Autoimmune manifestations of selective 7 S immunoglobulin deficiency of chickens

:‘LINICAL.

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

14, 334-347 (1979)

Autoimmune Manifestations of Selective 7 S lmmunoglobulin Deficiency of Chickens’ JOHN MONTERO, M. ERIC GERSHWIN,” JAN EKLUND, HANS ABPLANALP, KENT ERICKSON, LESLIE TAM, ALBERT A. BENEDICT, AND RICHARD M. IKEDA

Recently, an immunodeficiency analogous to acquired selective agammaglobulinemia of humans has been described in a line of University of California, Davis. chickens (UCD 140). This syndrome is characterized by normal immunoglobulin synthesis early in life followed by a variable expression dysgammaglobulinemia. Serial observation of these birds, and age matched controls, reveals, in addition to their abnormal levels of immunoglobulins, accelerated morbidity and mortaiity associated with the frequent development of Coombs’ positive hemolytic anemia, serum anti-y-globulin activity, and cryoprecipitates; though these traits are highly heritable, the exact mode of inheritance is as yet obscure. Nonetheless. there are statistically significant correlations between several of these features suggesting a common etiology. Dysgammaglobulinemic birds possess cryoprecipitate more frequently than unaffected birds, and autoimmune hemolytic anemia is frequently associated with the immunodeficiency. Additionally, males are more often Coombs’ positive and more frequently exhibit serum cryoprecipitate than females. The mechanisms for the defects are unclear, but an abnormality in a regulatory cell population is suggested.

INTRODUCTION

The frequent occurrence of autoimmune phenomena and/or pathology in association with primary immune deficiency states in man is well documented. Janeway rt al., in 1956, were the first to recognize the relationship between agammaglobulinemia, juvenile rheumatoid arthritis, and tenosynovitis (1). Shortly afterwards, Good et al. reported on the incidence of classical rheumatoid arthritis and thymoma in patients with a variety of immunodeficiencies including X-linked (Bruton’s) agammaglobulinemia, hypogammaglobulinemia, and common variable immunodeficiency (2). Similarly, autoimmune diseases, including Coombs’ positive hemolytic anemia, systemic lupus erythematosis (SLE), and allergic thyroiditis have been described in various forms of humoral immunodeficiencies, and have been carefully discussed (3-5). Cellular immunodeficiency disorders such as chronic mucocutaneous candidiasis, ataxia telangiectasia, and chronic ’ Supported in part by National Cancer Institute Grant 20816 (MEG) and Research Grant AI-05660 from the National Institute of Allergy and Infectious Disease. NIH (AAB). M.E.G. is recipient of Research Career Development Award AI-00193. L Reprint requests to M. Eric Gershwin, M.D.. School of Medicine. TB 192. University of Calitbrnia. Davis, California 95616. 334 0090- 1229/79/110334-14$01.00/O Copyright ‘c 1979 by Academic Press. lnc ,411 rights of reproductmn in any form rc~erved

AUTOIMMUNITY

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335

granulomatous disease are frequently associated with endocrinopathy as well as an increased incidence of autoantibodies (6). The genetic basis of such associations is suggested by studies of families exhibiting both immunodeficiency and autoimmunity (7). Indeed, asymptomatic family members may express a variety of autoantibodies, while affected patients display features of rheumatoid arthritis (RA), SLE, autoimmune hemolytic anemia, and dermatomyositis. Furthermore, thoughts have focused on the immunoregulatory role of suppressor lymphocytes and macrophages on the pathogenesis and relationship of both autoimmunity and immunodeticiency (8, 9). Benedict and co-workers have recently described a line of chickens with an inherited immunodeficiency analogous to acquired selective agammaglobulinemia of humans (10, 11). This deficiency, which may be similar to a mutation described earlier by Losch and co-workers (12), is characterized by a period of normal immunoglobulin synthesis early in life, followed by variable expression dysgammaglobulinemia. Affected birds, in addition to their immunoglobulin abnormalities, tend to have an accelerated morbidity and mortality, with frequent development of signs of anemia. Because of these phenomena, a prospective study, examining several aspects of autoimmune disease, was initiated. We report that dysgammaglobulinemic chickens frequently develop a constellation of immunologic abnormalities including a late onset Coombs’ positive hemolytic anemia, anti-y-globulin activity, and cryoprecipitates. This model provides a fertile avenue of examining the critical relationships of autoimmunity and immunodeticiency found in humans. MATERIALS

