1271 LYMPHOCYTE RESPONSE TO VIRUS ANTIGENS IN SYSTEMIC LUPUS ERYTHEMATOSUS ROBERT E. WOLF and MORRIS ZIFF The cell-mediated immune response of lymphocytes to rubella, measles, parainfluenza types 1,2, and 3, varicella-zoster, and herpes virus type 1 virus antigens was evaluated in 15 SLE patients and 15 matched controls by incorporating 3H-thymidine in whole blood cultures as a measure of blastic transformation. SLE patients were less responsive than normal individuals to six of eight virus antigens tested. Culture of washed SLE cells in AB plasma did not reverse the hyporesponsiveness.The results indicated that a functional impairment of the circulating lymphocytes appeared to be responsible for the in vitro hyporesponsiveness of SLE patients to virus antigens. From the Rheumatic Diseases Unit, Department of Internal Medicine, University of Texas Southwestern Medical School, and Veterans Administration Hospital. Dallas, Texas. Supported by Postdoctoral Fellowship and Clinical Study Center Grants from The Arthritis Foundation, USPHS Grants AM09989 and AM051 54, a Veterans Administration Research Associateship, and a grant from the Oklahoma Lupus Foundation. Robert E. Wolf, M.D., Ph.D.: Arthritis Foundation Postdoctoral Fellow. University of Texas Southwestern Medical School (presently Assistant Professor of Medicine, University of Texas Southwestern Medical School, and Research Associate, Dallas VA Hospital); Morris Ziff. Ph.D., M.D.: Professor o f Medicine, Chief, Rheumatic Diseases Unit, University of Texas Southwestern Medical School. and recipient, Research Career Award, National Institutes of Health. Address reprint requests to Robert E. Wolf, M.D., Rheumatic Diseases Unit, Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas. Texas 75235. Submitted for publication January 6, 1976; accepted April 22. 1976. Arthritis and Rheumatism, Vol. 19, No. 6 (November-December 1976) Consideration of viruses in the etiology and pathogenesis of systemic lupus erythematosus (SLE) is based on a number of observations. Tubuloreticular structures superficially resembling paramyxoviruses have been detected in various tissues from patients with SLE (1-4), but it has recently been concluded (5) that these structures are not specifically related to myxo- or paramyxoviruses and probably represent a nonspecific response to injury. Elevated antibody titers have been found in the serum (6,7); however they have not implicated a single virus and may represent nonspecific responses (8). More recently, evidence for enhanced type C RNA virus expression has been reported in SLE (9-1 1 ) as well as in the New Zealand mouse (12-14) and canine (1 5) animal models. A generalized, nonspecific diminution of cellmediated immunity in SLE has been indicated by decreased responses to plant mitogens and common bacterial and fungal antigens (16-29). However conflicting results have been reported (23-26,30,3 1 ). Low levels of circulating thymus-dependent (T) lymphocytes (23, 32-34) have also been observed. Although some investigations have demonstrated inhibitory effects of sera from SLE patients on normal T cells in vitro (26,35-37), serum factors d o not appear to be responsible for the decreased cell-mediated immunity (CMI) in SLE patients (26-28). Diminution of CMI appears to correlate best with disease activity (1 6,18,29,38). CMI to virus antigens has been little studied in SLE. In the only previous investigation, Utermohlen et WOLF AND ZIFF 1272 Table 1. Virus Antigens Used for Lymphocyte Stimulation a1 (39), using the leukocyte migration inhibition technique, observed specific hyporesponsiveness t o measles antigen in SLE patients. They observed t h a t this antigen failed t o inhibit migration, whereas inhibition of migration w a s normal in t h e presence of rubella and parainfluenza type 1 antigens. In t h e present studies t h e ability of lymphocytes from SLE patients t o u n d e r g o blastic transformation in t h e presence of rubella, measles, parainfluenza types I , 2, a n d 3, varicella-zoster, and herpes virus type 1 antigens w a s measured b y t h e incorporation of tritiated thymidine (3H-thymidine) during d e novo DNA synthesis. Lymphocytes from SLE patients w e r e hyporesponsive t o six of the eight virus antigens tested. In addition, there was no recovery of responses when washed SLE lymphocytes were cultured in plasma from a b l o o d g r o u p AB, R h positive individual. These results suggest t h a t a lymphocyte a b n o r m a l i t y is t h e main factor responsible for t h e hyporesponsiveness o f SLE lymphocytes t o