Immunologie Methods for the Detection of Humoral and Cellular Immunity

Symposium on Practical Immunology

Immunologic Methods for the Detection of Humoral and Cellular Immunity Ronald D. Schultz, Ph.D.,* and Lincoln S. Adams, B.S.t

Modern immunology is one of the most dynamic areas of basic science. The emphasis placed on immunologic studies during the past 15 years has generated a whole new area of technology which is currently being applied to diagnostic and therapeutic medicine. The application of immunology to clinical medicine is now recognized as a specialty of medicine known as clinical immunology. Clinical immunology has become an integral part of veterinary medicine. Clinical immunology laboratories are either present or are in the planning phase at almost every veterinary college in the United States and Canada. Clinical immunology units frequently are associated with an immunology research laboratory so that tests that utilize a great many of the principles and techniques of basic immunochemistry and immunobiology can be performed on samples obtained from clinic patients. The marriage between basic immunology and clinical immunology remains an essential feature of veterinary clinical immunology, since there currently are no immunologic procedures that should be considered to be routine diagnostic tests when applied to a veterinary specimen. The principal reason for not considering these procedures to be routine is the lack of experience and baseline information on the normal population. As with any new area of science and technology, time and experience are required to properly evaluate and interpret results. Although veterinary clinical immunology is still in the developmental stages, it is now possible to evaluate the previous three to five years of limited immunologic testing with the newer procedures applied to do*Professor of Immunology, Auburn University College of Veterinary Medicine, Auburn, Alabama timmunology Technician, James A. Baker Institute for Animal Health, New York College of Veterinary Medicine, Cornell University, Ithaca, New York

Veterinary Clinics of North America- Vol. 8, No.4, November 1978

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mestic species, namely the tests to detect autoimmune phenomenon (i.e., antinuclear antibody, rheumatoid factor) and the in vitro correlates of cell-mediated immunity (CMI). More importantly, at least for certain techniques, there now is a limited amount of information available for the domestic species; therefore, we no longer need to rely solely on results or interpretations obtained from tests on laboratory rodents or man. Many similarities are found between species but, more important, significant differences are now apparent. Likewise the technology that frequently has been developed with laboratory rodent models or for man require minor but critical modification before they can be applied to studies with domestic species. Important goals of the veterinary clinical laboratory are to improve the availability and the accuracy of laboratory tests, to assure the correct interpretation of results, and to assess the significance of new tests introduced into clinical veterinary medicine. With the marked proliferation of new tests employing immunologic principles and new veterinary laboratories, these methods of laboratory diagnosis have often been uncritically applied to clinical situations. This article briefly explains the techniques currently used in immunodiagnosis and where applicable comments on interpretation of results.

DETECTION OF ANTIGEN, ANTIBODY, OR EFFECTOR SUBSTANCES (HUMORAL) The immune system has been divided into two major components, one involving humoral immunity (antibody, complement, etc.) and the other, cellular immunity. In many ways this separation is artificial and there is an increased awareness that there is cooperation and interdependence between cellular and humoral immunity. However, separation of the immune response is done in this article for convenience of presentation and to provide conceptual and practical methods for laboratory evaluation of immunity in clinical practice.

Immunodiffusion or Precipitation This technique is based on one of the most fundamental principals of immunology - the direct reaction of antibody and a soluble antigen. The technique can be performed in tubes, on slides, or in a variety of media such as agar or agarose. The test is dependent principally on the relative concentrations of antibody and antigen employed in the system. If the test is run in a medium such as agar, salt concentration, buffer electrolytes, pH, temperature, and distance between reactants also become important factors in this test. IgG antibodies are more effective as precipitating antibodies than are other immunoglobulin classes.

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

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Figure I. Reaction pattern in agar gel immunodiffusion. Patterns suggest that A and B and A and C are totally unrelated (precipitin lines completely cross each other). A, is partially related (lines do not completely cross) to A, and C is unrelated to A and A 1 , and A is identical to A (lines interesect at a point or curve).

Double diffusion in agar (agar gel immunodiffusion) is the most commonly used technique of the immunodiffusion procedures. The medium employed is agar or agarose with wells punched in various patterns or arrangements, with the intention of allowing antigen and antibody to diffuse from their respective wells, reacting with each other and at equivalence forming a precipitin line. This technique is currently not used routinely for immunodiagnostic procedures involving diseases of the dog or cat, but is used experimentally in canine brucellosis, infectious canine hepatitis, canine distemper, feline syncytium forming virus infection, and several other microbial infections of dogs and cats. In veterinary diagnostic medicine this test is used routinely for the detection of antibody to viruses such as equine infectious anemia (EIA) virus, bovine leukemia virus (BL V), bluetongue virus, and pseudorabies virus. The technique has the advantage of simplicity, specificity, and reliability, but lacks the sensitivity required for certain systems and is only semiquantitative (Fig. 1). Single Radial Immunodiffusion (RID) has the added advantages of sensitivity and of being quantitative. Radial immunodiffusion differs from double diffusion in that the antibody or antigen (reversed RID) is incorporated in the agar medium. The most common use of this procedure in veterinary immunology is in the quantitation of immunoglobulins and complement components in the serum or secretions of clinically affected animals. It would serve as the most reliable method to detect an immunoglobulin (humoral) immunodeficiency disease in dogs and cats (Fig. 2). Elevated levels of IgG are often seen in chronic infections, connective tissue disorders (systemic lupus erythematosus, polymyositis), and gammopathies. Information currently available would not permit

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5 mg 10mg 20mg

<0©

STANDARD UNKNOWN

A

s.

8

c

Figure 2 Single radial immunodiffusion (RID) of dog IgG. Standards in the upper well are used to establish a standard curve for the relationship between the diameter of the precipitin ring and antigen (IgG) concentration.

D

a definitive diagnosis based solely on results of RID for any disease of the dog or cat except an absolute immunoglobulin deficiency; however, the results do help to narrow the diagnosis. When the test is run in a clinical laboratory, reference standards and age and sex matched controls should be simultaneously run with the serum of a suspected immunodeficient patient. In our laboratory selective immunoglobulin deficiencies have been found most often in dogs with canine distemper infections and in cats with leukemia. Colostrum-deprived pups and kittens have significantly decreased levels of Ig's for about two to three months, because of the lack of maternal immunoglobulin normally obtained from the colostrum. Normal immunoglobulin values from our laboratory are presented in Table 1. Electrophoresis This technique involves the separation of proteins on the basis of their charge in an electric field. Zone electrophoresis requires a support medium that theoretically is inert and does not impede or enhance the flow of molecules in the electrical field. Cellulose acetate currently is the most commonly employed support for zone electrophoresis in the clinical laboratory. With this technique, serum or other fluids (i.e., cerebrospinal fluid) are applied to the cellulose acetate membrane and electrophoresced for a specific time. The procedure which we routinely use, Microzone (Beckman Instrument Company) electrophoresis, permits the separation of eight samples, and the time of electrophoresis is about 20 minutes. As a tool for diagnosis of immunologic disorders, cellulose acetate has very limited use, namely the detection of monoclonal or polyclonal gammopathies (Fig. 3). ImmuTable 1. Immunoglobulin Values in Adult Dog Serum Determined by Radial Immunodiffusion (RID)* IGG

Total

1500 ± 500

IGM 150 ±50

IGA 100 ± 60

*Expressed in mg per 100 mi. Serum obtained from mixed purebred and mongrel dogs (pooled samples from over 1000 dogs have been used to calculate the values).