AND METHODS

Birds. The natural history and development of University of California, Davis (UCD) 140 chickens have been previously described (10, 11). UCD 140 chickens have been maintained by random matings with avoidance of full-sibling matings. Additionally, experimental line 144 and 145, produced by crossing UCD 140 to other lines, and backcross line 143 were used in this study. A control line (UCD 159), of dissimilar genetic background, but exposed to the same environmental conditions, was used throughout. All birds were bred and maintained by the Department of Avian Sciences, UCD. Hens were artificially inseminated and eggs were collected for 2 weeks. The eggs were transferred to the hatchery and kept for 21 to 22 days in a Jamesway 252 incubator. On the day of hatch, chicks were banded and moved to floor pens at the Hopkins poultry tract. Chicks were vaccinated against Marek’s disease on day 1, Newcastles disease at 4 to 6 weeks and again at 8 to 10 weeks, and vaccinated for fowl pox at 8 to 10 weeks. All birds were transferred to individual cages in evaporatively cooled houses, males at 14 weeks of age, and females at 16 weeks. The animals were provided with food and water ad libitum. Serial monitoring of groups of 30-50 birds from several hatches was performed for the parameters below. Hematology. Packed cell volumes (PCV), hemoglobin concentrations, and blood cell counts were determined from fresh heparinized blood samples obtained from the wing vein. PCV were performed in microhematocrit capillary tubes (Corning). Hemoglobin concentrations were assayed by the cyanmethemogiobin

336

MONTERO

ET

AL.

method. Twenty microliters of whole blood was added to 5.0 ml cyanmethemoglobin reagent (Hycel) and allowed to stand at room temperature for at least 10 min. The OD,,, of this reaction mixture was measured in a Coleman Junior spectrophotometer and the hemoglobin concentration was read from a previously prepared standard curve. Blood cell counts were made by a modification of the Rees-Ecker method utilizing the following staining solution: 3.8 g sodium citrate, 0.2 ml neutral formalin, 0.5 g brilliant cresyl blue, and 100 ml distilled water (13). Fresh blood samples were diluted l/200 in this solution in a blood-diluting pipet and the cells were microscopically counted in a Neubauer hemocytometer. The specific granules of chicken thrombocytes are stained by the brilliant cresyl blue allowing them to be distinguished from other white blood cells. Erythrocytes could be easily counted in the same preparation. Smears of unheparinized blood stained with a buffered Wright stain were used for differential counts. An autologous red cell survival study was performed using “‘Cr-labeled erythrocytes (14). Two milliliters of blood was drawn from the wing vein of five control UCD 159 and five Combs’ positive UCD 140 chickens into an equal volume of Alsevers’ solution. The cells were washed 3 x by centrifuging the suspension and resuspending the pellet in a solution of 50% Alsevers and 50% phosphate-buffered saline (PBS) pH 7.4. After the final wash, packed cells were incubated with 100 &i NaZlCrO, (New England Nuclear) at 37°C for 1 hr. The cells were then washed 3 x to remove unbound radiolabel and resuspended to original volume in PBS. The cell suspensions were injected intravenously into the same animals from which the samples were obtained. At 2 hr postinjection and various times over the next 10 days 0.2-ml samples were drawn from each test bird. Samples were counted in an automatic gamma scintillation counter (Nuclear Chicago). Coombs’ tests. Heparinized blood samples were obtained from the wing vein and red cells washed 3x in PBS, removing the buffy coat after each wash. The cells were suspended to a final concentration of 5% in PBS. Rabbit anti-chicken y-globulin, anti-chicken 7 S Ig (Fc specific), and anti-chicken IgM (p specific) were heat inactivated in a 56°C water bath for 30 min and absorbed with an equal volume of packed normal chicken red cells. A direct Coombs’ assay was performed in microtiter plates with U-shaped wells (Flow labs) by adding 25 ~1 cell suspension to 25 ~1 serially diluted antisera (15). Known positive and negative controls were included in each test. Different lots of antisera were used through the course of the study preventing comparison of titers obtained at different dates. Additionally, direct red cell agglutination tests were performed in microtiter by adding 25 ~1 5% red cell suspension from normal chickens to serially diluted test chicken sera. Sera were obtained from unheparinized blood samples allowed to clot for several hours at 38°C in glass siliconized tubes. Test plates were prepared in duplicate with plates incubated at both room temperature and 4°C. Immunochemistry. Chickens were judged dysgammaglobulinemic by quantitating serum immunoglobulin levels by radial immunodiffusion (10, 11). Immunoelectrophoresis was performed in 1.0% Noble agar (Difco) in barbitol buffer, pH 8.6. Samples were run for 2 hr at 30 V in an electrophoresis chamber (BioRad) filled with barbitol buffer. Developing antisera were allowed to react overnight in