virus antigens. mogenization of infected chorioallantoic membranes, but all others were cell-free culture supernatants. The rubella and measles antigens had been treated with Tween 80 and ether. Samples of uninfected tissue cultures were used as controls with herpes virus type 1 and varicella-zoster antigens. Although tissue culture controls were not available for the other antigens, specificity of stimulation by measles HA and CF antigens, and by parainfluenza types I , 2, and 3, was indirectly indicated by the degree of discordant stimulation between the two measles antigen preparations and between the three parainfluenza antigen preparations, which were grown in the same tissue substrates under similar culture conditions. Stimulation of Cells with PHA and Virus Antigens in Whole Blood Cultures. Blastic transformation in the presence of virus antigens was determined for lymphocytes from I5 SLE patients (14 with active disease) and 15 controls ( 1 1 healthy individuals and 4 outpatients, 2 with hypertension, 1 with unilateral ovarian enlargement, and 1 with a healed human bite). The controls were considered as a single group because no difference in stimulation by antigen or mitogen was detected between the healthy and patient controls. Pertinent clinical data are presented in Table 2. Patients and controls were matched for age within 3 years. Racial and sexual characteristics of the two groups were similar. A modification of the whole blood culture assay of Pauly el a1 (41) was used t o measure aH-thymidine incorporation. Each culture contained 0.1 ml of fresh, heparinized whole blood, glutamine (300 pg/ml) (Eastman Kodak Co, Rochester, N Y ) and RPMI-1640 (Grand Island Biological Co, Grand Island, N Y ) in a total volume of 3 ml. Virus antigens at I : 10 and 1 :50 dilutions were added in 0. I-ml amounts to triplicate cultures. Control cultures containing 0.1 ml of medium or 0.1 ml of PHA (equivalent to 5 pl) (Difco Laboratories, Detroit, MI) were carried out in parallel. After incubation for 7 days at 37°C in humidified air with a 5% CO, atmosphere, the cultures were pulsed for 20 hours with I pCi (6.7 Ci/mM) of SH-thymidine (New England Nuclear, Boston, MA) under the same conditions. Harvesting was accomplished with a Millipore 3025 Sampling Manifold (Millipore Corp, Bedford, MA) and 2.4-cm diameter 934AH glass fiber filters (Reeve Angel, Clifton, NJ). The samples were washed twice with 3% acetic acid, 10% trichloroacetic acid (TCA), and 0.12 M phosphate-buffered saline (PBS) at pH 7.2. MATERIALS AND METHODS Table 2. Clinical and Laboratory Data of S L E Patients and Controls Virus Strain Herpes virus I Varicella-Loster Rubella Measles Measles Parainfluenza I Parainfluenza 2 Parainfluenza 3 Maclntyre VZ-I0 B- 127 Philadelphia 26 Philadelphia 26 C-3s C-39 C-243 Type of Antigen* Culture Source? CF CF HA HA CF HA HA HA EE HELF BHK-21 PHAM PHAM PMK PM K PM K * CF: complement fixation; HA: hemagglutination inhibition. t EE: 8-day-old embryonated eggs: HELF: human embryonic lung fibroblasts; BHK: baby hamster kidney: PHAM: primary human amnion: PMK: primary rhesus monkey kidney. Patients. The SLE patients met the criteria of the American Rheumatism Association for the diagnosis of SLE (40) and were either inpatients or outpatients at Parkland Memorial Hospital. Other characteristics of the two groups are presented below. Virus Antigen Preparations. The virus antigens used in these studies were acquired from the National Center for Disease Control, Atlanta, Georgia, through the cooperation of Dr. Helen Casey. The antigens were prepared for either hemagglutination inhibition (HA) or complement fixation ( C F ) antibody assays. Table 1 presents the strain and antigen type for each of the virus antigens and the tissue culture substrate in which each was prepared. The herpes virus type I antigen was prepared by ho- SLE(n Age (years) Race Sex ESR* (mm/hr) No. with hypocomplemen temia No. with positive anti-nDN A test$ * Westergren = 15) 15-44 (mean: 30) 13 black, 2 white 14 female, I male 17-125 (mean: 73) Control(n ND 9 ND erythrocyte sedimentation rate. $ 2 2 . 0 pg nDNA bound per I ml of serum. 