Surface characteristics of synovial fluid and peripheral blood lymphocytes in inflammatory arthritis.код для вставкиСкачать
297 SURFACE CHARACTERISTICS OF SYNOVIAL FLUID AND PERIPHERAL BLOOD LYMPHOCYTES IN INFLAMMATORY ARTHRITIS ALAN I. BRENNER, MORTON A. SCHEINBERG, and EDGAR S. CATHCART I n order to characterize the T- and B-cell populations of inflammatory arthritides, synovial fluid and peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), and rheumatoid variant diseases (RV) were studied. Normal peripheral blood lymphocytes were examined as controls. T cells were identified by spontaneous sheep red blood cell (E) rosette formation. B cells were From the Arthritis and Connective Tissue Disease Section of the Evans Department of Clinical Research, University Hospital, the Thorndike Memorial Laboratory, Boston City Hospital, and the Department of Medicine, Boston University School of Medicine, Boston, Massachusetts. Investigations supported by grants from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases (AM-04599 and TI-AM 5285), from the General Clinical Research Centers Branch of the Division of Research Sources, National Institutes of Health (RR-533), from the Massachusetts Chapter of The Arthritis Foundation, from The Arthritis Foundation, and from the John A. Hartford Foundation. Presented in part at the VI Pan-American Congress on Rheumatic Diseases, June 19, 1974. Toronto, Ontario, Canada. Alan I. Brenner. M.D.: Clinical and Research Fellow in Medicine, Boston University School of Medicine; Morton A. Scheinberg. M.D.: Clinical and Research Fellow in Medicine, Boston University School of Medicine; Edgar S. Cathcart, M.D.: Associate Professor of Medicine, Boston University School of Medicine. Address reprint requests to Edgar S. Cathcart, M.D.: Arthritis Section, Boston University School of Medicine, 75 East Newton Street, Boston, Massachusetts 02118. Submitted for publication September 25, 1974; accepted January 27, 1975. Arthritis and Rheumatism, Vol. 18, No. 4 (July-August 1975) determined by complement receptor lymphocyte (EAC) rosette formation and by the presence of surface immunoglobulins (SIg) utilizing fluoresceinated polyvalent antiglobulin. Synovial fluids were analyzed in terms of duration, mucin quality, white cell count, and differential. Synovial fluid and peripheral blood lymphocytes from patients with RA and R V were distributed in T- and B-cell percentages similar to those found in normal peripheral blood. In contrast, significant T-cell depression was observed in the percentage of synovial fluid and peripheral blood lymphocytes of SLE and JRA patients. This depression was apparent in comparison with normal peripheral blood and with the synovial fluid and peripheral blood from R A patients. Bcell percentages were similar in all patient groups and in comparison to normal peripheral blood lymphocytes. N o differences were noted in B-cell percentages when the EAC and SIg techniques of identification were compared. The percentage of cells bearing neither T- nor Bcell markers (null cells) was enumerated for each patient group and found to be significantly elevated in the synovial fluid and peripheral blood of SLE and JRA patients. Though the mean synovial fluid and peripheral blood null cell percentages in R A patients were similar to those in controls, a definite bimodal distribution was found in the synovial fluids. These data suggest that evaluation of T-, B-,and nullcell populations may be clinically useful in differen- BRENNER E T AL 298 tiating patients with SLE and JRA from those with rheumatoid arthritis and variant diseases. T h e investigation of the inflammatory arthritides, especially the study of synovial fluids, has focused primarily o n fluid-phase reactants, ie immunoglobulins (l), rheumatoid factors (2), complement components (3), a n d proteolytic enzymes (4,5). Studies delineating T a n d B lymphocytes i n rheumatologic conditions are sparse a n