AUTOIMMUNITY

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331

a moist chamber at room temperature. Immunodiffusion (ouchterlony) tests were performed in 1.0% agar dissolved in 0.15 M saline. Cryoprecipitutes. To test for the presence of serum proteins that precipitate in the cold, the method of Weisman and Zvaifler was employed (16). Blood was drawn from the jugular vein of chickens into prewarmed syringes and transferred to siliconized glass tubes kept at 38 to 40°C. The blood was allowed to clot for 3 to 4 hr at this temperature and the serum was harvested by centrifugation at SOOg for 10 min at room temperature. Then, 1 ml of serum was dispensed into a 12 x 75mm tube and placed at 4°C for 72 hr. Samples were handled in as sterile manner as possible throughout the procedure. After washing five times in cold 0.15 M saline, select cryoprecipitates were solubilized in a two-step procedure (16). Precipitates were suspended in 0.5 ml 0.3 M saline and kept at 38 to 40°C for 1 hr; then 0.5 ml distilled water was added and the mixture was kept an additional hour at 38 to 40°C and any remaining precipitate was removed by centrifugation. Direct analysis of this solubilized cryoprecipitate was accomplished by immunodiffusion and immunoelectrophoresis using Ig class-specific antisera. In addition, an indirect analysis was attempted. Solubilized complexes were allowed to reprecipitate at 4°C and the resulting precipitates were washed and resolubilized as prescribed above. This protocol was employed to ensure that serum proteins were not nonspecifically trapped in these complexes. Solutions were emulsified in Freund’s complete adjuvant (Difco) and injected subcutaneously into female New Zealand White rabbits at several different sites. The rabbits were boosted twice with the cryosolution emulsified in Freund’s incomplete adjuvant at 2-week intervals and bled 7 days after the final boost. The resulting antisera were harvested and run against whole chicken serum in immunoelectrophoresis. Furthermore, the cryoglobulin isolates from several birds were reacted with chicken 7 S Ig in the following tests: (1) direct passive hemagglutination with CrClz coupled 7 S Ig-sheep red blood cells, (2) qualitative precipitin reaction in capillary tubes, and (3) double diffusion in agar-gel using heat-aggregated 7 S Ig. Similarly, binding of 1251-labeled chicken 7 S Ig by cryoglobulin in double radioimmunoassay was measured. 1251-labeled chicken 7 S Ig was mixed with solubilized cryoprecipitate preparations. Rabbit anti-chicken p-chain-specific antisera and sheep anti-rabbit y-globulin were added after appropriate intervals. Free and bound antigen was separated by microcentrifugation. The binding activity of the cryoglobulins was studied in the presence of isolated 7 S Ig Fab and Fc fragments; the chicken 7 S Ig was monospecific by immunoelectrophoresis. Anti--y-globulin assay. In order to quantitate anti-y-globulin activity, a number of assays were employed. Early assays were performed with Hyland RF latex reagent coupled with human IgG. Test chicken sera were serially diluted in glycine-saline buffer pH 8.2 beginning with a l/20 dilution. One drop of the latex reagent was added to 1 ml diluted sera in a 12 x 75-mm tube and incubated at 37°C for 15 min with occasional shaking. Tubes were centrifuged for 1 min at SOOg and read for degree of agglutination against a diffuse light source. To characterize the anti-y-globulin activity further, a modification of the Singer-Plotz latex fixation test (17) using chicken 7 S Ig was employed. The test reagent was prepared by

