Abnormal lymphocyte function in scleroderma: A study on identical twins

CLINICAL

IMMUNOLOGY

Abnormal

AND

IMMUNOPATHOLOGY

44, 20-30

(1987)

Lymphocyte Function in Scleroderma: A Study on Identical Twins

MEHER M. DUSTOOR,MARCIA M. MCINERNEY,DANIEL MARTHA K. CATHCART

J. MAZANEC,AND

Departments of Immunology and Cancer Research and Rheumatic and Immunologic Clelfeland Clinic Foundation, Cleveland, Ohio 44106

Diseases.

This paper describes immunologic studies on a set of identical twins discordant for the presence of scleroderma. The affected twin had a low absolute T-cell count, low numbers of T4 helper/inducer cells, and an increase in the T8 suppressoricytotoxic cell count. The T cells of the patient responded poorly to mitogens and to allogeneic and autologous stimuli. By contrast, T-cell-helper activity for pokeweed mitogen-induced IgM synthesis was markedly enhanced in the patient. Furthermore, activated mononuclear cell supernatants from the patient markedly enhanced the synthesis of collagen by normal cultured fibroblasts. The unaffected twin by contrast displayed normal responses in these assays. The results suggest that the immunologic defects in scleroderma are not entirely genetically determined. “8 1987 Academic Press. Inc.

INTRODUCTION

Progressive systemic sclerosis (PSS) is a disease of unknown pathogenesis characterized by connective tissue abnormalities (1). Its etiology is unknown and the cause for tissue fibrosis is not clear. Recent research has linked the observed collagen accumulation in PSS patients to altered immune effector function of their lymphocytes (2, 3). In addition several immunologic abnormalities including excessive T-helper-cell function have also been reported in PSS (4-9). Many immunological functions are genetically determined (lo- 12). Genetic components have been defined for several autoimmune disorders (13- 15), although in scleroderma no clear-cut familial or major histocompatibility complex (MHC)-linked associations have been described. Except for one report, no studies have been performed on the immune competence of family members in cases of familial scleroderma (16). This report describes our immunologic studies on a pair of identical twins, one with advanced diffuse progressive systemic sclerosis and one in good health with no evidence of disease. In this situation it was possible to examine the relationship between abnormal immunologic findings and the presence of disease without interference from variations in genetic background. MATERIALS

AND METHODS

Subjects. The patient was a 62-year-old white female who fulfilled the American Rheumatism Association criteria for the diagnosis of PSS. Clinical features included typical sclerodermatous skin manifestations (sclerodactyly, digital ulcers, Raynaud’s phenomenon), lower esophageal dysmotility, and bibasal pul20 0090-1229/87

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Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.

SCLERODERMA:

