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ASPIRIN AND DELAYED TYPE HYPERSENSITIVITY MATTHEW W. DUNCAN, DONALD A. PERSON, ROBERT R. RICH, and JOHN T. SHARP The effects of aspirin on delayed hypersensitivity were assessed in 40 healthy subjects who were randomly assigned to two equal groups. In a double-blind format, the individuals in one group were placed on 4 gm aspirin daily for 5 days, and individuals in the other group were given placebo. Lymphocyte proliferation was measured before and 72 hours after the initiation of drug, by using three mitogens and four antigens. The percentages of T and B lymphocytes were likewise measured before and during therapy. The subjects were skin tested with the same four antigens 72 hours after the initiation of drug, and the skin tests were read 48 hours later. No significant differences between the two groups were detected when skin tests, lymphocyte proliferation, and percentage of T lymphocytes were compared. From the Departments of Internal Medicine, Virology, Epidemiology, and Microbiology and Immunology, Baylor College of Medicine, Texas Medical Center, Houston, Texas. Supported in part by the Paul Kayser Foundation, The Arthritis Foundation and USPHS Grants RR-00350 and HL-17269. Dr. Person is a Senior Investigator of The Arthritis Foundation and Dr. Rich is the recipient of USPHS Research Career Development Award I-KO4-Al00006. Matthew W. Duncan, M.D.: Fellow in Rheumatology, Department of Medicine (presently at the Alexian Brothers Medical Clinic, San Jose, California): Donald A. Person, M.D.: Assistant Professor of Internal Medicine, Virology, and Epidemiology: Robert R . Rich, M.D.: Associate Professor of Microbiology. Immunology, and Medicine, and Acting Head of the Immunology Section: John T. Sharp, M.D.: Professor of Internal Medicine and Chief, Rheumatic Disease Section (presently Professor of Clinical Sciences (Medicine), University of Illinois College of Medicine, School of Basic Medical Sciences at Urbana-Champaign. Urbana, Illinois. and Chief of Medicine. Danville Veterans Administration Hospital, Danville, Illinois). Address reprint requests to Donald A. Person, M.D., Baylor College of Medicine. Houston, Texas 77030. Submitted for publication January 13, 1977: accepted March 2, 1977. Arthritis and Rheumatism, Vol. 20, No. 6 (July-August 1977)

It was recently observed that 20% of patients with rheumatoid arthritis were anergic to a battery of six skin test antigens (1). Since most of these patients were taking salicylates, the question arose whether ingestion of aspirin could have depressed delayed skin reactions. We were further interested in correlating the effect on skin test reactions with the effect on in vitro lymphocyte proliferation, because the former has been used traditionally as the definitive measure of cellular immunity and because there are reports that aspirin affects lymphocyte proliferation (2-7). This correlation was especially important because of conflicting reports in the literature about the effects of ingested aspirin on in vitro lymphocyte function (6-10). In spite of these previous studies, the crucial question remained: Does the ingestion of therapeutic doses of aspirin suppress cell-mediated immunity in vivo? It will be shown here that the ingestion of 4 gm of aspirin daily for 5 days did not affect delayed type hypersensitivity.

MATERIALS AND METHODS Study Plan. Forty healthy students in the third decade of life were randomly assigned to two groups of 20. Sixteen of the participants were female with a mean age of 23.9 years (range 21-30) and 24 were male with a mean age of 24.3 (range 21-29). All were Caucasian except for 1 Oriental woman. The participants were advised about the experimental nature of the study, consent forms were signed, and the study protocol had the prior approval of the Baylor Institutional Review Board for Human Research. Twenty subjects were placed on aspirin at a dose of 1 gm four times daily for 5 days, and the other 20 received placebo (lactose, hydrous, USP). The aspirin and placebo were dis-