338

MONTERO

ET

Al

adding 0.1 ml 1% suspension of latex beads (0.807 pm, Dow Chemical) and 0.5 ml of a 0.5% solution of chicken 7 S Ig to 9.4 ml glycine-saline buffer, pH 8.2. This mixture was stabilized by the addition of bovine serum albumin (Miles Laboratories) to a concentration of 0.4% (18). The 7 S Ig was obtained by passing chicken sera through a 1.5 x 40-cm DEAE cellulose anion exchange column equilibrated with 0.1 M phosphate buffer, pH 6.4 (19). Test sera were serially diluted as above and an equal volume was added to the latex reagent. This reaction mixture was incubated at 56°C for 90 min and the tubes were read for agglutination after cooling to room temperature. Very similar agglutination titers resulted from both latex fixation tests. Samples were considered positive when they exhibited a titer of l/SO or greater, a value statistically different from the mean of controls. Attempts were also made to measure anti--y-globulin activity by the RoseWaaler test (20). Sheep red blood cells (SRBC) were coated with chicken 7 S Ig by incubating with a l/4 subagglutinating dose of chicken anti-SRBC sera for 30 min at 37°C. The anti-SRBC was obtained by injecting normal chickens with a 30%~ SRBC suspension in PBS intravenously, boosting after 7 days, and bleeding 7 days after boost. This antiserum was enriched in 7 S Ig by DEAE column chromatography as described above. The coated cell suspension was washed 3x and resuspended in PBS to a final concentration of 5%. Test chicken sera were serially diluted in microtiter plates and 25 ~1 cells suspension was added to the test wells. Agglutination was examined after 2 hr incubation at room temperature. Antithymocyte antibodies. An indirect immunofluorescent test was performed on thymus cells obtained from 2- to 4-week-old normal chicks (21). Thymi were removed and the cells were teased from the organ into cold RPMI-1640 (Pacific Biologicals) with forceps and then pressed through a stainless-steel wire screen. The cell suspension was washed 3 x in cold media and adjusted to a concentration of 10 x 10” ml after the final wash. Then 1.0 ml of the cell suspension was delivered to 12 x 75-mm tubes, the cells were pelleted by centrifugation, and the supemate was removed. To the pellet, 0.1 ml undiluted test chicken sera was added and the cells were resuspended. This mixture was incubated at room temperature for 60 min and at 4°C for an additional 60 min. The cells were then washed 3 x in cold RPMI- 1640 and pelleted. After removal of the supernate, 0.1 ml diluted fluorescein-isothiocyanate-conjugated (FITC) anti-chicken y-globulin was added to the cells and incubated for 30 min at room temperature. The antisera were used at a l/16 dilution in RPMI-1640 medium which eliminated any nonspecific labeling of thymocytes. Cells were washed 3x to remove unbound label, suspended in a drop of buffered glycerol, mounted on a glass slide, and viewed in a fluorescent microscope (American Optical series 20 equipped with a vertical illuminator with a mercury vapor lamp, a FITC #2070-617 exciter filter and an OG 5 15 barrier filter). Conventional cytotoxicity tests were also performed on thymocytes from 2- to 4-week-old chicks. Cell suspensions were prepared as outlined above and adjusted to 20 x lo6 ml in RPMI-1640. Then 0.1 ml of the cell suspension was mixed with 0.1 ml undiluted heat-inactivated test chicken sera and incubated at room temperature for 60 min and at 4°C for another 60 min. Cells were washed 3x in RPMI-

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IN CHICKENS

1640 and resuspended in 0.1 ml fresh chicken sera as a cource of complement. After incubating at 37°C for 90 min the percentage of dead cells was determined by trypan blue dye exclusion. Complement controls were included in each assay. Anri-DNA. Native DNA binding assays were performed as previously described using Millipore filtered PHJDNA (22). Briefly, 5 ~1 test sera diluted l/3 in borate buffer pH 8.0 was mixed with 100 pl(25 ng ligand) PH]DNA. Tubes were allowed to incubate 60 min at 37”C, and overnight at 4°C. To each reaction tube, 5 ml citrate buffer was added and the precipitate was collected on 25mm Millipore filters with a sampling manifold. Test and control filters were dried in counting vials and counted in a beta scintillation counter. Antinuclear antibody. Test sera were screened for antinuclear antibody by indirect immunofluorescence using mouse connective tissue fibroblasts as substrate (Meloy). A drop of test serum diluted l/20 in PBS was delivered to the appropriate well and allowed to incubate 30 min in a moist chamber at room temperature. Slides were thoroughly washed in saline and 0.7 ml diluted FITC-anti-chicken globulin was added to each slide. A negative and positive control was included. RESULTS