DISCORDANT

FINDINGS

IN TWINS

21

monary fibrosis. Laboratory testing revealed normal blood count and renal function. The Westergren sedimentation rate was 35 mm/hi-. Rheumatoid factor was strongly positive with a value of 233 RLS (normal less than IO), corresponding to latex titer of greater than 15120. Antinuclear antibody was present at a titer greater than 1:320. Anti-Sm was positive and anti-RNP was negative. Clq binding assay for circulating immune complexes was elevated to 3322 units/cc (normal less than 62). The patient was not on corticosteroid or cytotoxic drug therapy at the time of study. The identical twin sister was in excellent health when these studies were conducted. Normal, unrelated donors used in the study were age, race, and sex matched with the patient. HLA typing. HLA typing of each of the twins and selected normals was performed at the tissue typing facilities of the Cleveland Clinic Foundation. Both the PSS patient and her unaffected twin sister were HLA Al, 24, B7, 8, C7, and DR 4. thus confirming monozygosity. Isolation and separation of mononuclear cells (MNC). MNC were isolated from heparinized peripheral blood by centrifugation of diluted blood (1: 1 in phosphate-buffered saline) over Ficoll-Paque (Pharmacia, Piscataway, NJ) at 400g for 30 min. MNC were recovered at the interface and washed three times with RPM1 1640. For separation of MNC into T and non-T cells an overnight sheep erythrocyte rosetting procedure was employed. T cells were recovered from the rosetted pellet by lysis with 0.14 M NH&l and were 95% E-rosette positive. Non-T fractions contained less than 2% E-rosette-positive cells. Enumeration of T lymphocyte subsets. MNC (1 x 106) were incubated with appropriate dilutions of OKT3, OKT4, or OKT8 monoclonal antibodies (Ortho Diagnostics, Raritan, NJ) on ice for 30 min. After washing, the cells were incubated with FITC-conjugated goat anti-mouse Ig (Cappel Labs, Downingtown, PA) for 30 min. The percentage of positively stained cells was determined on a fluorescence activated cell sorter (FACS). Pokeweed mitogen-induced proliferation. Unseparated MNC and T and non-T cell populations were assayed for their proliferative response in the presence and absence of pokeweed mitogen (PWM; 1: 1000 final concentration). MNC cells were plated in 96-well microtiter plates at 1 x lo5 cells/well. In experiments with fractionated lymphocytes, T cells and non-T cells were added at a concentration of 0.8 x lo5 and 0.2 x lo5 cells/well, respectively, to reflect the typical T:non-T ratio of 4:l in unseparated MNC. These cultures were incubated for 72 hr and were pulsed with 0.5 t&i [3H]thymidine (Schwartz-Mann, Irvine, CA: sp act 3 Cilmmol) for the final 6 hr of culture. Cells were collected on glass-fiber filters using a multiple automated sample harvester, allowed to air-dry, and quantitated using a Beckman 7500 scintillation counter. Mixed lymphocyte reactions (MLR). Responder T cells (1 x 105) and 1 x lo5 mitomycin C-treated (500 pg mitomycin C/5 x lo6 cells/ml) autologous or allogeneic stimulator non-T cells were cocultured in triplicate in individual wells of a 96-well round-bottom microtiter plate in a total volume of 0.2 ml. Culture medium was complete RPM1 supplemented with 10% heat-inactivated normal human AB

22

DUSTOOR

ET AL.

serum. Cultures were incubated for 6 days at 37°C in 5% CO* + 95% air. For the final 18 hr of culture, 0.5 $i of [3H]thymidine was included in the cultures. The cells were then harvested on glass-tiber filters and the incorporated radioactivity was measured as previously detailed. Assay

for helper

T-cell function

for PWM-induced

polyclonal

IgM synthesis.

Allogeneic non-T cells (6 x 104) were cocultured with increasing numbers of T cells (4, 8, 16, 32, or 64 x 104) from the PSS patient or her normal twin in 0.2 ml of complete medium. This consisted of RPM1 1640 containing 100 U/ml penicillin, 100 kg/ml streptomycin, 200 mM L-glutamine, and 0.1 M Hepes buffer. The medium was supplemented with 10% heat-inactivated fetal calf serum and a 1:lOOO dilution of PWM was added. Triplicate cultures were incubated at 37°C in 5% CO* in air for 7 days. At the end of the incubation, supernatants from each well were assayed for IgM by a sensitive solid-phase fluorometric assay (17). Supernatant preparation. Supernatants were made from peripheral blood MNC of the patient, her twin, as well as an age-, race-, and sex-matched normal volunteer. Cells were incubated at a concentration of 1 x lo6 cells/ml in serum-free Dulbecco’s modified Eagle’s medium for 48 hr in a humidified atmosphere with 5% CO2 + 95% air. Activated cell supernatants were prepared by mixing equal numbers of MNC with irradiated (3000 rads) allogeneic MNC under the same conditions. The cells were pelleted by centrifugation and the supernatants were stored at - 70°C. Fibroblast proliferation. The effect of MNC supernatants on subconfluent fibroblast proliferation was measured after exposure to mononuclear cell supernatants as previously described. During the final 6 hr of a 48-h culture period [3H]thymidine was added to the fibroblast cultures (5 &i/ml). The cell layer was rinsed with buffer and then solubilized with 0.2% sodium dodecyl sulfate (SDS). [3H]Thymidine incorporation was determined by mixing the solubilized cell components with 9 vol of Aquasol and then detecting beta emissions on a scintillation counter. Fibroblast protein and collagen assays. The effect of mononuclear cell supernatants on protein and collagen production was determined by incubating nearly confluent fibroblast cultures with a 1: 10 dilution of supernatants obtained from the mononuclear cell cultures. Twenty-four hours before the end of the 48-hr incubation period the cultures were supplemented with 50 &ml ascorbic acid and 5.0 l&i/ml [2,33H]proline. The fibroblasts retained normal morphology during these studies. Following incubation, total radiolabeled protein was detected in the cultures by adding 0.2% SDS and carrier protein and then precipitating the protein with cold 10% trichloroacetic acid (TCA). The samples were collected on glass-fiber filters and washed extensively with cold 5% TCA and after drying, the [3H]proline incorporation was quantitated by beta scintillation counting. The amount of [3H]proline incorporated into collagen in identical cultures was determined by the specific collagen solubilization and precipitation method of Webster and Harvey (18) using 0.5 mg/ml neutral salt soluble collagen as carrier. Statistical analysis of the data. The paired t test was used to analyze all data for statistical significance.