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

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ALBUMIN

Figure 3. Cellulose acetate electrophoresis is performed on eight samples simultaneously in the Beckman microzone electrophoresis apparatus (left). The densitometric tracing of one of the serum samples is sh own (right).

noglobulin deficiencies are not readily recognized by cellulose acetate electrophoresis unless they are absolute, because of the relatively wide range of values for gamma globulin (IgG) in cats and dogs, and because IgM and IgA are found in the region in which other serum f3 globulins would conceal their presence or absence. It is not sensitive enough for the detection of IgE ! Although elevations of alpha and gamma globulins have been recognized in certain autoimmune diseases, the elevations are not specific and are found in a variety of chronic infectious or other debilitating diseases. The technique can also be used to identify increased immunoglobulin in cerebrospinal fluid and urine. Immunoelectrophoresis This technique combines electrophoretic separation and immunodiffusion. The support medium is generally agar or agarose when the sample is applied to a well (hole cut in agar). The sample is separated in an electric field and antiserum is placed in a trough (slit cut in agar) that is cut after completion of electrophoresis. The antigen a nd antibody are allowed to diffuse for approximately 24 hours in a mann er analogous to the double diffusion reaction described previously (Fig. 4).

ALBUMIN

(+)

(-)

Figure 4. Immunoelectrophoresis of dog serum reacted with anti-whole canine serum (uppe r tro ugh) and anti-canine immunoglobu lin (lower trough).

726

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Figure 5. Crossed immunoelectrophoresis of canine serum has the advantage of being a quantitative technique.

This technique can be modified so that it will be a quantitative test by combining immunoelectrophoresis with immunodiffusion in gel. The technique is known as crossed-immunoelectrophoresis, or quantitative immunoelectrophoresis (Fig. 5). Immunoelectrophoresis is useful in recognizing severe immunodeficiency states or immunoproliferative diseases such as m yelomas or polyclonal gammopathies. It is also a useful technique to examine cerebrospinal fluid, particularly in subacute distemper encephalitis, in which an oligoclonal IgG (homogeneous) is formed having antibody activity to canine distemper virus. It is possible to distinguish the more homogeneous oligoclonal protein in canine distemper virus infection from the more heterogeneous serum IgG found if the blood-brain barrier were broken with subsequent transudation of serum proteins into cerebrospinal fluid found in other forms of encephalitis. Electroimmunodiffusion

Electrophoresis can be applied to one dimensional single immunodifFusion, a technique known as Laurell rocket electrophoresis (Fig. 6). It is a sensitive quantitative technique for the rapid detection of protein. Rocket electrophoresis has not been widely used to date in clinical immunology, but in the future it may find greater application since recent modification allows quantitation of I g's, complement components, and numerous other protein antigens. Electrophoresis can also be applied to one dimensional double immunodiffusion, a technique known as counterimmunoelectrophoresis (Fig. 7). T his technique is similar to double immunodiffusio n , but gives results faster and is more sensitive. However, it requires that the antigen have a net electronegative charge, allowin g movement toward the anode in a buffer system in which antibody moves toward the cathode. The cathodal migration of antibody occurs as a result of the phenomenon known as electroendosmosis.

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

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f"igure 6. Electroimmunodiffusion (Laurell rocket electrophoresis) is a rapid quantitative and sensitive technique for serum and body fluid proteins.

(+)

0

( -)

( +)

Ab (Immune serum)

0

(Non-Immune serum)

(-)

Figure 7. Counterimmunoelectrophoresis (immunoelectro-osmophoresis) is a combination of double immunodiffusion and electrophoresis. It does not require th at the antigen h ave a net electronegative charge.

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Immunofluorescence (Fluorescent Antibody Technique) This technique is a histochemical or cytochemical procedure most commonly used to locate or identify antigens in tissue (direct technique) or detect specific antibody in serum or other secretions (indirect technique) of the patient (Fig. 8). The fluorescent compound most frequently conjugated to antibody for use in the procedure is fluorescein isothiocyanate (FITC). FITC readily binds covalently to antibody at an alkaline pH. Microscopes used for visualizing immunofluorescent specimens are simple, but expensive, modifications of standard transmission microscopes. For the direct procedure, antiserum with specific antibody activity for the antigen is conjugated. On the other hand, if the indirect procedure is employed, antibody to the species immunoglobulins is produced in a heterologous species (i.e., anti-dog IgG produced in rabbits) and the anti-immunoglobulin serum conjugated with a fluorescent compound. The immunofluorescence technique has been applied to the diag-

A

B

Direct

Indirect

~

X

li<-FITC

Canine antibody w1th activity to distemper virus and labeled with FITC.

.-FITC

Rabbit anticanine lg

~

Canine Ig with antibody to distemper virus.

I~ I

Canine distemper virus (CDV) infected tissue culture cells (I) and non-infected cells ( 2)

CDV (/ J Positive

1~1

Canine distemper virus (CDV) infected tissue culture cells (I) and non -infected cells ( 2)

Non-infected (2) Negative

COV{I) Positive

Figure 8. Comparison of the direct immunofluorescence (IF) test for canine distemper virus (CDV) in which antibody to CDV is conjugated with fluorescein-isothiocyanate (FITC) and is reacted directly with infected or control cells (A) and the indirect test in which the rabbit anti-canine Ig conjugated with FITC is reacted with the CDVinfected and control cells after they have reacted with canine serum tested for antibody to CDV (B). The indirect test is more sensitive than the direct, but requires a longer time and additional controls. It, however, is the IF method used routinely for screening field samples for antibody activity.

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

Table 2.

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The Indirect Immunofluorescence Test for the Detection of Autoantibody in Immunologic Disorders

DISORDER

SUBSTRATE

ANTIBODY TO

Systemic lupus erythematosus

Nuclear proteins and nucleic acids

Tissue sections, imprints, or tissue culture cells

Autoimmune thyroiditis

Microsomal antigens, colloid antigens, other thyroid antigens

Thyroid

Pemphigus*

Intercellular cement substance

Esophagus, skin, or oral mucosa

Pemphigoid*

Epithelial basement membrane

Esophagus, skin, or oral mucosa

Primary Addison's Disease

Adrenal cortical cells

Adrenal gland

Primary Hyperparathyroidism

Parathyroid cell

Parathyroid

Myasthenia Gravis and polymyositist

Muscle

Muscle

*Direct technique with biopsy of affected tissue is the preferred method. tTo the authors' knowledge there has not been a demonstration of antibody to muscle in either myasthenia gravis or polymyositis of the dog.

nosis of a wide variety of diseases of dogs and cats. Antibody to a variety of tissue antigens is detected with the indirect procedure (Table 2). The critical factors in this test are the quality and specificity of the FITC conjugated antibody and the appropriate substrate. Appropriate negative and positive controls should be included to demonstrate accuracy of the reaction. For certain tests conjugated antiserum must have a specific fluorescein/protein (FIP) ratio for optimal results.