15) 15-47 (mean: 30) 10 black, 5 white I5 female NDt 9 t ND: not done. = 1273 LYMPHOCYTE RESPONSE IN SLE The filters were placed in counting vials and dried before addition of 10 ml of Aquasol (New England Nuclear). The samples were counted for 5 minutes in a Beckman liquid scintillation counter. The cpm were adjusted to cpm/2 X lo6 mononuclear cells by using a conversion factor based on the number of mononuclear cells per culture at the time of preparation. The data were then expressed as a stimulation index (SI), where SI = cpm stimulated culture/cpm medium control culture. SI 2 2.0 was considered positive. Stimulation of Cells with Virus Antigens in Whole Blood Cultures Containing Autologous or Normal A B Plasma. Whole blood from 8 active, untreated SLE patients was centrifuged at I200 rpm for 10 minutes. The cells were washed three times with warm (37OC) RPMI-1640 and were centrifuged at room temperature, 1200 rpm for 10 minutes, after each wash. Either fresh autologous plasma or fresh AB plasma from a single donor was then added to the original blood volume. These reconstituted mixtures were used to prepare whole blood cultures as described above. Rubella, measles-CF, parainfluenza type 1, and parainfluenza type 3 antigens at dilutions of 1 : 10 and 1 : 50 were added in 0. I-ml volumes. The cellular responses were analyzed as above. Statistical Analysis. The z test (42) was used to compare rates of PHA stimulation (S1 2 10) in the SLE and control groups. Cpm incorporated in unstimulated cultures were analyzed by the student's t test (42). The responses to individual virus antigens and the number of antigens to which there was a positive response were compared by x 2 analysis (42). Nonparametric multiple group comparison by the Kruskal-Wallis test (42) was used to assess the response of SLE lymphocytes in autologous and A B plasma. RESULTS Response of Whole Blood Cultures to PHA. I n order t o evaluate nonspecific lymphocyte responses, whole blood samples from 15 SLE patients a n d 15 controls were cultured with PHA, a n d incorporation of 3Hthymidine was determined. A stimulation index (SI) equal to or greater than 10 was observed in 93% of the controls but in only 46% o f t h e SLE patients. This difference w a s highly significant ( P < 0.001). These and other differences were not d u e t o increased incorporation of 'H-thymidine in unstimulated SLE cultures, because n o significant difference was found between the mean c p m incorporated in unstimulated cultures of the control (510 f 146) a n d SLE (433 f 81) groups. Response of Whole Blood Cultures to Virus Antigens. Responses to virus antigens were determined in parallel with the PHA stimulation studies. T h e number a n d percentage of SLE patients with positive responses (SI 2 2.0) when whole blood cultures were incubated with eight virus antigens are presented in Table 3. T h e SLE patients were significantly less responsive than control individuals to rubella, measles-CF, varicella-zoster, a n d parainfluenza types 1, 2, a n d 3 antigens. No significant differences were observed with measles-HA and herpes virus type 1 viruses. In the case of the measlesHA antigen, t h e failure t o observe a significantly decreased incidence of response in SLE patients appeared to be due t o the controls' decreased frequency of response to this antigen. In the case of the herpes antigen, lymphocytes from most individuals in both t h e SLE and control groups were stimulated. Specificity of stimulation by t h e virus antigen preparations was indicated by the absence of differences in incidence of positive responders to varicella-zoster a n d herpes virus, when medium controls or tissue culture controls were used to calculate t h e SI (Table 3). In addition, specificity was suggested by discordant responses to the two measles antigens in 6 of 13 responsive individuals a n d to the three parainfluenza antigens in 12 of 21 responders. In addition t o t h e occurrence of hyporesponsiveness t o most virus antigens individually, SLE patients also responded t o fewer antigens than did the controls Table 3. Stimulation of Lymphocytes of S L E Patients and Controls by Virus Antigens Group SLE No. positive$ per no. tested Control No. positive per no. tested p9 Rubella HA Measles HA Measles CF Varicella CF* Herpes CFt Paraflu-l HA Paraflu-2 HA Paraflu-3 HA 3/15 2/15 2/15 3/15 9/14 4/15 3/15 5/15 12/15 7/15 9/15 12/14 12/13 11/14 9/14 12/14 0.003 0.1 I 0.02 0.001 0.19 0.015 0.04 0.01 * Two of 15 SLE patients and 13 of 15 control individuals were positive when SI = cpm stimulated cultures/cpm tissue culture control cultures. I'No difference in the number positive when SI was calculated with medium or tissue culture controls. $ Stimulation index 2 2.0; SI = cpm stimulated cultures/cpm medium control cultures. 