SCLERODERMA:

DISCORDANT TABLE

ENUMERATION

OF T-CELL

FINDINGS

23

IN TWINS

1

SUBSETS IN PERIPHERAL

BLOOD

% Cells staining with

PSS twin Normal twin Unrelated normal Normal laboratory values

OKT3

OKT4

OKTS

53.4 68.4 72.5 77.6 k 7.5

38.3 61.6 57.4 49.5 + 9

37.0 8.9 22.2 24.6 k 5.3

RESULTS T-Cell Subset Enumeration

FACS analysis of monoclonal antibody stained preparations of peripheral blood MNC revealed that the PSS patient had lower than normal numbers of circulating T and T4 helper/inducer cells and an elevation of TS suppressor/cytotoxic cell numbers (Table 1). Pokeweed Mitogen Proliferative

Response

Unseparated MNC cells of the PSS patient demonstrated a marked deficiency in proliferation to PWM, whereas her healthy twin displayed a normal proliferative response (Table 2). Further experiments with separated T- and non-T-cell populations (Table 3) confirmed that the defective response of the PSS patient was not related to T-cell lymphopenia. In this instance, the bulk of the response could be attributed to T cells since the T:non-T ratio in the cultures was 4: 1. PSS T cells in combination with autologous non-T cells were very poor responders (A cpm of 661) and even in combination with normal twin non-T cells showed lower proliferative responses than did normal T cells (Acpm 2244 vs 9187). Furthermore, the patient non-T cells had a suppressive influence on the proliferation of normal twin T cells (Acpm 2043 vs Acpm 9187). Defects in both T- and non-T-cell populations were thus evident. TABLE POKEWEED

MITOGEN

(PWM)-INDUCED

2

PROLIFERATION

OF UNSEPARATED

MNC”

Proliferation (cpmjb Cell source

- PWM

+ PWM

PSS twin Normal twin Unrelated normal

137 +- 29 383 2 181 70 f 17

349 k 16 4136 -c 45 2272 + 1909

a 1 x IO5 MNC were incubated for 72 hr in the presence or absence of PWM (I: 1000). b Mean of duplicates 2 data range.

24

DUSTOOR TABLE

ET AL. 3

POKEWEED MITOGEN-INDUCED PROLIFERATION OF SEPARATED CELLS~

I.

2. 3. 4.

T cells from

Non-T cells from

PSS-twin NL-twin PSS-twin NL-twin

PSS-twin NL-twin NL-twin PSS-twin

Proliferation tcpmJ - PWM

83 488 429 155

Acpm

+ PWM

2 4 2 140 +- 68 -c 91

744 9675 2673 2198

k -c i k

IO 412 132 I64

661 9187 2244 2043

a Cultures consisted of 0.8 x IO5 T cells and 0.2 x IO5 non-T cells incubated alone or with PWM (I: 1000) for 72 hr.