Antinuclear Antibody Test One of the most commonly used immunofluorescence tests is the antinuclear antibody test. A variety of substrates including mouse and rat liver and kidney sections, tissue culture cells, and homologous white blood cells have been used as substrates for the antinuclear antibody test. The two substrates most commonly used for the detection of antinuclear antibody test in canine and feline serum are tissue culture cells and liver sections. The procedure used in our laboratory is as follows: 1. Serum from the patient diluted 1:5 is placed on V ero cells and incubated for 30 minutes. Note: the Vero cells are grown on 8-

730

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SCHULTZ AND LINCOLN S. ADAMS

chamber slides (Lab-Tek) until 50 per cent of the cell layer is grown in. The cells are then fixed for 15 minutes with cold (4° C) methanol or acetone. 2. The cells are washed in phosphate-buffered saline (PBS) for 10 minutes with at least three changes of buffer. 3. The slides are flipped to remove as much buffer as possible, and anti-canine IgG or anti-feline IgG labeled with fluorescein isothiocyanate (FITC) is placed on the sections of the slide and incubated for 30 minutes. 4. Step 2 is repeated. The slides are dipped in distilled water and then air-dried. 5. A counterstain (e.g., Evans Blue) is used if desired. 6. A suitable solution such as phosphate-buffered glycerol or elvanol is placed on the slide, a coverslip is applied, and the slides are viewed for fluorescence with a microscope equipped for fluorescence microscopy. The procedure is essentially the same for the other substrates. Antibody from the patient is serially diluted or screened at a specific dilution calculated by the particular laboratory to be significant if a positive reaction is obtained. Interpretation is not always simple and results from inexperienced personnel or laboratories not familiar with the domestic species should always be suspected. Submitting an occasional normal sample or known positive sample to the laboratory are measures the practicing veterinarian can employ to test the reliability or at best the reproducibility of results from the laboratory testing the samples. Practicing veterinarians should also understand that laboratory results will not always confirm the clinical diagnosis. Certainly in rare instances the clinical diagnosis will be incorrect and the laboratory results will unfortunately help confirm that misdiagnosis. We currently do not know what percentage of dogs with systemic lupus erythematosus will test positive. Furthermore, we do not know what diseases other than systemic lupus erythematosus will present with a positive antinuclear antibody test. We would estimate from our results that approximately 90 per cent of dogs with systemic lupus erythematosus will have antinuclear antibody at some time during the course of disease. With the criteria we have set for considering a sample positive we have found antinuclear antibody in a few cases of polymyositis with no evidence of systemic lupus erythematosus. We have found a few dogs with rheumatoid arthritis to have antinuclear antibody (1:5 titer), and we have also found a few dogs with systemic lupus erythematosus to have rheumatoid factor (Table 3). The correlation between systemic lupus erythematosus cells and antinu· clear antibody would suggest that approximately 60 per cent of animals with a positive antinuclear antibody test have systemic lupus erythematosus cells. The presence of SLE cells can be considered to be

.....

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Table 3. Estimated Percentage of Positive Tests for Autoimmune Responses in Immunologic Disorders

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r"'

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IMMUNE

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t=i

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l:j

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ATOPY

t"l

TEST

NORM.

Antinuclear antibody

I

90

20

10

10

0

10

0

20

10

30

10

Rheumatoid factor (Rose Waaler)

0

10

10

60

10

0

0

0

0

0

5

0

Antiglobulin (Coombs')

0

20

90

5

20

0

5

0

0

0

5

0

::r::

Thyroid antibodies or sensitized cells

5

5

0

0

10

0

50

0

0

?

10

10

0

Lupus erythematosus cell test

I

60

15

0

0

0

5

0

0

0

30

5

z>I:)

Skin-antibody

?

?

0

0

?

0

0

90

?

?

0

?

(")

Muscle-antibody

0

0

0

0

0

0

0

0

0

0

0

0

"1

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N

tJO

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Complement (C3 ) concentration

N

N

N

N

t10

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uo

Lymphocyte blastogenesis (mitogens)

N

N to t

N

N

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Immunoglobulin concentration

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a positive diagnosis of systemic lupus erythematosus; however, their absence should not be considered to be diagnostic for the absence of systemic lupus erythematosus, since only 60 per cent of animals with systemic lupus erythematosus might be expected to be positive on any one sample, with possibly as many as 75 to 80 per cent being positive if multiple samples are tested during the course of disease. The antinuclear antibody test is preferred for screening animals suspected of having systemic lupus erythematosus. It is of interest to note that dogs have relatively low titers of autoantibody as compared with titers reported for man. This has been particularly apparent in autoimmune skin diseases such as pemphigus in which the indirect immunofluorescence test has been negative in certain cases, requiring direct immunofluorescence of a biopsy sample to make the diagnosis. Additional tests employing the indirect immunofluorescence technique in immunodiagnosis are the detection of antibody to toxoplasma in cats and the recently developed procedure to detect antibody to feline infectious peritonitis virus. The most commonly used immunodiagnostic procedures that use the direct immunofluorescence technique are detection of antibody and/or complement in kidney, thyroid and skin biopsies, detection of feline leukemia virus in peripheral blood leukocytes, and canine distemper virus in conjunctival smears, peripheral blood cells, or foot pad biopsies. The use of conjunctival smears or peripheral blood cells for the detection of canine distemper virus appears to be an unreliable method and cannot be highly recommended. Unfortunately a suitable alternative, readily adaptable to specimens shipped by surface mail, is not available. The development of a rapid and sensitive detection method for the diagnosis of canine distemper virus infections remains a challenge to the veterinary researcher. Numerous viral, bacterial and protozoan:, and helminthic parasites can be detected in tissue specimens using the direct or indirect immunofluorescence technique. Immunofluorescence is used to enumerate B cells in peripheral blood. Employing a technique to detect membrane immunoglobulin on lymphocytes, it is possible to determine the relative percentage of these cells. It should be noted that immunoglobulin is also present on monocytes, and immune complexes can attach to T cells, occasionally giving erroneous results for the percentage of B cells. Furthermore, in autoimmune diseases such as systemic lupus erythematosus and viral infections such as canine distemper, antibody to leukocytes can be produced. The antileukocyte antibody attached to the membrane would be recognized as membrane Ig positive cells, therefore called B cells. This would give erroneous results and falsely elevated B cell values. Methods are available to remove immune complexes or an-

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IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

tibody to membrane in which cells are first treated with trypsin or papain, incubated for 12 to 24 hours, and then tested for membrane Ig. B cells can regenerate their membrane immunoglobulin, whereas immune complexes and antibody for lymphocyte membrane will be washed away. The Ig positive non-B cells on subsequent testing will be negative for membrane Ig, allowing proper enumeration of the B cells.

Enzyme-Linked Immunosorbent Assay (ELISA) The technique is similar in certain aspects to the immunofluorescence test. Antibody is conjugated with an enzyme, most often peroxidase or alkaline phosphatase. Indirect or direct techniques are available and would be similar to those described above for the immunofluorescence test. Some of the advantages of this test over the immunofluorescence test are greater sensitivity, and a special microscope is not necessary. Some readily apparent disadvantages are the presence of endogenous enzyme in tissue samples that activate the substrates, lack of experience with the test in many systems, and the carcinogenicity of certain of the chemical reactants. This technique, however, is quickly being adapted to numerous immunodiagnostic tests in veterinary medicine, and in the near future will be used as a routine test for detection of antibody as well as antigen (Fig. 9).

Radioimmunoassay (RIA) This technique, although relatively new, has won wide acceptance in clinical medicine because of its exquisite sensitivity. It has been particularly useful in clinical endocrinology in spite of its high cost and relative level of sophistication. It shares some similarities with the immunofluorescence and enzyme techniques in that there is one method that detects antigen and a second that detects antibody. Antibody or

Figure 9. Enzyme linked immunosorbent assay (ELISA) is a method of detecting antibody to Brucella canis. Antigen is attached to wells of microtiter plate, and the plate with antigen is reacted with serum which is believed to contain antibody to B. canis antigen. The plates are washed and enzyme labeled anti-canine IgG is added, washed, and substrate is added and plates are incubated. A positive reaction is recognized by a visual change (becomes dark) in the color of the substrate.

anti-canine IgG enzyme conjugate E-

Peroxidase or alkaline phosphatase

~

Brucella canis antibody (dog serum)

* * -Brucella

canis antigen

s-s -Substrate Microtiter plate

734

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It

A

~~

~ Cortisol

• ••• =

12~I

SCHULTZ AND LINCOLN S. ADAMS

~Cortisol

1n serum

'\f II

Antibody to Cortisol Figure 10. Competitive radioimmunoassay for cortisol in canine serum. The unlabeled antigen (A) competes with labeled antigen (*A) for a limited number of antibodycombining sites and decreases the amount of labeled antigen bound to the antibody. This is then translated into the amount of cortisol present in the serum.

antigen is conjugated not with a fluorescent chemical or an enzyme, but rather with a radioisotope, frequently, but not always, 125iodine. The two methods most frequently in use would be a competitive binding assay, which utilizes a radiolabeled antigen of known activity, in which the unknown is added to compete with the radiolabeled substance for antibody combining sites, and a second system in which the antibody is labeled with radioisotope to measure antigen in an unknown system. Both techniques can employ either a liquid or soluble phase assay; or, in the more recent modification of the technique, the solid phase, certain reactants are absorbed onto a solid matrix (Fig. 10). In addition to its use for measuring hormones and drugs, it is currently being used to measure antibody to DNA in systemic lupus erythematosus, specific IgE antibodies (RAST), total IgE (RIST), and antibody to certain viral antigens in canine serum. Complement Fixation The principle upon which this technique is based states that complement components are utilized when an antigen-antibody reac.tion occurs. The complement components are not available for the subsequent lysis of antibody-sensitized red blood cells. The test can presumably be used for the detection of antibody to any antigen (Fig. 11).