Hematology and Coombs’ Tests Serial monitoring of UCD 140 birds, but not control birds between the ages of 6 to 12 months, revealed abnormally weak chickens displaying pale combs and green bilimbinous feces. Analysis of sick birds indicated large numbers of immature erythrocytes, early and late polychromic rubricytes. Active erythropoiesis was evident in smeared bone marrow samples from sacrificed birds. However, these smears were depleted of mature red blood cells. Quantitation of this phenomenon disclosed a significant anemia (P < 0.01) when packed cell volumes (PCV), hemoglobin concentration, and red cell counts from line 140 birds were compared to age- and sex-matched controls (Table 1). A more pronounced anemia (P < 0.001) was observed when blood count values from Coombs’ positive line 140 birds were compared to controls. Indeed, bone marrow from these birds revealed a lymphocytosis and severe depletion of erythrocytic series cells. To determine whether anemia was caused by increased loss or decreased pro-

PACKED

CELL

TABLE 1 VOLUME, HEMOGLOBIN, AND BLOOD CELL COUNTS FOR 8- TO ~-MONTH-OLD FEMALES OF UCD 140 AND UCD 1.59, SPRING HATCH 1977 Number tested

PCV

(%)

Hb Wdl)

RBC

(x lO”/mm”)

WBC

(x lO?mm”)

UCD

159 controls

21

29.55 k2.16”

10.88 21.53

2.71 t .42

27.1 ~3.2

UCD

140

18

26.46 23.98

8.22 ~2.19

2.33 + .48

32.91 2.51

23.37 23.79

7.95 k-2.01

1.90 i .28

32.50 t 10.85

UCD 140 Coombs’

(+)

6

340

MONTERO

ET

AL.

D.Caombs I+) fS.E. w-~D.Coombs(-1fS.E.

‘. ‘\\

%_

.I.

Time

(days)

1. Survival of autologous >‘Cr-labeled RBCs in peripheral blood of 140 birds and 5 Coombs’ negative control UCD 159 birds. FIG.

5

Coombs’ positive

UCD

duction of red cells, an erythrocyte survival study was performed. 5’Cr-labeled red cells were cleared significantly faster from the peripheral blood of Coombs’ positive line 140 chickens as compared to Coombs’ negative control line birds (P < 0.005 for all sampling times beyond Day 1). For example, at 7 days postinjection, approximately 70% of the label remained in the blood samples from control chickens while only 30% remained in Coombs’ positive birds (Fig. 1). The erythrocyte survival curve obtained for the Coombs’ positive chickens deviates from linearity, indicating a nonrandom loss of cells, as might be expected with in vivo lysis or increased clearance of antibody-coated cells. It was found that UCD 140 chickens became direct Coombs’ positive at about 7 to 8 months of age (Table 2). The frequency of occurrence increased from 10% at TABLE INCIDENCE

OF POSITIVE

DIRECT CONTROL

Hatch I. UCD 140 Fall 1976 Spring 1977 Fall 1977 II. Control UCD 159 Spring 1977

COOMBS’

2 Tts-r

UCD 159

wrrH

Aot

IN

UCD 140 ANI)

CHICKENS

Age (days)

Number positive/ number tested

Percentage positive

287-320 362-404 511-541 195-211 330-370 183-21 I

4123 5/13 418 3147 8162 0140

17.4 38.5 50 6.4 13.4 0

211 370

o/22 0120

0 0

AUTOIMMUNITY

IN

TABLE INCIDENCEOF

SERUM

CRYOPRECIPITATES"

UCD Hatch I. UCD 140 Fall 1976 Spring 1977 Fall 1977 11. Control UCD 159 Spring 1977

341

CHICKENS

3

WITH AGE IN ABNORMAL 159 CHICKENS

UCD 140 ANDCONTROL

Age (days)

Number positive/ number tested

Percentage positive*

348-378 413-443 330-370 180-211

7/8 213 25135 31142

87.5 66.7 71.4 73.8

211 370

1116 3120

6.25 15

f1Cryoprecipitates defined as serum proteins that precipitate on exposure to cold and redissolve on warming (see text); tests performed only on fresh sera. ’ Purified normal chicken IgM, at 15 mg/ml, does not form a cryoprecipitate under these conditions.

onset to approximately 50% in birds over 18 months of age. In immunodeficient subjects, the immunoglobulin class detected on the surface of the red cell corresponded in all birds with the dysgammaglobulinemia. That is, chickens with elevated levels of serum IgM tended to have IgM-coated cells, while birds with normal to elevated 7 S Ig were shown to have 7 S Ig attached to their erythrocytes. As yet, only rare examples of free antierythrocyte antibody have been detected in the serum of anemic chickens (Table 2). Cryoprecipitates