Autologous

and Allogeneic

Stimulation

Table 4 shows the results obtained with autologous MLR (AMLR) cultures containing different combinations of patient and normal twin T and non-T cells. The autologous MLR response was clearly depressed in the patient compared to that of her normal twin (P < 0.01). The allogeneic response to third-party unrelated normal non-T cells was also significantly lower in the patient (P < 0.005). The non-T cells of the patient also appear to be defective stimulators of the AMLR since they were unable to stimulate even the normal twin’s T cells to proliferate (391 cpm with patient non-T cells vs 2335 cpm with twin non-T cells). Even in the allogeneic MLR the PSS patient’s cells were significantly weaker stimulators than the normal twin’s cells (P < 0.005). Further experiments were performed where additional patient or twin non-T cells were added to patient, twin, or third-party AMLR cultures (Fig. 1). Additional patient non-T cells did not enhance patient AMLR responses (culture 3). When PSS twin non-T cells were added to cultures of PSS twin + normal twin non-T cells, 59% suppression was seen (culture 2 vs 4). Similarly, when PSS twin or normal twin non-T cells were added to normal twin AMLR cultures (cultures 7 and 8), the additional normal twin non-T cells enhanced AMLR responses (culture 8), whereas additional PSS twin non-T cells suppressed the response by 40% (culture 5 vs 7). Similar suppression by PSS twin non-T cells did not extend to AMLR reTABLE

4

AUTOLOGOUS MLR BETWEEN PSS PATIENT AND THE NORMAL TWIN SISTERS Stimulator non-T cells Responder T ceils

PSS twin

Normal twin

PSS twin Normal twin Unrelated normal

327 -c 67 391 ?I 219 24468 4 2834

925 + 89 2335 k 790 42372 -c 710

Unrelated normal

(cpm) 11194 f 2154 29977 a 5154 9970 2 1980

121 x IO5 T cells were cultured with 1 x IO5 mitomycin C-treated non-T cells for 6 days.

SCLERODERMA:

DISCORDANT

FINDINGS

2.5

IN TWINS

sponses of third-party unrelated individuals (culture 12). Although the response in cultures containing added patient non-T cells was about half that of cultures containing normal twin non-T cells (culture 11 vs 12) it is most likely a reflection of the lower allogeneic stimulatory capacity of the PSS twin non-T cells. Helper

T-Cell Function

Figure 2A shows a typical IgM synthesis curve obtained from I2 nornal subjects when increasing numbers of T cells are added to 6 x lo4 allogeneic non-T cells. IgM synthesis increases to an optimum at a 1: 1 to 2: 1 ratio of T:non-T cells followed by a decrease at the higher cell numbers. The initial portion of the curve reflects the activity of T helper cells, whereas the decrease at higher ratios is thought to be due to T suppressor cells. As seen in Fig. 2B, T cells from the normal twin could provide help to allogeneic normal non-T cells in inducing IgM synthesis in vitro. This curve was not significantly different from that obtained with normal T cells. In contrast, the twin with diffuse progressive systemic sclerosis showed considerably increased helper-T-cell function compared to her normal twin over a broad range on this curve and this difference was statistically significant (P < 0.001) at the latter two 4c

5

:

2!

i-

2c

)-

0 ‘;

I5

D IO

I-

5

P

P

STIMULATOR WON-T CELLS, P T w P ADDITIONAL -PP CELLS ,NOr+T CELLS, -

RESPONDER ITCELLS,

P

Tw

CULTURE# I

P

2

3

Tw

N

N

N

Tw

T w T w Tr P

Tw Tw

N

N

N

N

-

-

P

Tw

-

N

Tw

P

5

6

7

a

9

IO

II

12

4

AUTOLOGOUS

N

ALLOGENEIC

FIG. 1. Effect of addition of extra PSS or twin non-T cells to AMLR cultures. Cultures consisted of I x IO5 responder T cells and 1 x 10’ mitomycin C-treated stimulator non-T cells. Additional cells consisted of 1 X 10’ mitomycin C-treated non-T cells. P-PSS twin, Tw-normal twin. N-unrelated third-party normal individual.

26

DUSTOOR ET AL.

NUMBER

OF T CELLS/CULTURE

(x

lo-‘)

FIG. 2. (A) Effect of adding increasing numbers of allogeneic normal T cells on polyclonal IgM synthesis by 6 x 10“ non-T cells. This is a composite curve obtained from 12 normal individuals in 12 separate experiments. (B) Effect of adding increasing numbers of PSS patient (A) or normal twin (0) T cells to 6 x IO4 allogeneic normal non-T cells.