d limited in several respects. Although several investigators (6-8) have studied rheumatoid synovial fluid and peripheral blood lymphocytes, each series has been limited i n t h e number of surfacemarking techniques a n d there has been no general agreement on the comparisons between rheumatoid a n d normal peripheral blood lymphocytes, or between rheumatoid peripheral blood a n d synovial fluid lymphocytes. Furthermore there has been no work comparing various inflammatory arthritides for Ta n d B-lymphocyte percentages i n blood a n d synovial fluid. Because of the presence of d e novo immunoglobulin synthesis (9), a B-cell function, a n d macrophage migration inhibitory factor production (lo), a predominantly T-cell function, i n inflammatory synovial fluid, a n d because of the suggestion that peripheral blood T-lymphocyte depression may correlate with certain pathogenic features of systemic lupus erythematosus (1 1), the surface characteristics of peripheral blood a n d synovial fluid lymphocytes from patients with inflammatory arthritides have been studied. These studies demonstrated that T-cells are depressed i n JRA, as well as in SLE, whereas the proportions of T a n d B lymphocytes from patients with RA a n d its variants were not significantly different from those of controls. MATERIALS AND METHODS Clinical Characteristics of the Patient Population. T h e peripheral blood and synovial fluids of 30 patients with a variety of inflammatory arthritides as well as peripheral blood lymphocytes from 11 normal volunteers were studied. Rheumatoid Arthritis All of the 14 patients studied had classic disease by the criteria of Ropes et a1 (12), and all had a positive sheep cell agglutination test (SCAT). A t the time of the study, none of the patients was being treated with corticosteroids, and none had ever received cytotoxic drugs. One patient had both a positive antinuclear antibody test (ANA) (homogeneous pattern) and hypocomplementemia (C3 89 mg%; normal: 120-160 mg%). This patient, a 59-year-old man with evidence of cutaneous vasculitis, had nodular deforming rheumatoid arthritis and none of the classification criteria for SLE other than positive ANA. Systemic Lupus Erythematosus Five patients, selected only for the presence of joint effusions, were examined. A t the time of study, all were separable from the RA patients by the preliminary classification criteria for SLE (13). Four patients had positive ANAs in a homogeneous or peripheral pattern; none had hypocomplementemia or exhibited renal failure. None of the patients had received cytotoxic drugs, but 1 of the 5 was receiving corticosteroids (10 mg of prednisone per day) for control of her polyarthritis. Juvenile Rheumatoid Arthritis Six patients were studied. Five of the 6 had the onset of their oligo- or polyarthritis before age 16, although only 2 were less than 16 years of age at the time of study. The sixth, although 34 years old at the onset of his illness, had a febrile presentation with rash and polyarthritis typical of those patients described by Bywaters (14) and by Bujak et a1 (15) as having adult Still's disease. One patient had a positive ANA and a positive SCAT. She was a 62-year-old woman who had had continuous deforming polyarthritis since age 6. None of these patients demonstrated hypocomplementemia, none had evidence of polyserositis, and only 1, a 9-year-old boy with JKA and secondary amyloidosis, had evidence of renal disease. Rheumatoid Variants This group of 5 patients included 3 with psoriatic arthritis, 1 with Reiter's syndrome, and 1 with inflammatory bowel disease, ankylosing spondylitis, and peripheral polyarthritis. Lymphocyte Studies. Peripheral blood and synovial fluid samples were obtained simultaneously from each patient. Lymphocytes for all surface marker studies were separated from heparinized blood or synovial fluid by FicolHypaque centrifugation according to the method of Boyum (16). Heparinized