4 xz test. WOLF AND ZIFF 1274 8r had received either antiinflammatory or immunosuppressive drugs before the study. The mean SIs in both autologous plasma and AB plasma were in the nonstimulatory range (<2.0) for all four virus antigens. Although the mean values tended to be higher for cells cultured in autologous plasma than in AB plasma, the differences were not statistically significant for any of the virus antigens by multiple group comparison based on Kruskal-Wallis nonparametric analysis. a " 4- DISCUSSION 0 1 2 3 4 5 6 7 8 NUMBER OF ANTIGENS RESPONDED TO Fig 1. Number of viral antigens to which lymphocytes from SLE patients (solid bars) and control individuals (cross-hatched bars) showed positive responses. Each bar represenrs the number of subjecrs having an SI 2 2.0 to each of the eight viral antigens tested. (Figure I ) . These differences were highly significant (P < 0.001). Among the SLE patients, 1 1 responded to less than three virus antigens, and 4 to three or more antigens. Among the controls, on the other hand, only 1 responded to less than three antigens, and 13 responded to three or more antigens. Effects of Prednisolone on Responsiveness to Virus Antigens. Because 8 of the 15 SLE patients had received some prednisolone, the possible effects of therapy on responsiveness were also analyzed. The SLE patients were divided into two treatment groups: a ) 7 patients who had received no prednisolone, and b ) 8 patients who were taking prednisolone. Table 4 shows that hyporesponsiveness was observed in both untreated and treated patients with no significant difference between them. Thus the decreased responses in the SLE patients as a group did not appear to result from the effects of prednisolone on the lymphocytes. Because 14 of the 15 patients had active disease, it was not possible to correlate hyporesponsiveness to virus antigens with disease activity. Responses of Washed S L E Cells in Autologous and Normal AB Plasma. The responses of washed peripheral blood cells from 8 additional patients with active SLE to rubella, measles-CF. parainfluenza type 1 , and parainfluenza type 3 virus antigens were simultaneously compared in fresh autologous plasma and fresh AB plasma to evaluate the influence of serum factors such as virus antibody or antilymphocyte antibodies (Table 5 ) . All patients were clinically active, and none The cellular immune response of patients with SLE to rubella, measles, parainfluenza types 1, 2, and 3 RNA viruses, and varicella-zoster and herpes virus type 1 DNA viruses was evaluated in a whole blood culture technique that used incorporation of 3H-thymidine as a measure of blastic transformation. Decreased responses were detected with rubella, measles-CF, parainfluenza types I , 2, and 3, varicella-zoster virus antigens, and PHA, but not with measles-HA and herpes virus type 1 antigens. SLE patients also responded to significantly fewer antigens than did the controls. These results are consistent with a generalized hyporesponsiveness of SLE lymphocytes to virus antigens and are in accord with the reported decrease of cellmediated immunity (CMI) to plant mitogens and common bacterial and fungal antigens (16-29) in SLE, rather than implicating any of these viruses as a specific pathogen. Moreover, it cannot be concluded from the data presented that the hyporesponsiveness to virus antigens was peculiar to SLE, because other chronic inflammatory diseases were not studied. Several phenomena that might contribute to the observed hyporesponsiveness of SLE patients to virus antigens were evaluated. The administration of corticosteroids may decrease the number of circulating T lymTable 4. Relationship of Prednisolone Dose to Response of SLE Patients' Lymphocytes to Viral A ntigens Therapy No. of Antigens Responded To Not Receiving Prednisolone* Receiving Prednisolonet I 2 23 5 2 6 2 * One patient received aspirin, 650 mg q 4 hours. None was receiving other antiinflammatory or immunosuppressive drugs. t Six patients were taking 10-20 mg/day. Two had received I or 2 doses of 6 0 mg at the time of initial diagnosis. A blood sample was drawn 16 and 18 hours after last dose. LYMPHOCYTE RESPONSE IN SLE 1275 Table 5 . Responses of Lymphocytes from SLE Patients t o Virus Antigens in Whole Blood Cultures with Autologous and Normal A B Plasma Mean SI* f SE Plasma Source Rubella Measles-CF Parainfluenza Type I Parainfluenza Type 3 Autologous I .7 f 0.5 0.9 f 0.1 NSt 1.9 f 0.4 I.OfO.l NS I .4 f 0.2 0.9 f 0.1 NS I .6 f 0.3 0.9 f 0.I NS AB P * SI: stimulation index. t NS: not significant by multiple group comparison based on Kruskal-Wallis nonparametric analysis. phocytes (43,44) and alter in vitro correlates of CMI (45,46). In the present study the administration of prednisolone did not appear