There are several disadvantages to the test. Considerable standardization of reagents is required before running the test, and many serum samples are anticomplementary. Also, there is little or no specificity to the reaction, thus if purified antigens are not used one cannot determine the specificity of the antigen antibody reaction. An additional disadvantage is that the standard complement fixation test utilizing guinea pig complement and rabbit antibody to sensitized sheep red blood cells does not work well with cat serum, because guinea pig serum is not readily fixed by feline immunoglobulins.

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

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The complement fixation test, although employed for the detection of antibody to numerous microbial antigens in human and certain domestic species serum, is seldom used with canine and feline serum. The complement fixation test should not be confused with tests to detect hemolytic complement levels or complement components in serum of dogs or cats with possible complement deficiencies. Complement assays in autoimmune disease and certain complement deficiency diseases determine the concentration of hemolytic complement (CH50 ) in the serum or synovial fluid or determine individual complement components (e.g., C3 ). (These procedures are described later in this article.) Virus Neutralization Test

The virus neutralization test is commonly employed to determine the relative amount of blocking or neutralizing antibody to a specific virus or for the identification of an unknown virus. For many viral infections of the dog and cat, the virus neutralization titers can be used as a correlate of protective immunity to monitor the efficacy of vaccination or to determine the cause of a specific disease. For certain

B

A Antigen

0

•

+

Complement

t y Anti body

RBC

Antigen-Ab Complement

with Complement

•

no lysis Figure II. Complement fi xation. A positive test is based on the consumption of complement in part A. and the lack of lysis because complement would not be available for the indicator system (B). If the antibody was not presented to the antigen in part A, complement would be available and lysis would occur in the indicator system B. This would be interpreted as a negative test.

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viruses (such as herpes virus), the detection of neutralizing antibody cannot be directly correlated with protection against reinfection. The viral neutralization test requires the ability to prepare and maintain cell cultures, a procedure rarely available in most small laboratories or hospitals. The virus neutralization procedure has been simplified by microtiter equipment and disposable plasticware; however special facilities and cell cultures are the limiting factors in providing this test. The basis of the test is the ability of specific antibody at a particular dilution to react with a known quantity of virus particles, preventing the virions from infecting susceptible cells. The antibody titer is a relative term which relates to the highest dilution of antibody capable of neutralizing (preventing infection) of a standard quantity of virus (50 to 100 TCID50 ). There are numerous methods used to determine if the serum has neutralized or prevented viral infection. They include looking for inhibition of cytopathic effect (such as cell death, syncytium formation), inability of the infected cells to absorb red blood cells (hemabsorption), inability to detect virus by the direct immunofluorescence test, or lack of infectivity of the virus antibody complex in animals. Viral neutralization assays are commonly used to detect antibody to most of the viruses that infect dogs and cats.

Agglutination Agglutination reactions between antibody and particulate antigens form the basis of the most commonly used techniques in the immunology laboratory. Unlike the precipitin reaction which requires a soluble antigen and generally involves IgG antibodies, the agglutination reaction requires a particulate antigen or stable cell that will not spontaneously agglutinate, and generally IgM antibody is most effective. Agglutination reactions are classified as direct, requiring only the presence of antigen and antibody, or indirect (passive), requiring antigen to be absorbed or conjugated to particulate matter. The indirect or passive agglutination test can be performed with antigens that are readily attached to particulate matter such as red blood cells, bentonite, or latex particles. Agglutination tests can be performed in tubes, on slides, or in microtiter trays. Direct agglutination tests are performed with red blood cells, bacteria, fungi, protozoa, and a variety of other particulate antigens. Direct agglutination tests with a bacterial suspension is used to detect antibody to Brucella canis. Several agglutination tests are available including a slide agglutination test as well as tube agglutination. The slide agglutination test, like all serologic tests, is plagued by certain problems of false positive or false negative reactions. Bacteria that are antigenically similar to Brucella antigens cause false positive tests. If the slide agglutination test is utilized as a screening test for canine brucellosis, it is recommended that simultaneous blood cultures and

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

737

additional serologic tests (i.e., tube test) be used to confirm positive slide agglutination results. In general false negative results are rarely observed, but they may be, if the animal is infected but has not developed antibodies at the time of the test. The antiglobulin test (Coombs' test) is a simple modification of the agglutination test which detects the presence of nonagglutinating levels of antibody on red blood cells, bacteria, or virus. The antiglobulin test increases the sensitivity of the agglutination test and has additionally made it possible to detect immunoglobulins (i.e., IgG, IgA) which normally are not good agglutinins. This test forms die basis of the diagnostic test for autoimmune hemolytic anemia, in which antibody-coated red blood cells are agglutinated in the presence of anti-immunoglobulin and complement antisera. It also forms the basis of the Rose Waaler test for the detection of rheumatoid factor.

Antiglobulin (Coombs' Test) Autoimmune hemolytic anemia is a hemolytic state in which antibody can be demonstrated on erythrocytes (Fig. 12). These antibodies are most often warm antibodies, either agglutinating or hemolytic antibodies that can be detected by the antiglobulin (Coombs') test. However, on rare occasions these antibodies may be cold antibodies; that is, they can only be identified if the test is performed in the cold. The laboratory procedure we use to detect warm antibodies is as follows: Blood is allowed to clot (preferred for shipment by mail) or collected in EDT A. Cells from the clot or from the EDT A tube are washed four times to ensure that plasma protein is not coating cells nonspecifically. The washed erythrocytes are suspended in saline to a final concentration of 2 per cent. The antiglobulin (Coombs') reagents are diluted in a microtiter U plate and equal volumes (0.025 ml) of

Figure 12. Schematic representation of the antiglobulin (Coombs') test for the detection of antibody-coated red blood cells in autoimmune hemolytic anemia of the dog.