ASPIRIN AND HYPERSENSITIVITY

pensed in capsules, were identical in appearance, and were coded by one investigator who did not participate in the skin testing or in dispensing the capsules. The code was not broken until all studies, including skin test results, lymphocyte tests, salicylate blood levels, and t test analyses, were completed. On the first day of the study, before drug ingestion, blood was drawn for in vitro lymphocyte studies. Seventy-two hours after the drug or placebo was started, blood was again drawn for repeat lymphocyte studies, and the plasma was stored for determination of blood salicylate studies. At that time (72 hours) the skin test antigens were injected, and 48 hours later the skin tests were read, the medication was stopped, and the study was terminated. Skin Testing. The skin test antigens included Streptokinase-Streptodornase (SK/SD, Varidase, control no. 359528, Lederle Laboratories, Pearl River, NY) 200 units and 50 units/ml respectively; Mumps Skin-Test Antigen (control no. 8FL49A, containing at least 20 complement-fixing units/ml, Eli Lilly, Indianapolis, IN) diluted 1 :80 of the original stock solution; Dermatophytin (Trichophyfon gypseum, T purpureum, and T interdigitule, lot no. 161595IM, Hollister-Stier Laboratories, Spokane, WA) diluted 1 :200 of the original stock; and Dermatophytin “0” (Cundidu [moniliu] ulbicans, lot no. 56 1631 IM, Hollister-Stier Laboratories) diluted 1 :200 of the original stock. All dilutions were made with sterile saline. The skin tests were administered by intradermal injection of 0.1 ml of each antigen into the volar aspect of the forearms-two antigens on the right and two on the left. Forty-eight hours later, two diameters of induration at each test site were measured. In the case of the mumps skin test, two diameters of erythema were measured in addition to induration. Lymphocyte Proliferation. Thirty to forty milliliters of blood were drawn, mixed with preservative-free heparin (approximately 10 IU/ml blood) and centrifuged at 900 X g for 15 minutes. The buffy coat was removed and layered over sodium metrizoate/Ficoll solution at a density of 1.077 g/ml (Lymphoprep, Nyegaard & Co, Oslo, Norway) and centrifuged at 900 X g for 30 minutes at ambient temperature. The band of mononuclear cells at the interface between the plasma and Lymphoprep was collected and the cells were washed twice in Hanks’ balanced salt solution. An aliquot of the cells was suspended in medium TC 199 with 20 mM HEPES buffer (Microbiological Associates, Bethesda, MD) and 10% fetal calf serum (FCS) for assessment of sheep erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosettes. The remaining cells were suspended in medium TC 199 with 20% heat-inactivated, pooled AB positive plasma containing sodium bicarbonate (0.75 g/L) and gentamicin (50 pg/ml). Cells were cultured in microculture plates (Linbro, 1S-FB-96-CT, International Scientific Industries Inc, Cary, IL) in quadruplicate groups in 0.2 ml medium at a concentration of 2.5 X 1od cells/ml. Cultures were incubated at 37°C in a humidified 5% C0,-95% air incubator for 4 days, at which time each well was pulse labeled with 1 pCi of tritiated thymidine ([3H]TdR, sp act 2.0 Ci/mM, New England Nuclear, Boston, MA). The plates were reincubated for 16 hours, the cells were harvested by aspiration onto glass fiber filters, and radioactivity was assayed in a liquid scintillation spectrometer. Net counts per minute (Acpm) were calculated by subtracting