synovial fluid was first treated with hyaluronidase (Wydase, Wyeth Laboratories, Philadelphia, Pennsylvania, Lot No. 4732403) according to a modification of the method of Hedberg (17); otherwise the techniques were similar for corresponding fluid and blood lymphocytes. Following aspiration, 5 ml of blood or synovial fluid were layered onto 3 ml of Ficol-Hypaque and centrifuged at 1200 rpm for 40 minutes. The lymphocyte-rich interface layer was then carefully removed and washed twice in Hanks balanced salt solution (HBSS). The cells were then counted in a hemocytometer chamber and, for E and EAC rosette studies, adjusted in HBSS to a concentration of 4 x 106 cells/ml. T lymphocytes were detected by sheep red blood cell (E) rosette formation according to the method of Jondal et a1 (18). 1 x lo6 lymphocytes in 0.25 ml of HBSS SURFACE CHARACTERISTICS OF SYNOVIAL FLUID 299 Table 1. Synovial Fluid Characteristics of Study Population Much Patient Number Diagnosis Rheumatoid arthritis Systemic lupus erythematosus Juvenile rheumatoid arthritis Variants Good Fair Poor 14 5 0 1 5 4 6 1 0 2 2 9 0 3 3 5 were combined with an equal volume of a 0.5% washed sheep red cell suspension, incubated at 37°C for 15 minutes, centrifuged at 1000 rpm at 4°C for 5 minutes, and then allowed to incubate as a pellet at 4°C for a further 8-12 hours. B lymphocytes were identified by the complement receptor technique described by Bianco et a1 (19). Washed sheep red blood cells (E) were first incubated with a sublytic concentration of 19s amboceptor (A) and then with mouse complement (C) to form the complement carrier system 19s EAC. 1 x 106 lymphocytes in 0.25 ml of HBSS were combined with 0.25 ml of a 0.5% suspension of 19s EAC and incubated at 37°C for 30 minutes. One drop of a suspension of latex particles (Dow Diagnostics, No. 54217) was added to each reaction in order to identify, by phagocytosis, macrophages and any remaining polymorphonudear leukocytes that also carry surface complement receptors. The percentage of E or EAC rosettes formed was determined by counting a total of 200 rosetting and nonrosetteforming lymphocytes in each reaction. Each determination was performed in duplicate, and the results were expressed as the average percent of rosetting cells. B lymphocytes were identified by the presence of surface immunoglobulins according to a modification of the method of Aisenberg and Bloch (20). Following FicolHypaque centrifugation, lymphocytes were resuspended in HBSS containing 10% fetal calf serum and 0.1% Na Azide. 2 x 106 lymphocytes in 0.1 ml of medium were then combined with 0.1 ml of a fluorescein-conjugated polyvalent goat antihuman antiserum (Meloy Laboratories, Springfield, Virginia, Lot No. C201-22 031) and incubated at 4°C for 30 minutes. The lymphocytes were washed twice in the same medium, resuspended in one drop of glycerol/ HBSS (50/50 v/v). and read in a Leitz fluorescent microscope through an FITC primary and -65 and +45 barrier filters. WBC Mean Lymphocytes, Mean Percentage 10500 4260 48 56 39 21 20530 21940 RESULTS Synovial Fluid Studies. The results of routine synovial fluid analyses are presented in T a b l e 1. Mucin tests were recorded as good, fair, or poor; white counts and percentages of lymphocytes are given, as means of the groups studied. When compared to all other groups, patients with SLE exhibited better mucin tests, significantly lower white counts, and higher percentages of lymphocytes. Lymphocyte Studies. T- and B-lymphocyte determinations by E and EAC rosette formation a n d surface immunoglobulin studies were carried o u t o n all patients ( T a b l e 2). Because of their small number, the patients with rheumatoid variant diseases were combined a n d the results expressed as the means of t h e group; by this method, t h e results obtained were similar to those f o u n d in rheumatoid arthritis. For statistical comparisons, the studies have been limited to the RA, SLE, and J R A