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erythrocytes are added to the antiglobulin reagent and the patient's serum, followed by incubation of the plate for 30 minutes at 37° C and an additional 30 minutes at room temperature. The plat<': is read microscopically and macroscopically for evidence of agglutination. It is essential that the antiglobulin reagent has, as a minimum, antibody activity to IgG and C3 and that it does not react with normal red blood cells. A positive test would be diagnostic of antibody-coated red cells, a phenomenon most often associated with autoimmune hemolytic anemia, but also recognized in dogs with multiple myeloma and some cats with hemobartonella infections and leukemia. Special precautions should be used in interpreting results from feline antiglobulin (Coombs') test, since a common cause of anemia is hemobartonella, and a less common cause of antiglobulin positive test is feline leukemia virus infection. Cold agglutinin disease in man is the most common type of cold antibody autoimmune hemolytic anemia. It can occur as an acute or chronic condition, the former being associated with Mycoplasma pneumonia infections and the latter seen often in older patients, sometimes associated with Raynaud's phenomenon (blue appearance in skin) and hemoglobulinuria, particularly in cold weather. Blood samples collected in EDTA should be incubated at 37° C to elute the IgM antibody. Complement will remain on the surface which can be detected by the antiglobulin (Coombs') test. The plasma from patients with cold agglutinin disease is then diluted and tested against the washed red blood cells. The test is incubated at 4° C in saline as a diluent and 30 per cent albumin as diluent. Cold agglutinins are usually present at very high titers (i.e., > 1:1 00) if the animal has the disease. Titers of less than 1: 100 can be found in sera of certain normal dogs and cats. A few cases of cold agglutinin disease have been reported in the dog. Rose-Waaler Test for the Detection of Rheumatoid Factor

The Rose-Waaler test is similar to the antiglobulin test described above (Fig. 13). Sheep red blood cells are coated with a subagglutinating dose of rabbit or dog anti SRBC antibodies. There does not appear to be a difference in the species of antibody used to sensitize the cells. These sensitized cells are added to dilutions of the patient's serum. Incubation is for a minimum of 1 hour at 37° C and 1 hour at room temperature. Agglutination is determined microscopically and macroscopically. We have established that a titer of 1: 16 or greater is considered positive, a titer of 1:8 suspect, and almost all dogs will have a titer of 1:4 or 1:2; therefore, these are considered negative. Approximately 60 per cent of the dogs with other criteria of rheumatoid arthritis are positive in this test. The screening test used widely in human medicine to detect

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fi'h~WI?'!ctt't>;,:/

r<7elor (RFj

Figure 13. Schematic representation of the Rose-Waaler test for the detection of canine rheumatoid factor associated with rheumatoid arthritis.

rheumatoid factor is the latex slide agglutination test. The human latex reagent cannot be used to detect canine rheumatoid factor. Recently a canine latex reagent was developed to detect rheumatoid factor in canine arthritis. The results of the test on a large number of samples, suggested such a reagent was not suitable for detection of rheumatoid factor in canine rheumatoid arthritis. The reasons for these results are not presently known.

Skin Test There are several different skin tests that can be used to identify certain classes of antibody. The immediate hypersensitivity or type I reaction is commonly used to determine the offending allergen in allergic inhalant dermatitis of dogs. The reaction is characterized by a wheal and flare and appears within minutes. Normally reaction sites would be measured at 15 and 30 minutes after intradermal administration of the antigen. Skin testing of cats is not normally done, as atopic diseases are rare in this species. There are two passive tests to detect IgE antibodies in a number of species. They are the Prausnitz-Kustner (P-K) test and passive cutaneous anaphylaxis (PCA) test. It has been reported that neither technique is particularly effective in the dog. Skin tests to detect type III hypersensitivity reaction or the Arthus reaction involve the subcutaneous injection of antigen into animals which have antibody capable of precipitating the antigen (IgG) and fixing complement. An acute inflammatory reaction develops at the site of injection within several hours, starting as an erythematousedematous swelling, but as it develops, local hemorrhage and thrombosis occur and frequently progress to necrosis in a few days. Arthus reactions can be produced in both dogs and cats and may be an Important mechanism in certain bacterial hypersensitivity reactions, im-

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mune complex mediated glomerulonephritis, infectious canine hepatitis uveitis (blue eye), and joint lesions in rheumatoid arthritis.

Assays for Complement The complement system is one of the mam humoral effector mechanisms in protective immunity against a variety of infectious agents. Additionally complement is involved in immune complexinduced tissue damage (type III hypersensitivity). Clinically useful assays for complement would consist of total hemolytic complement or CH50 and specific functional or immunochemical assays for various individual components. Because of a paucity of information on the complement system of dogs and cats and the lack of purified complement components or antisera to them in these species, little or nothing is known about their levels in immune mediated diseases and complement deficiencies have not been recognized in the dog or cat. Using techniques to measure CH50 in dogs and radial immunodiffusion to quantitate the C3 component of complement, we have not regularly recognized significant alteration of complement in systemic lupus erythematosus, rheumatoid arthritis, or a variety of other diseases. The significance of these results is unknown, but they do not correlate with results for other species (e.g., man). This area has been neglected, and therefore should receive greater attention in the future.

Assays for IgE (Reagenic Antibody) Radioallergosorbent Test (RAST), Radioimmunosorbent Test (RIST). Immunoglobulin E, the antibody involved in the immediate hypersensitivity reaction (type I) as well as in immune responses to vermin infections, is difficult or impossible to detect with conventional tests because serum concentrations are very low. A number of laboratories interested in immediate hypersensitivity (atopy) are currently attempting to modify the RAST to measure specific IgE in dogs. In the technique, serum IgE is measured by adding the dog's serum to allergenlinked cyanogen bromide-activated sepharose (dextran). After thorough washing, the absorbed IgE antibodies are measured by the uptake of radioisotope-labeled purified anti-canine IgE (Fig. 14). The reaction is extremely sensitive, as are other radioimmunoassay (RIA) procedures, but it does require a specialized laboratory. The test could be considered an in vitro correlate of skin testing for type I hypersensitivity. Another test which is an RIA test to measure IgE is the radioimmunosorbent test (RIST). This test is a competitive binding assay to measure the quantity of IgE in serum and is not a measure of the specific antibody activity of IgE with regard to a specific allergen (antigen) (Fig. 15).

IMMUNOLOGIC DETECTION OF HUMORAL AND CELLULAR IMMUNITY

0 0

~~

+

Cyanogen-bromide sephadex with allergen (i.e. ragweed J (A)

741

~))cq Patient's serum containing Antibody(IgE) to ragweed ( B!

+

,. Jf. 14 ...,

I labeled anti canine IgE ( C J

Figure 14. The radioallergosorbent test (RAST). The test is an RIA procedure that detects IgE antibody to various allergens. If the patient has IgE antibody in its serum (B) it reacts with the allergen (A) and provides an insoluble complex which is reacted with 125 I labeled anti-canine IgE. The mixture is processed and counted in a gamma counter to determine whether it is positive or negative.

Goat or rabbit Insoluble anti canine IgE

+

Anti canine Ig£

Dog serum Ig£

Figure 15. The radioimmunosorbent test (RIST) is an RIA procedure that measures the total amount of IgE in serum. Conventional tests (electrophoresis, RID) are not sensitive enough to detect IgE!

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Total IgE levels in the serum are also measured by a radioactive single RID procedure. Rabbit anticanine IgE is incorporated into agar and standard or unknown sera placed in wells. (See discussion of RID earlier in this article.) An invisible precipitin ring forms, which after removing the nonreacted antisera is visualized by incubating with 125 Ilabeled goat anti-rabbit IgG followed by radioautography. The concentration of IgE is determined by measuring the diameter of the ring and comparing it with the standard preparations. The clinical significance of quantitation of IgE is currently not known, because the range of values for normal dogs is large and because intestinal parasitic infections greatly increase the levels of IgE, making it difficult to relate values to allergic conditions. In addition, the IgE level does not identify offending allergens; therefore, skin tests or the RAST must be used for that purpose. The experimental nature of IgE tests in veterinary medicine and the lack of suitable reagents have greatly limited their clinical value; thus they are currently not available to the practicing veterinarian.