Complexes that precipitate from serum when exposed to cold and dissolve upon rewarming were detected in more than 50% of UCD 140 chickens greater than 6 months of age (Table 3). A maximum of 15% of control UCD 159 chickens were shown to exhibit similar precipitates at approximately 1 year of age. Macroscopitally, these complexes varied from a fibrillar or floccular mass that broke up upon agitation, to a fine granular material. Occasionally, a gelatinous mass, or cryogel, would form in the collection tube almost immediately after sampling blood from UCD 140 birds. Direct analysis of extensively washed and solubilized cryoproteins by immunodiffusion or immunoelectrophoresis invariably revealed the presence of immunoglobulins. For example, in a study of 14 cryosolutions, all were found to contain IgM, three had detectable 7 S Ig, and only one appeared to have IgA. Indirect analysis by immunoelectrophoresis, utilizing an anticryoprecipitate prepared by injecting rabbits with solubilized complexes, indicated the presence of other unidentified serum proteins, probably complement and lipoprotein. In addition, rheumatoid factor activity was detected in a small number of these cryosolutions. This activity was weak compared to that detected in serum, however, these solutions contained only 0.1 to 0.5 mg/ml protein. The specific nature of this rheumatoid factor, as measured by radioimmunoassay, is discussed below. Anti-y-globulin

Activity

Serum anti-y-globulin

activity was detected by latex fixation assay in the sera of

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ET Al-.

‘TABLE INCIDENCE

AND

MEAN

Tn-ttt

OF RHEUMA-IOID CONT.ROI

UCD

Number Age (days)

Hatch 1. UCD 140 Fall 1976

Spring 1977 Fall 1977 II.

Control

spring

UCD

1977

159

At

IIVI.~>

IN ABNOHMAL

UCD

140 .~NIJ

CHICKENS

positive:

number

348 - 378 413-443 5llG541 330-370 142- 163 180-21 I

4

FACWR

Percentage positive

tested

119 4ih 919

77.x 66.7 100 52.8

I9136 8154 I l/42

14.8 26.2

Reciprocal titer (geometric mean) r SD 145 160 373 IO0 147 IOX

L -+ i :t -c e

I6 26 21 IS 18 16

ICY

211

ii16

370

O/20

6.3 0

x0 0

chickens from UCD 140 as young as 6 months of age. In an age-dependent study, it was found that frequency and titer both generally increased as chickens became older (Table 4). This immunoprotein reacted with latex beads coupled with either chicken 7 S Ig, or remarkably, human IgG. This is noteworthy, in that human rheumatoid factors rarely cross-react with chicken 7 S Ig. Activity in sera from control UCD 159 chickens was infrequent and of low titer when present (~1/40) (Table 4). In order to define more specifically the binding characteristics of this antiglobulin activity in sera and cryoprecipitates, a radioimmunoassay was employed. In this study, of one isolated cryoprecipitate containing only IgM, binding of lz51labeled chicken 7 S Ig at both 38.5 and 4°C was noted (Fig. 2). The affinity for 7 S Ig, however, was weak and binding was inhibited by Fab fragments and not Fc, suggesting that the rheumatoid factor reacted with light chains. An unusual curve was obtained when an isolated mixed cryoprecipitate, containing both IgM and 7 S Ig, was reacted with labeled 7 S Ig in a triple antibody radioimmunoassay. There was little or no binding measurable at high protein concentration (low dilution) and maximal binding at a l/IO dilution of the cryoprecipitate (Fig. 3). It was also demonstrated that anti-y-globulin activity from serum of immunodeficient chickens, as well as the G-200 IgM fractions from serum, shows the same type of

2 53

2. Binding radioimmunoassay FIG.

of serial diluted at 38.5 and 4°C.

I27

isolated

0.63

0.31

cryo-IgM

0 158 Img 17S/ml)

for

‘251-labeled

7 S Ig in a triple

antibody

AUTOIMMUNITY

Reciprocal

IN CHICKENS

Dilution

FIG. 3. Binding of serial diluted isolated mixed cryoprecipitate antibody radioimmunoassay.