groups. Unless noted all statistical evaluations were performed by the t test. Peripheral Blood Studies D a t a on the peripheral blood lymphocytes from 11 normal subjects a n d from the patients with RA, SLE, a n d JRA are presented i n Table 1. W h e n normal lymphocytes were compared with the patient groups for the mean percentage of E rosettes formed, 0.05) difference between there was significant (P 3%) a n d SLE (55 f 11%) lymphocytes, normal (66 < +_ Table 2. Results of T - and B-Lymphocyte Studies ~~ ~~~ ~ ~~ B Cells T Cells, E Rosettes Diagnosis Rheumatoid arthritis Systemic lupus erythematosus Juvenile rheumatoid arthritis Rheumatoid variants* Normal EAC Rosettes SIg Cells Peripheral Blood Synovial Fluid Peripheral Blood Synovial Fluid Peripheral Blood Synovial Fluid 6 9 2 8% 55+ 11% 58 k 4% 58 f 20% 66+ 3% 76+ 11% 44+ 7% 43+ 14% 54 + 15% 17f 9% 18 + 10% 23* 9% 1 7 k 10% 1 8 2 5% 13+6% l3+8% 19+5% 25-C8% 1 8 k 10% 17+ 5% 16+ 9% 9 + 4y0 1 9 2 11% 9 + 2% *Variant results are not included in statistical studies. - - 2 0 k 8% 14+ 6y0 16C 4% - BRENNER E T AL 300 NULL CELLS MEAN PERCENTAGES IN SYNOVIAL FLUID 100 Fig 1. Mean percentages of null cells (see text) in the synovial fluid of patients with RA, SLE, and JRA. as well as a significant ( P < 0.001) depression in the E rosettes formed by JRA peripheral blood lymphocytes (58 f 4y0). A significant depression in E rosette formation was also noted when the lymphocytes of RA patients (69 f 8y0)were compared to those from patients with SLE ( P < 0.05) and with JRA ( P < 0.01). EAC rosette formation demonstrated no significant differences either when normal lymphocytes were compared to patient populations, or when the various patients groups were compared with each other. Synovial Fluid Studies Table 1 shows that 76 2 11% E rosettes were found in rheumatoid synovial fluids, whereas the 44 f 7y0 found in SLE and the 43 f 14y0 found in JRA fluids were significantly ( P < 0.001) depressed. A further finding was the complete lack of overlap when E rosette percentages from RA fluids were compared to those of SLE and JRA fluids. EAC determinations again differed little between the three groups. When compared with normal peripheral blood lymphocytes, however, rheumatoid synovial fluid EAC appeared significantly depressed ( P < 0.05). EAC vs Surface Immunoglobulin as a B-Cell Marker As can be seen in Table 1, synovial fluid studies revealed no significant differences between simultaneous EAC and surface immunoglobulin determina- tions. Similar results were obtained in peripheral blood studies, in both normal lymphocytes and in cells from all the patient groups. Null Cells Null cells were defined as the percentage of lymphocytes bearing neither T- nor B-cell surface markers. T h e mean null cell percentage from 11 normal subjects was 18%. Wide variations in standard deviation made comparisons by mean and standard deviation unsuitable; however by use of the MannWliitney U test, in comparing synovial fluid null cells from rheumatoid patients (mean: 13'%) with those from patients with SLE (mean: 43Y0) and from JRA patients (mean: 38y0),significant elevations in SLE ( P < 0.002) and JRA ( P < 0.02) synovial fluid null cell percentages were evident (Figure 1). A bimodal distribution was noted in the synovial fluid null cell percentages from patients with RA, with one group (5 patients) having 0-4% null cells, and another group (5 patients) having 15-30y0 null cells. No clinical or laboratory marker, including Westergren sedimentation rate, the presence of rheumatoid or antinuclear factor, the degree of clinical activity at the time of study, or routine synovial fluid analysis, could differentiate these two subgroups within the rheumatoid population. DISCUSSION Functional lymphocyte studied by mitogenic stimulation and mixed lymphocyte culture reactions have indicated abnormal 'r- and B-cell activity in SLE ( 1 1). Moreover, several researchers have commented