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mean cpm in control cultures from mean cpm in mitogen- or antigen-stimulated cultures. The mitogens tested included phytohemagglutinin-P (PHA, 1 pg/ml, Wellcome Reagents Limited, Beckenham, England), concanavalin A (Con A, 1 pg/ml, Nutritional Biochemicals Corporation, Cleveland, OH), and pokeweed mitogen (PWM, 1 pg/ml, Grand Island Biological Company, Grand Island, NY). The same antigens used for skin tests were used for lymphocyte studies after exhaustive dialysis against phosphate-buffered saline (pH 7.4,0.007 M phosphate in 0.15 M NaCI) and filtration through a 0.45 millipore membrane (Millipore Corp, Bedford, MA). The antigens were stored in aliquots at -20°C before use with the lymphocyte cultures. The final antigen concentrations in tissue culture were SK/SD, 40 U/10 U and 8 U/2 U; Dermatophytin “0”,1: 100 and 1 :400; mumps antigen, 1 :20; and Dermatophytin, 1 : 100. Lymphocyte Rosettes. The percentages of B and T lymphocytes were determined by using sheep red blood cell (SRBC) rosetting techniques (1 I). The percentage of T cells was estimated by using E rosettes. Two million lymphocytes in 0.2 ml of TC 199 with 10% FCS were mixed with 0.2 ml of washed 1% SRBC and centrifuged at 200 X g for 5 minutes. The pellets were undisturbed and the tubes were stored in an ice water bath at 4°C overnight. On the next day the pellets were gently resuspended and the rosettes were counted in a hemocytometer. A minimum of 200 lymphocytes was counted, and a positive rosette was scored when three or more SRBCs were attached to a lymphocyte. The number of B cells was determined by the number of positive EAC rosettes. One milliliter of a 5% suspension of washed SRBC was mixed with 1.0 ml of a 1: 100 dilution of rabbit IgM antiSRBC (Cordis Laboratories, Miami, FL) and incubated 30 minutes in a 37°C water bath. The cells were washed two times and resuspended in 1.O ml of 0.15 M NaCI. An equal volume of undiluted mouse complement was added and the mixture was incubated for 30 minutes at 37°C. The cells were again washed two times and finally resuspended in 10 ml TC 199 with 10% FCS. An equal volume of EAC was added to 0.2 ml media containing 1 X l(P lymphocytes. The suspension was incubated at 37°C for 2 hours in a water bath with periodic mixing, then centrifuged at 200 X g for 5 minutes at 4°C. The pellets were then resuspended and the EAC rosettes were counted. These methods accounted for the identification of 90-93% of the lymphocytes and their classification as either T cells (mean 73.4%) or B cells (mean 18.1%). Salicylate Level. The plasma salicylate level from the 72-hour blood was estimated by the colormetric method of Trinder (12). Statistical Methods. The t statistic for two means, linear regression, and x2 analyses were performed on a programmable calculator (HP65, Hewlett-Packard Company, Cupertino, CA).

RESULTS There were no significant differences in the magnitude of delayed hypersensitivity responses between placebo and aspirin groups after 120 hours of drug

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pared before and after 72 hours of drug ingestion, before the injection of the skin test antigens (Figure 2). Each group of 20 subjects thus served as its own control. The results at zero time did not differ significantly from the results after 72 hours of drug within either group. When the two groups were compared after 72 hours of drug ingestion, the proliferative response of the placebo group was significantly less than the response of the aspirin group to Dermatophytin (t = -2.67, df = 37, P < 0.02). However this difference is difficult to interpret because only 2 in the placebo group had positive skin tests to Dermatophytin in contrast with 5 in the aspirin group. More important was the fact that aspirin had no inhibitory effect on the proliferative response to Dermatophytin. No significant differences were observed in the responses of either group to the mitogenic effects of PHA, Con A, or PWM (Figure 3). There was no evidence that aspirin affected the percentage of T lymphocytes. There was a small but statistically significant increase in B cells from 16.8 f 1.13% (mean f SEM) before, to 21.33 f 0.87% after 72 hours of aspirin ingestion ( t = -3.18, df = 36, P < 0.005). The mean salicylate blood level was 0.47 f 0.37 mg/dl (range 0-1.8 mg/dl) for subjects on the placebo, and 14.34 f 4.92 mg/dl (range 7.75-28.2 mg/dl) for subjects on aspirin. The design of this study provided an opportunity to test the correlation between in vivo skin test reactivity and in vitro lymphocyte proliferation in response to the

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Fig 1. Skin test reaciions to the four aniigens (mean diameter f S E M ) in the two groups. Open bars are the placebo group and solid bars ihe aspirin group. The number of positive skin iesis is shown below each bar. The mean diameiers of eryihema at ihe mumps skin siie were not significantly differeni (14.2 f 1.5 mm us 14.1 f 2.1 mm).