points on the curve. Furthermore, the downward trend in IgM synthesis at the higher T:non-T ratios was not present with the patient’s cells, suggesting a T-suppressor cell defect in addition. Stimulation

of Fibroblast

Activity

The results presented in Table 5 indicate no significant difference in the ability of activated supernatants from the patient or the normal in enhancing fibroblast proliferation. By contrast, both protein and collagen synthesis by fibroblasts were elevated by culture supernatants prepared from the PSS patient (Table 6). DISCUSSION

In this study we have investigated several immunological functions in a set of identical female twins discordant for the occurrence of scleroderma. Our results demonstrate a distinct dichotomy of responsiveness between the patient and her normal twin, suggesting strongly that the immunologic abnormalities seen in scleroderma are not dictated by genetic predisposition. The pathogenesis of PSS is unclear but increasing evidence suggests that immunological mechanisms are involved. The occurrence of autoantibodies (8. 9) and rheumatoid factors (19) and defective cell-mediated immune responses together with studies establishing immune system control of connective tissue me-

SCLERODERMA:

DISCORDANT

FINDINGS

TABLE 5 MNC SUPERNATANTEFFECTSONFIBROBLAST

PROLIFERATIONS [3H]Thymidine incorporation (cpmlwelBb

Cells used for supematant preparation None Unrelated normal PSS twin

27

IN TWINS

7015 t 237 12353 f 2089 13436 t 524

cl:10 dilution) t 1: 10 dilution) t 1: 10 dilution)

n Data are from a representative experiment. h Mean of duplicates s data range.

tabolism all point strongly to an immune component in the pathogenesis of the disease (2, 20, 21). Our PSS patient had a low absolute T-cell count, lower numbers of OKT4+ cells, and an increase in OKT8+ cell numbers. Several studies have documented a reduction in the T-cell count in PSS (6, 22). Conflicting results have been obtained when T-cell subsets have been enumerated. Two studies have reported decreased Tp (helper) cells together respectively with normal or increased Ty (suppressor) cells (5, 23). Another study found OKT8+ cells to be lowered in PSS patients (24). Our own study with a group of approximately 25 PSS patients did not show any overall statistically significant differences in T-cell-subset number (unpublished data). The data on the PWM-induced proliferation of both unseparated mononuclear cells and the separated T- and non-T-cell populations confirm the inability of patient T cells to respond appropriately to PWM. This may, in part, be due to greater numbers of T8 suppressor cells in this patient. This finding is in agreement with other studies on PSS documenting decreased mitogen responsiveness (6, 25, 26). In addition, from Table 3 it appears that the patient non-T cells may also have a suppressive influence on T-cell proliferation. Among non-T cells, monocytes have potent suppressor function and have been shown to be responsible for immunoregulatory defects in certain diseases (27, 28). A similar patient response was seen in the MLR where patient T cells were unable to respond to an autologous or allogeneic cell stimulus. Here also, patient non-T cells had a supTABLE 6 MNC SUPERNATANTEFFECTSOFFIBROBLASTCOLLAGENANDPROTEIN Cells used for supernatant preparation

PRODUCTION"

[3H]Proline incorporation Protein

Collagen (cpm/well)*

None Unrelated normal PSS twin

(1: 10 dilution) ( 1: 10 dilution) (I: 10 dilution)

u Data are from a representative experiment. b Mean of duplicates 2 data range.

22173 k 107 24839 -e 905 37774 t 6736

6581 k 515 6397 f 995 10156 +- 598

28

DUSTOOR

ET AL.

