. C.. and Anderson, R.. S. A,fi. Med. J. 59, 661. 1981. Received May 18. 1984: accepted with revision November 16. 1984