on peripheral blood T-cell depression in SLE, using both sheep red blood cell rosette formation and rabbit antihuman thymocyte serum as markers (21-23). Scheinberg and Cathcart have correlated this T-cell depression both with clinical activity at the time of the study and with the degree of lymphopenia (11). Lymphocyte studies in rheumatoid arthritis, on the other hand, have produced widely divergent results. In terms of functional measurements Stratton (24) as well as Panayi (25) has reported poor synovial fluid response to phytohemagglutinin and pokeweed mitogen. Stratton however reported a normal rheumatoid peripheral blood response to several mitogens, whereas Panayi found rheumatoid peripheral blood lymphocyte responses to be depressed. Several authors have studied the percentages of T and B lymphocytes in the peripheral blood and SURFACE CHARACTERISTICS OF SYNOVIAL FLUID synovial fluid of patients with rheumatoid arthritis. Again the results appear contradictory. Keith and Currey, employing 30-minute 4OC incubation, found 20-4OOj, E-rosetting T cells in the peripheral blood and synovial fluid of rheumatoid patients, and obtained similar results from normal peripheral blood (6). Froland et al (8) studied peripheral blood and synovial fluid lymphocytes from a rather heterogenous group of inflammatory arthritides. They reported higher T-cell percentages in the synovial fluid and peripheral blood than in normal peripheral blood, though the mean percentages were low (20-300/,) and the standard deviations high. Papamichail and coworkers (26) showed a marked increase in rheumatoid peripheral blood SIg staining, notably in IgG-bearing cells (31%). In contrast Mellbye et al (7) reported a decrease in rheumatoid peripheral blood B lymphocytes by both SIg and EAC markers. I n synovial fluid studies in rheumatoid patients, Froland et al (8) reported less than 1% SIg-bearing cells whereas Mellbye et al (7) found fluid SIg cells present in higher percentages than in either rheumatoid or normal peripheral blood. T h e wide variations in reported results of Tand B-lymphocyte percentages have several possible explanations. Jondal et a1 (18) have clearly shown that prolonged 4OC incubation is required for complete E-rosette formation. Immunoglobulin capping, endocytosis, and loss of surface marker have been studied at 37OC; studies at 4"C, using Na Azide to inhibit cellular metabolism, should result in more complete surface immunofluorescent staining (27). Recently, Winfield et al, working at 4OC, have found very high percentages of immunoglobulin-bearing lymphocytes i n the peripheral blood of patients with RA and SLE. They have shown that the bulk of these immunoglobulins may be removed by overnight incubation at 37°C and by elution techniques, and that they represent lymphotoxic antibodies adherent to the cell surfaces (28). T h e present studies reveal a relative depression in rheumatoid synovial fluid B lymphocytes by EAC rosette formation, as intimated by Mellbye et al (7), but fail to confirm any significant difference between simultaneous EAC and SIg determinations, whether in peripheral blood or synovial fluid. A significant depression in the percentage of T lymphocytes in the peripheral blood of patients with SLE was confirmed. It was also found that T cells are significantly depressed in the synovial fluid of patients 301 with SLE and in both the peripheral blood and synovial fluid of patients with JRA. T o our knowledge these are the first reported studies of T- and B-lymphocyte abnormalities in JRA. Panush et al (29) as well as Hoyeraal (30) have studied cellular immune mechanisms in J R A by both in vivo skin testing and in vitro techniques of lymphocyte stimulation and MIF production. Both groups found impaired skin test responses in JRA patients which would be in keeping with our finding of T-cell depression. T h a t this depression is not a manifestation of an excess of immunoglobulin-producing