DETECTION OF CELLULAR IMMUNE FUNCTION Numerous in vitro correlates of cell-mediated immunity have been developed during the last 5 to 10 years. Many of these techniques have been adapted to veterinary immunology and have been used for clinical diagnosis. An understanding of their applicability to clinical disease and their limitations is essential. Delayed Hypersensitivity (Type IV) Skin Tests The in vitro correlates of CMI assume particular significance as clinical tests for the dog and cat, because typical DTH skin responses are not readily detected in these species. Our experience as well as that of numerous other veterinary researchers is that DTH skin testing in dogs and cats is characterized by a predominant infiltration of polymorphonuclear neutrophils, rather than mononuclear cells, into the site and the induration and erythema classic of DTH in other species is minimal or absent even in dogs and cats sensitized with or infected with mycobacterial species and skin tested with the classical purified protein derivatives (PPD). Poor DTH skin reactions have been particularly frustrating in clinical medicine, in that the standard panel of skin test antigens commonly used to monitor cellular immunity in man cannot be duplicated in cats or dogs, employing antigens which should be useful for that purpose. Helminth antigens (Ascaris canis), tissue antigens, viral antigens (CDV), bacterial antigens (PPD), fungal antigens, and protein antigens have been employed experimentally and clinically in dogs and cats with limited reproducibility or irreproducible results.

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Sensitization with chemicals such as dinitrochlorobenzene used successfully to demonstrate a DTH-like reaction in man and rodents was reported to be useful in the dog only if biopsies of the skin were examined microscopically. A similar technique applied to cats did not appear to us to be useful to measure DTH even when biopsies were examined, in that a classic acute inflammatory response was noted rather than a DTH. We also have found this test to have limited usefulness in the dog. Several years ago we reported on the intradermal use of mitogens to assess the immune responsiveness of dogs immunosuppressed by canine distemper virus or in dogs with generalized demodectic mange. Although we found the response to phytohemagglutinin (PHA) and concanavalin A (Con A) to be reduced in these two diseases both associated with a secondary immunosuppression, histologic examination of biopsies from inoculated sites appear to be similar to a classical acute inflammatory reaction rather than a classic DTH reaction. Reactions to skin mitogens peak at 24 to 36 hours rather than 48 to 72 hours and are characterized by infiltration with neutrophils. Recently a detailed comparative histologic and ultrastructural study was completed by Dr. Helen Greisen of our laboratory. Skin reactivity to mitogens was tested in the dog, rabbit, and guinea pig to determine if cutaneous basophil hypersensitivity Qones-Mote) reaction, believed to be a manifestation of a T cell-mediated release of basophil chemotactic factor, was similar in these three species. The results clearly suggest that unlike the rabbit and guinea pig in which a good DTH and CBH can be demonstrated, the dog had no CBH reaction in the skin biopsies. The reason for the poor DTH and CBH in the dog is currently not known, but it should not be interpreted as a lack of CMI in this species. The poor delayed type skin reactivity is also manifested clinically in that there are very few documented cases of contact dermatitis in dogs and there have been no cases of contact dermatitis to my knowledge demonstrated to occur naturally in the cat. Many of the contact dermatitis cases demonstrated in the dog are direct contact irritants that have not been demonstrated to be due to a T cell mediated type IV hypersensitivity. One method to demonstrate a DTH or CMI response in dogs and cats is skin allograft rejection. Skin allografts can be readily performed in dogs and cats to assess a diminished or suppressed cellular immune response. Normal rejection times for dogs and cats are 12 ± 2 days. Delays of 5 to 7 days would suggest a significant impairment of CMI. In our experience the delay of skin allograft rejection time is one of the last immune functions to be lost; therefore this procedure would not be very sensitive and would only measure very severe immunodeficiency or immunosuppression. Significant delay in time of skin allograft rejection has been recognized in cats with leukemia.

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Because of the inability to readily demonstrate a DTH reaction in dogs and cats a greater reliance on the in vitro tests has required the adaptation of these techniques to the dog and cat. Separation of Mononuclear Cells from Blood

The most commonly employed method to separate mononuclear cells (monocytes and lymphocytes) from polymorphonuclear granulocytes and re,d blood cells is density gradient centrifugation on ficollhypaque or ficoll-isopaque (Fig. 16). The ficoll can be purchased from Pharmacia Fine Chemicals, Piscataway, New Jersey. The hypaque or isopaque can be purchased from a variety of sources (hypaque Winthrop Laboratories, New York, New York; isopaque- GallardSchlesinger, Carle Place, New York). We have found the commercially prepared Ficol-Paque (Pharmacia Fine Chemicals), density 1.077, to work suitably for the separation of dog and cat mononuclear cells if you do not wish to prepare the material in your laboratory. Separation is readily accomplished by carefully layering the heparinized (preservative free) blood on the top of the density gradient solution and centrifuging at room temperature for 15 minutes in a swinging bucket type rotor at 1300 xg (2200 to 2500 RPM). Analysis of the cells would suggest that a successful separation would result in an 85 to 95 per cent mononuclear cell preparation. Approximately 80 to 85 per cent of the mononuclear cells are lymphocytes and 15 to 20 per cent are monocytes. It is possible to reduce the monocytes by first incubating the blood with carbonyl iron (GAF Corp., Binghamton, New York). Add 0.5 gm carbonyl iron to 10 ml of heparinized blood, then incubate for

---Plasma

Blood----

~Wll2!t- -Mononuclear cells

Fico// Hypaque-Red blood cells and Polymorphonuclear cells Before Figure 16.

After

Separation of blood mononuclear cells by density gradient centrifugation.

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one hour at 37° C with constant or intermittent mixing. Separation is then performed as described previously by applying blood to the density gradient and centrifuging. This additional step reduces but does not completely eliminate monocytes. The Lymphocyte Blastogenesis (Transformation) Technique The test has proved extremely helpful in the laboratory diagnosis of CMI deficiency in the dog and cat. The technique is subject to a variety of trials and tribulations; however, after optimal conditions for the test are established, it is an excellent immunodiagnostic tool in a variety of clinical conditions and promises to have wider applicability in the future. A number of modifications of the technique are available. Optimal conditions established for mitogenic stimulation of canine and feline peripheral blood cells in the macro test were as follows: heparinized blood was centrifuged at 160 xg for 10 minutes to obtain a huffy coat and leukocyte-rich plasma fraction, devoid of as many erythrocytes as possible. The white cell suspension was diluted so that 0.1 ml contained between 5 X 10 5 to 5 X 106 leukocytes. Cells were placed into 21 X 70 mm screw-capped glass vials which contained 1 ml of RPMI-1640 media with 100 units of penicillin, 100 f-tg of streptomycin, 10 per cent fetal bovine serum (FBS), and an optimal amount of mitogen. Optimal conditions for the micro test differed slightly from the macro test. Heparinized blood was centrifuged on a ficoll-isopaque gradient for 15 minutes at 1300 g. The mononuclear cell layer was removed and the cell suspension was adjusted so that 0.1 ml contained between 5 X 10 4 and 5 X 105 cells. Cells were placed in wells of a flat bottom 96-well microtiter tissue culture plate which contained 0.1 ml of the media used in the macro test. The mitogens included phytohemagglutinin (PHA), concanavallin A (Con A), pokeweed mitogen (PWM), E. coli lipopolysaccharide (LPS), and lipid A and streptolysin 0 (SLO). Culture tubes with loosened caps and microtiter plates were incubated for 72 hours at 39° C in 5 per cent C02 and air. Two ~-tCi of 3 H thymidine was added in a volume of 0.5 ml to the tubes and 1 ~-tCi in 0.1 ml was added to the wells. For the macro tube test the cells were centrifuged, washed, precipitated with trichloroacetic acid, dissolved in formic acid, and the radioactivity determined by scintillation spectrophotometry. The cells in the micro plate test were harvested with a MASH II (Microbiological Associates, Bethesda, Maryland). The lymphocyte blastogenesis test was used to evaluate a large number of dogs and cats admitted to the Small Animal Clinic of the New York State College of Veterinary Medicine, as well as dogs and cats on various research experiments at the James A. Baker Institute for Animal Health (Fig. 17). The following should be used as a guideline when performing the test on clinical samples. Samples should be