pressive influence. Evidence for monocyte suppression of the MLR is present in the literature (29, 30). In contrast to T cells involved in mitogen and allogeneic responses, there was a marked increase in T-helper activity for immunoglobulin synthesis. This is in accordance with our results on PSS patients and those of others (4, 31). The decrease in IgM synthesis seen with normal cells at the higher T:non-T ratios is due to the activities of suppressor T cells (32). Thus, since IgM synthesis with patient’s cells continues to increase rather than decrease at the higher T:non-T ratios as the normal curve does, there appears to be a T-suppressor-cell defect in the patient as well. Another indicator of increased helper cell function in the patient was the enhanced secretion into culture supernatants of factors capable of inducing fibroblast collagen and protein synthesis. We have shown previously (2) that scleroderma lymphocytes produce soluble factors which induce enhanced collagen synthesis by normal tibroblasts. The increase in helper cell function seen in the patient was not present in her normal twin sister. The pathogenesis of most of the rheumatic diseases is probably multifactorial. The relationship of several HLA antigens with scleroderma has been suggested. The HLA make-up (Al, 24. B7, 8, C7, DR4) of our set of twins deserves comment relative to the association of the various alleles with disease. Of particular interest is HLA-B8 which is linked with a number of disorders displaying features of autoimmunity or abnormal immune responsiveness (33). Hughes et al. (34) reported an increased incidence of HLA-BS in patients with scleroderma who had extensive visceral disease together with depressed T-lymphocyte numbers and low mitogen responses. The HLA-B8 phenotype in normals has also been associated with increased reactivity to alloantigens. DR 4 positivity has also been linked to sero-positive rheumatoid arthritis (12). In a study of scleroderma in a kindred, four of five affected siblings expressed the HLA-DRw4 antigen (35). In addition, the Al allele has also been shown to be associated with autoimmune disease (36). Thus with the exception of C7, the twins possess HLA alleles which have documented associations with autoimmune disease and in some reports, with scleroderma as well. Familial scleroderma is a rare occurrence. In a review of 727 cases Tuffanelli and Winkelmann found no familial occurrences (37). Many reports contain only partial documentation. A few well-documented reports of family members with scleroderma have been published (35. 38-40). Studies of immunological function in such families are very scarce. Soppi et al. (16) carried out an immunological analysis in eight siblings, two of whom had PSS, and found no uniform disturbance in any immunological function common to the patients or to all siblings. In a study of PSS in the highly inbred Brandywine population, Greger (38) estimated the frequency of PSS to be 250 times that in the normal population. In addition, Emerit et al. (41) have observed a high incidence of chromosomal breakage in patients with PSS as well as in asymptomatic siblings and children. These studies are therefore suggestive of a genetic component in the pathogenesis of PSS. The studies described herein show markedly differing immunologic responses from a patient with scleroderma and her healthy identical twin. It is not clear

SCLERODERMA:

DISCORDANT

FINDINGS

IN TWINS

29

whether the immune defects precede or are the result of the disease. It does appear from these studies, however, that these defects are not entirely genetically predetermined yet do correlate with the presence of the disease state. ACKNOWLEDGMENTS The authors thank Dr. Claudio Fiocchi for helpful discussions and reviewing the manuscript and Nijole Mazelis for excellent secretarial support. This project was funded by a Research Program Committee grant from the Cleveland Clinic Foundation and by a grant from the Scleroderma International Foundation.

REFERENCES I. LeRoy, E. C., In “Textbook of Rheumatology” (W. N. Kelley, E. D. Harris, Jr., S. Ruddy, and C. B. Sledge, Eds.). pp. 121 l-1230, Saunders, Philadelphia, 1981. 2. Cathcart, M. C., and Krakauer, R. S., C/in. Immunol. Immunopat/~ol. 21, 128, 1981. 3. LeRoy, E. C., J. Clin. Invest. 54, 880, 1974. 4. Krakauer, R. S., Sundeen. J., Sauder, D. N., and Scherbel, A., Arch. Dermafol. 117, 80, 1981. 5. Alarcon-Segovia. D., Palacios, R., and lbanez de Kasep. G., J. C/in. Lab. [mmuno/. 5, 143. 1981. 6. Hughes, P.. Holt. S.. Rowell. N. R., and Dodd, J., Brit. J. Dermnrol. 95, 469. 1976. 7. Cooper, S. M., Harding, B., Mirick, G. R., Schneider. J., Quismario, F. P., and Friou, G. J., C/ipl, Exp.

fmmunol.

34, 235, 1978.

8. Tan, E. M., Rodnan, G. P.. Garcia, I., Moroi. Rheum.

Y., Fritzler, M. H., and Peebles, C., Arrhr/t[.r

23, 617, 1980.