B cells may be inferred by the stable percentages of both EAC and SIg lymphocytes, and by the proportionate increase in null cells in both of these conditions. I n terms of pathogenesis one important feature of T-cell depression may be the loss of normal T-cell mediated B-cell suppression and the subsequent production, by B cells, of excessive antibody products that are especially prominent in SLE. T h e striking difference between T-cell percentages in RA and those in SLE and J R A also suggests the possibility either that the pathogenic mechanisms are dissimilar, or that host responses to similar insults are handled in dissimilar fashion. T h e high percentage of null cells i n the peripheral blood of patients with SLE has been reported previously by this laboratory (11). Similar high null cell percentages in SLE synovial fluid as well as in both peripheral blood and synovial fluid from patients with JRA are newly demonstrated by these studies. T-cell depression and a concomitant rise in the percentage of apparently unreactive null cells may explain the observation by Hoyeraal (30) of a negative correlation between impaired skin reactivity and the number of circulating peripheral blood lymphocytes in patients with JRA. I n contrast to the present findings, recent studies by Winchester et al demonstrate an increased percentage of null cells in RA but not in SLE synovial fluid. These results may be explained either by differences in disease activity, by exposure to steroids or cytostatic agents, or by technical variations that await further standardization (31). T h e bimodal nature of the null cell population in rheumatoid synovial fluid has been commented on by Williams and coworkers (21). Although we did not observe two statistically separable populations of T cells as did Williams, a distinct depression of synovial fluid B lymphocytes by EAC rosette formation was found, and this probably contributed to the null cell population. This finding could not be cor- BRENNER E T AL 302 related with any of the usual clinical or laboratory parameters of disease activity; however, synovial fluids were not studied for the presence of antigen-antibody complexes or for variations i n complement levels. T h u s it has been demonstrated that i n clearly defined patient populations significant T-cell depression a n d null cell elevation do occur in systemic lupus erythematosus a n d juvenile rheumatoid arthritis. Further studies of large numbers of patients are necessary before these differences i n lymphocyte populations can be considered clinically useful. ACKNOWLEDGMENTS T h e authors would like to thank Dr. Alan C. Aisenberg for his expert advice on the surface immunoglobulin staining technique, Dr. Hebert Kayne for statistical analysis, and Mr. David Feigenbaum for photographic assistance. We also gratefully acknowledge the technical assistance of Ms. Lindy Burnett. REFERENCES 1. Bennett JC: The role and character of immunoglobulins in rheumatoid inflammation. Fed Proc 32: 138-142, 1973 2. Zvaifler NJ: The immunopathology of joint inflammation in rheumatoid arthritis. Adv Immunol 16:265-336. 1973 3. Ruddy S, Austen KF: Activation of the complement system in rheumatoid synovitis. Fed Proc 32: 134-137, 1973 4. Harris ED Jr, Evanson JM, DiBona DR, et al: Collagenase and rheumatoid arthritis. Arthritis Rheum 13: 83-94, 1970 5. Oronsky AL, Ignarro LJ, Perper RJ: Release of cartilage mucopolysaccharide-degrading neutral protease from human leukocytes. J Exp Med 138:461472, 1973 6. Keith BI, Currey HLF: Rosette formation by peripheral blood lymphocytes in rheumatoid arthritis. Ann Rheum Dis 32:202-207, 1973 7. Mellbye OJ, Messner RP, DeBoard JR, et al: Immunoglobulin and receptors for C,3 on lymphocytes from patients with rheumatoid arthritis. Arthritis Rheum 51:371-380, 1972 8. Froland SS, Natvig JB, Husby G: Immunological characterization of lymphocytes in synovial fluid from patients with rheumatoid