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3-5day

+ Small Lymphocytes

Incubation

2J

Mitogen or Antigen

-

Loter label for 12-18 hours 3 H thymidine

and Monocytes Figure 17. Lymphocyte blastogenesis (transformation test). Lymphocytes and monocytes or macrophages are isolated and cultured with mitogen or antigen for three to five days. Radioisotope, generally 3 H-thymidine, is added to the culture for an additional 12 to 18 hours and the cells are harvested and radioactivity determined with a liquid scintillation spectrophotometer.

run in duplicate or triplicate and repeated at least one time on a second day before any conclusions are drawn from results. Lymphocytes should be cultured in autologous serum or plasma in addition to fetal bovine serum to detect immunosuppressive factors (i.e., generalized demodectic mange, vitamin E deficiency). Values should be compared to normal mean values established for a large number of control animals, but more important, should be compared to normal control samples run simultaneously with the animal being evaluated. Under the conditions outlined we have found the test to be a very useful diagnostic tool. Clinical and experimental conditions in which we have found significant suppression of canine lymphocyte responses to phytomitogens have been: canine distemper, generalized demodectic mange (autologous serum or plasma present), certain nutritional deficiencies, a percentage of dogs with lymphosarcoma, a small number of dogs with aspergillosis, a percentage of dogs older than nine years of age, and animals treated with certain drugs. Suppression in the cat has been associated with feline leukemia virus infection, clinical leukemia, panleukopenia virus infection, and an occasional animal without specific disease. It is our experience that feline lymphocytes in general respond more poorly to PHA than does the dog or other species. The cat lymphocytes respond well to Con A and PWM. It is not currently known whether this reflects a difference in lymphocyte subpopulations of the cat as compared with other species, or if it is a technical artifact. It is also of interest to note that steroid treatment of the dog does not affect the lymphocyte response to phytomitogens. Based on a number of experimental studies, numerous cell manipulations and drug treatment of dogs and cats we would suggest that the following populations of cells are stimulated by mitogens: PHA

Predominantly T cells (this population or subpopulation may be absent in most cats)

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Con A

T cells (perhaps more than one subpopulation or a population different from that stimulated by PHA) PWM T and B cells (early response two and three days after stimulation predominantly T cells) T and B cells SLO LPS (lipid A) B cells (most dog and cat peripheral blood lymphocytes do not respond to E. coli LPS; however, their milk or splenic lymphocytes will) Inhibition of Cell Migration This test, considered to be an in vitro correlate of CMI or DTH, measures the production of the lymphokine migration inhibition factor (MIF). If this factor inhibits the migration of macrophages it is called "macrophage migration inhibition factor" and the factor that inhibits the migration of blood monocytes and neutrophils is called "leukocyte migration inhibition factor" (Fig. 18). The basic technique we have found suitable for the dog is one in which peripheral blood leukocytes are packed into a capillary tube or placed in a well of an agarose plate and antigen or mitogen added to the cells. If MIF is produced by the lymphocytes little or no migration occurs. If the lymphocytes are unable to produce MIF, normal migration occurs. The inability to produce MIF would be correlated with a deficiency of lymphocyte function and presumably a deficiency of CMI. There are also modifications of the technique which use dog and cat lymphocytes to produce MIF and guinea pig peritoneal macrophages as the target cells. The indirect procedure is necessitated by the difficulty of obtaining peritoneal macrophages in the dog and cat. Standard techniques successfully used to stimulate large numbers of peritoneal macrophages in laboratory species have been of limited value in stimulation of peritoneal macrophages in dogs or cats. More

A

G) Canine Herpes Virus Immune Dog

8

No Antigen Immune Dog

c

Canine Herpes Virus Non-Immune Dog

Figure 18. Leukocyte migration inhibition test. Sample A is the inhibition of migration of neutrophils and monocytes in a peripheral blood sample of a dog immune to canine herpes virus. Samples B and C are controls, demonstrating no inhibition of cells if the antigen is not present and no inhibition with a nonimmune dog's cells.

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than 50 per cent of the cells in peritoneal exudates of dogs and cats stimulated with a variety of substances will be polymorphonuclear leukocytes. Delaying the harvest of cells for approximately 7 to 9 days after stimulation of peritoneal exudates has resulted in a somewhat better harvest of macrophages with a concurrent decrease in polymorphonuclear leukocytes, but results with the dog and cat never approach results of this technique in laboratory rodents. The MIF technique, because of numerous factors affecting reproducibility, has not found wide acceptance in the clinical laboratory. Lymphocyte Rosette Assays forT Cells

Several years ago it was found that human T lymphocytes spontaneously rosetted with sheep red blood cells. Investigations were initiated to determine if similar tests could be used to detect canine and feline T cells: It became immediately apparent that neither canine nor feline lymphocytes rosetted with sheep erythrocytes; however, several species of erythrocytes were found to rosette with lymphocytes from dogs and cats. It was reported that human and guinea pig erythrocytes rosette with dog cells and guinea pig erythrocytes rosette with cat cells (Fig. 19). Several studies have recently appeared suggesting that erythrocyte rosetting may not be specific for T cells in the canine species or the feline species. The reason for this skepticism results from several of the following observations: (1) in several laboratories only 20 to 30 per cent of the peripheral blood lymphocytes of dogs and cats formed rosettes as compared with 60 to 80 per cent of blood lymphocytes from man; (2) the observation that approximately 3 per cent of cells from the thymus of the dog rosette, whereas more than 90 per cent of cells from the human thymus rosette; (3) the lack of appreciation of the knowledge that cells such as polymorphonuclear leukocytes and monocytes rosette and this is not a phenomenon restricted to lymphocytes; and (4) the previous inability to correlate rosetting results in dog or cat with other functional tests or additional techniques to identify T and B cells. Studies in our laboratory would suggest that human erythrocytes

Figure 19. Erythrocyte rosette forming cells (ERC). A subpopulation of canine and feline T cells form ERC with human and guinea pig red cells, respectively.

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are better than guinea pig erythrocytes for the demonstration of lymphocyte rosetting in the dog. Unlike results from a number of other laboratories we find under optimal conditions, 40 ± 10 per cent of peripheral blood lymphocytes from the dog to form rosettes. These results are obtained with a preparation of mononuclear cells with no polymorphonuclear leukocytes and less than 5 per cent monocytes. The most important and convincing finding that the rosetting cells are lymphocytes is that if the rosetting cells are isolated by ficoll-hypaque density gradient, they can be demonstrated to respond to phytomitogens in the blastogenesis test, a characteristic that cannot be demonstrated for polymorphonuclear leukocytes or monocytes. However, unlike the situation in man in which all or most T lymphocytes in the blood rosette, we find that only a certain subpopulation comprising perhaps 40 to 50 per cent of the total lymphocytes of the dog rosette. This subpopulation, which we refer to as T 2 , is a lymphocyte that has acquired a receptor for human red blood cells after the lymphocyte has migrated from the thymus to the peripheral tissue. The lymphocytes of the thymus referred to here as T 1 cells have not acquired a receptor for red blood cells. A similar situation may hold for the cat since as with the dog only a small percentage of cells rosette. This finding limits, to some extent, the clinical usefulness of direct lymphocyte rosetting in that only a certain subpopulation rosettes and that there can be considerable variation in populations presumably without significant alteration of immune function. Specific membrane antigens have been found on the T lymphocytes in certain species. In mice these antigens are referred to as thy antigens, (in man as TLA) and are usually detected by immunofluorescence. Similar antigens occur on dog T cells and antisera have been produced to thymocytes and to brain suspensions (also known to contain a similar antigen). These antisera have to date been used predominantly in canine research only. With these antisera we find 60 to 75 per cent of blood lymphocytes to be positive, further evidence to suggest that the direct erythrocyte technique measures only a subpopulation of canine T lymphocytes. EAC (Erythrocyte-Antibody-Complement) Rosettes for B Cells

A certain percentage of lymphocytes contain surface receptors for complement (CR). These cells, known to be a subpopulation of B cells in certain species and believed to be B cells in dogs and cats, can be identified by sensitizing sheep red blood cells with antibody and complement. Complement deficiency in the C5 or C6 component or mouse complement is used so that lysis of the antibody red cell does not occur spontaneously. The rosette formed in this assay is morphologically identical to the one found in the direct erythrocyte assay described previously. The clinical value of this test is unknown at. this

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time because of the limited application of the test with canine and feline lymphocytes.