343

u

for ““I-labeled

7 S Ig in a triple

binding characteristics, with no binding of 7 S Ig seen at low dilution. In addition, IgM preparations from normal chickens exhibit similar binding activity for lz51labeled 7 S Ig in the radioimmunoassay, suggesting that this antiglobulin activity may be the result of nonspecific binding of 7 S Ig by IgM. However, this activity was not present in normal serum or normal IgM preparations in latex fixation assays. Finally, the binding characteristics of this chicken anti-y-globulin factor in a Rose- Waaler Ig-coated sheep cell agglutination test was studied. In this assay, no reactivity for 7 S Ig could be detected in serum or solubilized cryoprecipitates from line 140 chickens. Antithymocyte Activity Naturally occurring antibody directed toward thymus cells of young chicks could rarely be demonstrated in sera of older UCD 140 chickens by indirect immunofluoresence. This activity was infrequently detected and weak. Antithymocyte activity was not detected in the cytotoxic assay described herein. Antinucleic Acid Antibodies In a survey of sera from serially aged birds from lines 159, 140, and backcross and outcross lines, no significant binding of DNA was detected. Similarly, antinuclear antibodies were not found when chicken sera were reacted with mouse fibroblast cells. Correlations A number of statistical associations can be made between the dysgammaglobulinemia and other immunologic abnormalities found in these chickens. For UCD 140 birds from the Fall, 1977 hatch, there is a positive but not significant correlation between rheumatoid factor and dysgammaglobulinemia (Table 6). In addition, cryoprecipitates are found significantly more often in the sera of immunodeficient UCD 140 chickens. However, this association is not absolute and a number of UCD 140 birds with normal serum immunoglobulin levels also possess cryoprecipitates. In UCD 140 a positive direct Coombs’ reaction is found with increased incidence in dysgammaglobulinemic chickens over 1 year of age. Furthermore, though no sex dependence is noted for the immunodeficiency, UCD 140 males are more frequently Coombs’ positive and possess cryoprecipitates more often than females (Table 5).

344

MONTERO

ET AL.

TABLE SEX

DEPENDENCE

OF DYSGAMMAGI.OBUL

IN~MIA, IN

IX II

2

ANU

CUOMBS’

Rt A< ilob

10 13

Cryoprecipitate”

Coombs‘ test”

t

+

28 6

1.78 <0.20

P

IP~I,AI~S.

UCD 140 CHIcKENS

Dysgamma” ~. .~~.. + Male Female

C CR>O~R~~

0 13

I0 6

26.48
IO ‘2 4.28 -: 0.05

--

n UCD 140 chickens, Fall hatch 1977. 6 to 7 months of age. ’ UCD 140 chickens greater than I year of age.

DISCUSSION

In this study, the development of certain autoimmune manifestations in a line of mutant chickens, previously shown to have an inherited immunodeficiency, is reported. The birds described herein, besides a late onset dysgammaglobulinemia, display a direct Coombs’ positive hemolytic anemia, cryoprecipitates, and serum rheumatoid factor. Statistically, there is a significant positive association between some of the autoimmune features and immune deficiency, suggesting a common etiology. For example, dysgammaglobulinemic birds more frequently possess serum cryoprecipitates and are more often direct Coombs’ positive. Interestingly, cryoprecipitates and antibody-coated erythrocytes are found more often in male line UCD chickens, even though dysgammaglobulinemia occurs independently of sex. The increased incidence of certain human autoimmune diseases in females is also known. SLE, rheumatoid arthritis, Sjogrens’ syndrome, and primary biliary cirrohosis are found more frequently in women (23). It is of particular notoriety that in mammalian species the female is the homogametic sex (XX) while in the chicken, it is the male that possesses two sex chromosomes (ZZ). This suggests that the sex chromosome, or other sex-linked products, may play a role in modification of the immune response. The presence of Coombs’ positive hemolytic anemia was indicated by the deTABLE ASSOCIATION

6

OF RHEUMATOID FACTOR, CRYOPMCIPITAX, DYSGAMMAGLOBULINEMIA IN

Rheumatoid factor”

Dysgamma

+ x P

POSITIVE

Cryoprecipitate”

+

-

+

8 3

13 17

22 8

2.52 10.20

AND

COOMBS’

Tt.sr wt I I+

UCD 140 CHICKENS Coombs’ test”

1 11 12.36 <
” UCD 140 chickens, Fall hatch 1977, 6 to 7 months of age. b UCD 140 chickens greater than 1 year of age.