arthritis. Scand J Immunol 2: 67-73, 1973 9. Smiley JD, Sacks C, Ziff M: In vitro synthesis of immunoglobulin by rheumatoid synovial membrane. J Clin Invest 47:624-632, 1968 10. Stastny P, Cooke D, Jasin H, et al: Production of a macrophage migration inhibitory factor in rabbits with experimental arthritis. Fed Proc 31:797, 1972 (abstr) 11. Scheinberg MA, Cathcart ES: B cell and T cell lymphopenia in systemic lupus erythematosus. Cell Immunol 12309-314, 1974 12. Ropes MW, Bennett GA, Cobb S, et al: Revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 9:175-176, 1958 13. Cohen AS, Reynolds WE, Franklin EC, et al: Preliminary criteria for the classification of systemic lupus erythematosus. Bull Rheum Dis 21 :643-648, 1971 14. Bywaters EGL: Still's disease in the adult. Ann Rheum Dis 30:121-133, 1971 15. Bujak JS, Aptekar RG, Decker JL, et al: Juvenile rheumatoid arthritis presenting in the adult as fever of unknown origin. Medicine 52451-444, 1973 16. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononudear cells by one centrifugation and sedimentation of Ig. Scand J Clin Lab Invest 21:97, 1968 (suppl) 17. Hedberg H: Studies on synovial fluid in arthritis. Acta Med Scand 479:85-86, 1967 (suppl) 18. Jondal M, Holm G, Wigzell H: Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med 136:207-215, 1972 19. Bianco C, Patrick R, Nussenzweig V: A population of lymphocytes bearing a membrane receptor for antigen, antibody, complement complexes. I. Separation and characterization. J Exp Med 132702-720, 1970 20. Aisenberg AC, Bloch KJ: Immunoglobulins on the surface of neoplastic lymphocytes. N Engl J Med 287: 272-276, 1972 21. Williams RC, DeBoard JR, Mellbye OJ, et al: Studies of T and B lymphocytes in patients with connective tissue diseases. J Clin Invest 52:283-295, 1973 22. Messner RP, Lindstrom FC, Williams RC Jr: Peripheral blood lymphocyte cell surface markers during the course of systemic lupus erythematosus. J Clin Invest 52:3046-3056, 1973 23. Yu DT, Clements PJ, Peter JB, et al: Lymphocyte characteristics in rheumatoid patients and the effect of azathioprine therapy. Arthritis Rheum 17:37-45, 1974 24. Stratton JA: Response of synovial fluid and peripheral blood lymphocytes to in vitro stimulation. Arthritis Rheum 15:457, 1972 (abstr) 25. Panayi GS: Response of rheumatoid synovial fluid lymphocytes to non-specific mitogens. Lancet 2 5 1 1-513, 1973 26. Papamichail M, Brown JC, Holborow EJ: Immunoglobulins on the surface of human lymphocytes. Lancet 2:850-852, 1971 27. Ross GD, Rabellino EM, Polley JM, et al: Combined SURFACE CHARACTERISTICS OF SYNOVIAL FLUID studies of complement receptor and surface immunoglobulin-bearing cells and sheep erythrocyte rosetteforming cells in normal and leukemic human lymphocytes. J Clin Invest 52:377, 1973 28. Winfield JB, Winchester P, Wernet P, et al: Different types of antilymphocyte antibodies in the sera of systemic lupus erythematosus (SLE) patient. Clin Rezearch 23:432, 1974 (abstr) 29. Panush RS, Bianco NE, Schur PH, et al: Juvenile rheu- 303 matoid arthritis. Cellular hypersensitivity and selective IgA deficiency. Clin Exp Immunol 10203-215, 1972 30. Hoyeraal H: Impaired delayed hypersensitivity in juvenile rheumatoid arthritis. Ann Rheum Dis 32331-336, 1973 31. Winchester RJ, Winfield JB, Siegal F, et al: Analyses of lymphocytes from patients with rheumatoid arthritis and systemic lupus erythematosus. J Clin Invest 54: 1082-1092, 1974 BACK ISSUES OF ARTHRITIS AND RHEUMATISM The Arthritis Foundation has arranged for The Heritage Press to store and handle the sales of back issues of ARTHRITIS AND RHEUMATISM from Volume 1 (1958) through Volume 17 (1974), including supplements. Heritage will reprint out-of-stock back issues of the journal and other complete volumes and sets as well as single issues. Therefore, those issues of ARTHRITIS AND RHEUMATISM that had gone out of print will now be available. 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