Membrane Immunoglobulin Assay for B Cells Lymphocytes with readily demonstrable immunoglobulin on their surface are believed to be B cells. The procedure to detect membrane Ig-positive cells is generally direct or indirect immunofluorescence (Fig. 20). Immunoglobulin will be found as a rim of fluorescence, as patches of fluorescence, or at one or both caps of the cell depending on the conditions of the assay. We find approximately 15 ± 5 per cent of peripheral blood lymphocytes to be membrane-Ig positive in apparently healthy dogs. The predominant membrane Ig is IgM. In a variety of diseases the number of Ig-positive cells will change. It is essential to determine if this change is the result of an increase in the B cell population or is caused by the presence of immune-complexes or antilymphocyte antibody on T cells (see discussion previously in this article).

Neutrophil Function If a deficiency in nonspecific immunity is suspected, such as cyclic neutropenia, chronic granulomatous disease, or defective bactericidal activity, the following laboratory tests may be used as an aid to diagnosis: Phagocytic and Bactericidal Function Tests. Phagocytic index and bactericidial activity tests measure the ability of cells to phagocytize and subsequently kill bacteria, a function of the lysozomal enzymes. The test is performed by mixing a suspension of bacteria (i.e., staphylococci or E. coli) with a suspension of leukocytes from the patient and leukocytes from a normal control animal in the presence of fresh

------- ,-.-------....._____ ____

.....____----

~,

Rim

Patch

Cap ( Polar)

Figure 20. Types of membrane Ig fluorescence recognized on canine B-lymphocytes. If a metabolic inhibitor (i.e., sodium azide) and cold temperatures are maintained, most of the cells will have rim and patch fluorescence.

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serum. Heparinized blood is suitable for the test. After an appropriate period of time the number of viable bacteria is determined for the patient and the control by direct bacterial plate count techniques. The inability to phagocytize and/or kill bacteria is indicative of a deficiency in neutrophil function and rarely monocyte function. Nitroblue Tetrazolium Test. This test is particularly useful to detect nonfunctional neutrophils in chronic granulomatous disease. Neutrophils from normal dogs or cats rapidly reduce nitroblue tetrazolium during in vitro phagocytosis. Neutrophils from animals with chronic granulomatous disease are unable to reduce NBT. The test has been applied to a large number of samples from dogs and to date only two have been found to be deficient, one clinically normal dog and one with pyoderma. It is essential that samples for this test are not collected in EDT A because it interferes with the rest. Chemotaxis. Measurement of directed locomotion of neutrophils and macrophages is possible using special chemotactic chambers. In this assay the cells are placed in one part of the chamber (generally the upper portion) and are separated from a source of chemotactically active material by a filter with a small pore size. After a period of incubation, the filter is removed and stained to determine the numbers of migrating cells. This test can be used to assess chemotactic factors or the ability of neutrophils or monocytes to be attracted to specific or nonspecific stimuli. Histocompatibility Tests for Dog Leukocyte Antigens and Dog Erythrocyte Antigens Two tests are currently available to type dog leukocytes. One is a serologic test which uses antisera developed in dogs by the inoculation of leukocytes from another dog. The antisera in the presence of complement will kill the leukocytes of nonrelated (incompatible) dogs. Killing is generally detected by uptake of trypan blue by nonviable cells. A number of laboratories throughout the world have the ability to serologically type dog leukocytes. An additional test for typing leukocytes is the mixed leukocyte reaction. The test is similar to the lymphocyte blastogenesis test (described previously); however, instead of mitogen, lymphocytes from two dogs are mixed together to determine if one stimulates the other. It is possible to inhibit DNA synthesis in one animal by irradiating the cells or treating them with mitomycin C. The test is then referred to as the one way mixed leukocyte reaction. A combination of the serologic tests and MLR can be used to determine the relative compatibility between individuals for organ transplants. Additionally the association between certain serologically or lymphocyte defined leukocyte antigens and disease is the subject of investigation in numerous species including the dog. The dog erythrocyte antigens assume clinical importance in repeated blood transfusions. Although numerous blood groups have

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been recognized in the dog, the one with greatest importance is blood group A (At. A2 ). Although the first transfusion with blood from an A-positive dog to an A-negative recipient will have little or no clinical consequence, additional transfusions can be fatal, because of the immunologic hypersensitivity reaction that can occur. This adverse reaction can be prevented by keeping an A-negative blood donor in your clinic.

SUMMARY A variety of immunologic techniques have been introduced during the past few years. Many of these techniques are being applied to clinical specimens in an attempt to help the practicing veterinarian make a diagnosis. The introduction of new techniques requires extensive testing with clinically normal and diseased patients. It is essential for the practicing veterinarian to understand that the techniques available for the detection of immunologic disorders in the dog and cat are not routine diagnostic procedures and that adequate information has not been developed for any of the techniques described to assure the clinical significance of either positive or negative results (Table 3). This should not discourage the practitioner from submitting samples, but should encourage him or her to question the significance of those results and to attempt to correlate them with history and clinical signs before arriving at a final diagnosis. ACKNOWLEDGMENTS

The authors would like to acknowledge Mr. George Batik for the preparation of all figures except 12 and 13, which were kindly prepared by Ms. Patricia Barker.

SUPPLEMENTAL READING 1. Bach, F. H., and Good, R. A. (eds.): Clinical Immunobiology, Vol. 3. New York, Academic Press, 1976. 2. Bloom, B. R., and Glade, P. R. (eds.): In Vitro Methods in Cell-Mediated Immunity. New York, Academic Press, 1971. 3. Gel!, P. G. H., Coombs, R. R. A., and Lachmann, P. ]. (eds.): Clinical Aspects of Immunology. Edition 3. Oxford, England, Blackwell Scientific Publications, 1975. 4. Halliwell, R. E., Lavelle, R. B., and Butt, K. M.: Canine rheumatoid arthritis- A review and case report.]. Small Anim. Pract., 13:239-243, 1972. 5. Hurvitz, A. I., and Halliwell, R.: Veterinary clinical immunology. Scientific proceeding. Am. Anim. Hosp. Assoc.,2:3, 1975. 6. Lewis, R. M.: Spontaneous autoimmune diseases of domestic animals. Int. Rev. Exp. Pathol., 13:55, 1974. 7. Osburn, B. I., and Schultz, R. D. (eds.): Advances in Veterinary Science and Comparative Medicine, Vol. 23. New York, Academic Press, 1978.

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8. Rose, N. R., and Bigazzi, P. E. (eds.): Methods in Immunodiagnosis. New York, John Wiley & Sons, 1973. 9. Rose, N. R., and Friedman, H.: Manual of Clinical Immunology. Washington, D. C., American Society for Microbiology, 1976. 10. Schultz, R. D.: Immunologic disorders in the dog and cat. Vet. Clin. North Am., 4:153-174, 1974. 11. Tizard, I. R.: An Introduction to Veterinary Immunology. Philadelphia, W. B. Saunders Co., 1972. 12. Vyas, G. N., Stites, D. P., and Brecher, G. (eds.): Laboratory Diagnosis of Immunologic Disorders. New York, Grune and Stratton, 1974. Department of Microbiology College of Veterinary Medicine Auburn University Auburn, Alabama 36830