to be responsible for the decreased response of SLE lymphocytes to virus antigens, because equally depressed responsiveness was detected in both treated and untreated patients. A similar lack of relationship between corticosteroid therapy and hyporesponsiveness to mitogens or bacterial and fungal antigens in vitro and in vivo has been reported (27-29,33). In fact, Rosenthal and Franklin (29) have reported recovery of CMI in SLE patients receiving corticosteroid therapy by in vitro and in vivo parameters. Both cytotoxic (47-49) and noncytotoxic (50) antibodies which may be T-cell specific (51) have been detected in the serum of SLE patients. These and other possible SLE serum factors may nonspecifically decrease in vitro CMI responses of normal lymphocytes to mitogens (26,33,37) and allogeneic leukocytes (36,37,52), and they may impair spontaneous rosette formation with sheep erythrocytes (33). In addition, elevated virus antibody titers in autologous SLE plasma may have neutralized the virus antigens. In order to evaluate the influence of virus antibody or antilymphocyte antibody on the response to virus antigens, the cells were processed at room temperature and then washed three times with warm (37°C) medium prior to culture in the presence of AB serum from a normal donor. The failure to increase the responsiveness of lymphocytes by this treatment argues against a significant role for serum factors in the observed hyporesponsiveness to virus antigens. Winchester et a1 (50) have reported a marked reduction of cold-reactive, surface-bound immunoglobulin after washing at 37"C, and Wernet and Kunkel (52) have demonstrated at least a partial recovery of response to allogeneic leukocytes by SLE responder lymphocytes in AB serum, whereas total suppression of reactivity was observed in autologous serum. Because overnight incubation before culture has been reported to alter murine lymphocyte subpopulations (53), the washing procedure used avoided this potential source of error. Utermohlen et a1 (39), using a leukocyte migration inhibition assay, observed a decreased response of CMI to measles-CF antigen but not to rubella and parainfluenza type 1 antigens in SLE patients. These investigators raised the possibility that the decreased reactivity to measles antigen might have been indicative of a generalized defect of CMI. This interpretation is consistent with the results of the present study. The fact that negative responses to rubella and parainfluenza type 1 antigens were observed in the present study and not by Utermohlen and coworkers may be attributable to differences in the method of evaluating responses to virus antigens. In the present study CMI was measured by 3Hthymidine uptake of lymphocytes in culture, whereas Utermohlen et a1 measured inhibition of leukocyte migration. Specificity of the responses to the virus antigens and not to constituents of the culture media employed was indicated by two observations. First, herpes virus type 1 and varicella-zoster antigens showed no significant differences in the number of positive responders when uninfected tissue culture control material was compared with medium controls to calculate the stimulation index in the case of these two antigens. Second, although supernatants containing the measles-CF and measles-HA antigen were both obtained from infected primary human amnion cells, stimulation indices of these two antigens were discordant. Parainfluenza antigens were all obtained from infected primary monkey kidney cells, but the responses to these three antigens were also discordant. The present findings of a diffuse hyporesponsiveness of lymphocytes from SLE patients to virus antigens appear to be best explained by a basic functional defect of the lymphocytes of these patients, rather than by the WOLF A N D ZIFF 1276 effects of serum factors or therapy. Utsinger (27) a n d Lockshin et af (28) h a v e reached a similar conclusion regarding t h e response of SLE lymphocytes to mitogens. 16. ACKNOWLEDGMENT The authors thank Ms. Olivia White for her very capable technical assistance and Ms. Terrie Harris for manuscript transcription. REFERENCES I . Gyorkey F, Min KW, Sincovics JG, et al: Systemic lupus erythematosus and myxovirus. N Engl J Med 280:333, I969 2. Hurd ER, Eigenbrodt E, ZiK M: Cytoplasmic tubular structures in kidney biopsies in systemic lupus erythematosus. Arthritis Rheum 12541-542, 1969 3. Kwano K, Miller L, Kimmelstiel P: Virus-like structures in lupus erythematosus. N Engl J Med 281:1228-1229, 1969 4. 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