therapy (Figure 1). All individuals in both groups had positive skin tests to mumps antigen. The number of positive tests with the other antigens was consistent with the established frequency of exposure to the antigens and not different between the two groups. In the assessment of lymphocyte pro!iferation to the same skin test antigens, the two groups were com35

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ASPIRIN A N D HYPERSENSITIVITY

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test antigens as two related measurements of cell-mediated immunity. Multiple linear regression analyses of skin test reactivity (mean induration in mm) and lymphocyte proliferation (Acpm) for each of the skin test antigens at all dilutions were performed, and no significant correlations were found with the exception of SK/SD. In this instance the in vitro reactants at both 1 : 10 and 1 :50 dilutions gave low but significant correlation coefficients (r = 0.32 and r = 0.38 respectively). When skin test reactivity was compared to lymphocyte proliferation in terms of positive and negative results, a correlation was found. For example, the results with SK/SD were concordant in 93%of the tests. The overall concordance for all antigens was approximately 70%(x' = 20.35, P < 0.0005).

DISCUSSION The main purpose of this study was to determine what effect, if any, ingested aspirin has on delayed type hypersensitivity. As noted above, the classic method of assessing delayed type hypersensitivity has been to measure delayed skin reactions to ubiquitous skin test antigens. We previously observed that 20%of patients with rheumatoid arthritis were anergic when skin-tested with six antigens. Those results were highly significant when compared with 2% anergy in matched controls (1). Because most patients with rheumatoid arthritis ingest significant quantities of salicylates and because the literature contains conflicting reports about the effects of

1177 aspirin on lymphocyte function in vitro (2-lo), it was essential to establish whether aspirin does or does not affect delayed type hypersensitivity. Ideally, one should skin test each individual before and during aspirin and placebo therapy. With such a study plan, each individual would serve as his or her own control. This method was not used because it was considered possible that pretreatment antigenic challenge could influence responses to the second test on aspirin or placebo. As an alternative it was reasoned that if enough persons were randomly assigned to test or control groups, the effect of treatment could be detected. T o prevent bias the study was conducted in a double blind fashion. With this study design, aspirin administered for 3 days before skin testing (and continued until the tests were read 48 hours later), in doses large enough to produce blood levels between 7.75 and 28.2 mg/dl, did not affect the skin test results. It was further shown that the ingestion of aspirin did not affect in vitro lymphocyte proliferation in response to the skin test antigens, and did not affect the mitogenic affects of PHA, Con A, and PWM, or the percentage of T lymphocytes. A number of investigators have reported the inhibitory effects of aspirin added directly to lymphocyte cultures. The suppressive effects included inhibition of protein and DNA and RNA synthesis (3,4), diminished proliferative responses to PHA (2,4-7), Con A, and PWM (6,7), and decreased response to antigenic stimuli, both microbial (4,6,7) and HLA-D antigens as assessed in the mixed lymphocyte reaction (5,7). These effects of aspirin have been reported to be dose-dependent and reversible by washing (5-7). Although there has been considerable speculation about the mechanism of the in vitro effects of aspirin on lymphocytes, the factors responsible for the action of aspirin have not been defined. Opelz et a1 suggested on the basis of in vitro observations that aspirin might suppress cellular immune responses in vivo (5). We contend that the only test for such an effect is that described here, namely delayed type skin testing. Nevertheless, in an attempt to explore their suggestion, several investigators have studied the effects of ingested aspirin on in vitro lymphocyte function and the results have been conflicting (6,8-10). Crout et a1 have reported suppression of lymphocyte proliferation to PHA and PWM after aspirin ingestion in 19 normal volunteers (8). Panush and Anthony also reported suppression of lymphocyte proliferation to antigens and mitogens in 2 normal individuals taking therapeutic doses of aspirin (6). In contrast, two groups have reported no suppression of lymphocyte proliferation in vitro by the prior ingestion