9. Moroi, Y., Peebles, C.. Fritzler, M. J., Steigerwald, J., and Tan, E. M., Proc. Nut/. Acad. Sci, USA 77, 1627, 1980. 10. Sasazuki, T.. Nishimura, Y., Muto, M., and Ohta, N.. Immunol. Rev. 70, 51, 1983. Il. Ambinder. J. M., Chiorazzi. N.. Gibofsky, A., Fotino, M., and &n&l, H., C/in. [mmuno/. Immunopathol. 23, 269, 1982. 12. Stastny. P.. N. Engl. J. Med. 298, 869. 1978. 13. Remertsen, J. L., Klippel. J. H., Johnson, A. H., Steinberg, A. D., Decker, J. L., and Mauer, D. L.. N. Engl. J. Med. 299, 515. 1978. 14. Stokes, P. L.. Asquith, P, Holmes, G. K. T., Mackintosh, P.. and Cooke, W. T., Lancer 2, 162, 1972.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Bluestone, R., and Pearson, C. M., N. Engl. J. Med. 297, 1060, 1977. Soppi, E., Lehtonen, A. and Toivanen, A., C/in. Exp. Immune/. 50, 275, 1982. Sundeen, J. T., and Krakauer, R. S., J. Immunol. Methods 26, 229, 1979. Webster, D. F., and Harvey. W., Anal. Biochem. 96, 220, 1979. Clark, J.. Winkelmann, R. K., McDuffte, F. L.. and Ward, V. E., Mayo C/in. proc. 46, 97, 1971, Johnson, R. L., and Ziff, M., J. C/in. Invest. 48, 240, 1976. Wahl. S. M., Wahl, L. M., and McCarthy, J. B., J. Immunol. 121, 942, 1978. deJesus, D. G., and Clancy, R. L., J. Rheumatol. 2, 336, 1975. Gupta. S.. Malaviya, A. N., Rajagopalar, P., and Good, R. A., Clin. Exp. Immunol. 38,342, 1979. Whiteside. T. L., Kumagai. Y., Roumm, A. D., Almendinger, R., and Rodnan, G. P., Arr1wi~i.s Rheum.

26, 841, 1983.

25. Salem, N. B., and Morse, J. H.. Arthritis Rheum. 19, 875, 1976. 26. Horwitz, D. A., and Garrett, M. A., C/in. Exp. Immunol. 27, 92, 1977. 27. Becker. M. J., Dricker. I., Farkas, R., Steiner, Z.. and Klajman, A., C/in. EXP. Immunol. 45,439, 1981. 28. Goodwin, J. S., Messner, R. P.. Bankhurst, A. D., Peake, G. T., Saiki, J. H.. and Williams. R. C., N. Engl. J. Med. 297, 963, 1977. 29. Dustoor, M. M.. Ransohoff, R. M., and Krakauer. R. S.. Fed. Proc. 41, 808, 1982. 30. Katz, P., and Fauci, A. S., J. Immunol. 123, 2270, 1979. 3 1. McInemey, M. M., Dustoor, M., and Krakauer, R. S., Fed. Proc. 42, 951, 1983.

30

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ET AL.

32. Thomas, Y., Sosman, J., Irigoyen. 0.. Friedman, S., Kung, P. C., Goldstein, G., and Chess, L., J. Immunol. 125, 2402, 1980. 33. Braun, W. E.. “HLA and Disease. A Comprehensive Review.” CRC Press, Boca Raton. FL, 1979. 34. Hughes, P., Gelsthorpe, K., Daughty, W.. Rowell, N. R., Rosenthal, F. D., and Sneddon, L. B., Clin. Exp. Immunol. 31, 351, 1978. 35. Sheldon, W. B., Lurie, D. P., Maricq, H. R., Kakeleh, M. B., DeLustro. F. A., Gibofsky, A., and LeRoy, E. C., Arthritis Rheum. 24, 668, 1981. 36. Valenta, L. J., Bull, R. W., Hackel, E., and Bottazzo, G. E, Acta Endocrinol. 100, 143, 1982. 37. Tuffanelli, D. L., and Winkelmann. R. K., Arch. Dermatol. 84, 358, 1961. 38. Greger, R. E., Arch. Dermatol. 111, 81, 1975. 39. Gray, R. G., and Altman, R. D., Arthritis Rheum. 20, 35, 1977. 40. Flores, R. H., Stevens, M. B., and Amett, E, J. Rheumatol. 11, 3, 1984. 41. Emerit. I., Housset. E.. and Feingold, J., J. Lab. C/in. Med. 88, 81, 1976. Received August 22, 1986; accepted with revision March 9, 1987