$ ____-.14 2

-~ _--.18 14 4.69 co.05

.-

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tection of antibody-coated erythrocytes, decreased PCV, hemoglobin concentration, and red cell count, and an increased loss of radiolabeled red cells from peripheral blood. Hemolytic anemia has previously been reported in chickens experimentally rendered agammaglobulinemic by bursectomy (24). This phenomenon was not considered true autoimmune disease even though Ig-coated red cells were detected. Instead, it was felt that some unknown infectious agent had absorbed to the erythrocyte surface and antibody was directed toward this agent rather than red cell antigens. A pathogenic mechanism such as this has not been ruled out in this study, despite the fact that control chickens, exposed to the same environment as dysgammaglobulinemic birds, do not develop signs of anemia. It is possible that UCD 140 chickens have an inherited susceptibility, or are more prone to infection because of their immune deficiency. Cryoprecipitates, cold-insoluble serum proteins, found in these experimental birds may be a nonspecific in vitro phenomenon enhanced by abnormally high serum protein concentrations. Alternatively, precipitation may be relatively specific and indicative of in vivo immune complexes. Direct immunochemical analysis reveals the presence of immunoglobulins, while indirect analysis with an antisera produced against solubilized precipitate indicates the presence of other serum components. At least one isolated cryo-IgM has been found to react rather specifically with radiolabeled IgG, and did not bind dinitrophenyl-human serum albumin, bovine serum albumin, arsenylated keyhole limpet hemocyanin, and copolymer GAT (A. A. Benedict, unpublished data). Three types of cryoprecipitates, based on heterogeneity of the constitutents, have been described in man (25). Type I and monoclonal, consisting of only a single homogeneous immunoglobulin class. Type II are mixed, containing both homo- and heterogeneous Ig components. Type III are polyclonal and consist of one or more immunoglobulin class, none of which is homogeneous. Antiglobulin activity, complement proteins, and other compounds such as DNA have been detected in these complexes, especially those of Type III. The presence of these additional components, and the frequent occurrence of cryoprecipitates in diseases complicated by vasculitis and nephritis, suggests that these may represent immune complexes (25, 26). The appearance of serum anti-y-globulin in this immunodeticient line of birds is an age-dependent characteristic, increasing in both frequency and titer with time. This activity is detectable by latex fixation but not by Ig-coated sheep cell agglutination tests. In a triple antibody radioimmunoassay, reactivity is generally atypical in that there is no binding of lz51-labeled 7 S Ig at high concentrations of serum or isolated IgM preparations. In addition, IgM from normal serum was found to exhibit similar binding curves to that obtained with samples from immunodeficient birds. The reason for this reactivity is not known at this time. In the latex fixation test, sera or IgM from normal chickens rarely show positive titers. Also of interest, was the fact that binding of 7 S Ig is inhibited by Fab fragments and not Fc, indicating that these rheumatoid factors react with light chains of the immunoglobulin molecule. It should be noted, as emphasized earlier by Mandy et al., that in infrahuman species (e.g., rabbits), reaction with Fab characterizes antiantibody (” homoreactant”), whereas rheumatoid factor reacts with Fc (27).

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In man, classical rheumatoid factor is an IgM molecule that reacts with the heavy chain portion of IgG. It is now recognized that these antiglobulins are a heterogeneous group with a broad spectrum of reactivities. For example, IgG and IgA rheumatoid factors have been discovered, and it has been demonstrated that certain high titered antiglobulins react with light chains (28). This light chain reactivity can be inhibited by autologous light chains but not by intact IgG molecules, suggesting that these factors are directed toward sites uncovered by isolation methods (28). There are other examples of rheumatoid factor activity toward buried IgG determinants. It has been noted that some rheumatoid factors react to internal configurations revealed only by partial digestion (29). Hyperimmunization of animals with bacteria, or denatured IgG will induce the formation of antiglobulins (30, 31). In the latter cases, the appearance of these antibodies is thought to be due to stimulation by IgG molecules, altered chemically or by combination with antigen, rendering it autoimmunogenic. Indeed, besides their occurrence in rheumatoid arthritis, rheumatoid factors are associated with chronic infections in man (32). Again, the inherent immunodeficiency could leave these chickens more susceptible to foreign antigens that initiate anti-y-globulin activity production when combined to IgG. Chickens have been demonstrated to produce anti-y-globulins when infected by mycoplasma (33). It is clear that certain forms of immunodeficiency and autoimmunity are associated with immunoregulatory abnormalities (23, 34-36). The exact nature of the relationship of suppressor cell deficiency of enhancement and the development of autoimmunity or hypogammaglobulinemia is not known. Questions such as these may be answered through study of the autoimmunity and immune deficiency demonstrated in UCD 140 chickens. Further study is necessary to determine more precisely the relationship between the various features, to identify a specifically abnormal lymphoid cell subpopulation( to resolve the genetic basis of disease, and to ascertain whether infectious agents play a causative role. REFERENCES 1. Janeway,

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