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of aspirin (9,lO). Yazici et a1 reported that aspirin did not affect PHA-stimulated lymphocyte proliferation (9), and in a carefully controlled study, Smith et a1 reported no suppression of lymphocyte reactivity after the administration of aspirin for 14 days (10). The results reported here are in accord with this latter report. Without detailing the many vagaries of lymphocyte culture techniques, it is generally recognized that substantial differences may be observed in the absolute responsiveness of lymphocytes from the same individual obtained and cultured on different days. This technical problem, which may lead to inappropriate conclusions, was obviated in the latter study (10) and in the study reported here by comparing proliferative responses of statistically adequate populations of drugtreated and control subjects tested in a single day. In contrast, studies reporting suppression of lymphocyte responses by aspirin ingestion were performed by comparing lymphocyte responses on different days (6,8). We conclude that ingestion of aspirin for 3 days does not affect the proliferation of washed lymphocytes stimulated by mitogens or antigens. Although it has been reported that salicylate present in cell culture inhibits lymphocyte proliferation (2-7), the relevance of this observation to the effect of aspirin in vivo is not clear. The effect of ingested aspirin on in vitro lymphocyte function, if any, is apparently completely reversible. We considered setting up lymphocyte cultures by using autologous plasma. But since lymphocyte proliferation tests are commonly performed in 10 or 20% plasma, even this procedure would not have given conclusive data if proliferation was not inhibited, because the salicylate level in the lymphocyte cultures would have been one-fifth to one-tenth that contained in whole plasma. Performing the tests in 100% plasma was not seriously considered because serum concentrations from 25 to 50% have been shown to inhibit the labeling of lymphocyte DNA (13). More important, short-term aspirin ingestion in therapeutic doses sufficient for substantial antiinflammatory and antipyretic effects had no effect on skin tests manifesting delayed type hypersensitivity. Furthermore, we suggest that the anergy observed in patients with rheumatoid arthritis

(1) cannot be explained solely on the basis of salicylate

ingestion.

ACKNOWLEDGMENTS The authors acknowledge the expert technical assistance of Juanita McKeever, Chris Arhelger, and Carolyn Leatherwood.

REFERENCES 1. Andrianakos AA, Sharp JT, Person DA, et al: Cell me-

diated immunity in rheumatoid arthritis. Ann Rheum Dis 36: 13-20, 1977 2. Gantner G E Jr, Zuckner J: Salicylate suppression of lymphocyte transformation. Arthritis Rheum 8:443, 1965 3. Forbes IJ, Smith JL: Effects of anti-inflammatory drugs on lymphocytes. Lancet 2:334-337, 1967 4. Pachman LM, Esterly NB, Peterson RDA: The effect of

salicylate on the metabolism of normal and stimulated human lymphocytes in vitro. J Clin Invest 50226-230, 1971 5. Opelz G, Terasaki PI, Hirata AA: Suppression of lymphocyte transformation by aspirin. Lancet 2:478-480, 1973 6. Panush RS,Anthony CR. Effects of acetylsalicylic acid on

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8.

9.

10.

11.

12. 13.

normal human peripheral blood lymphocytes. Clin Exp Immunol 23:114-125, 1976 Panush RS: Effects of certain antirheumatic drugs on normal human peripheral blood lymphocytes. Arthritis Rheum 19:907-917, 1976 Crout JE, Hepburn B, Ritts R E Jr: Suppression of lymphocyte transformation after aspirin ingestion. N Engl J Med 292:221-223, 1975 Yazici H, Saville PD, Chaperon ED. Aspirin suppression of delayed hypersensitivity. Clin Res 22:645A, 1974 Smith EM, Hoth M, Davis K: Aspirin and lymphocyte transformation. Ann Intern Med 83:509-51 I, 1975 Aiuti F, Cerottini JC, Coombs RRA, et al: International Union of Immunological Societies (IUIS) Report of July 1974. Identification, enumeration, and isolation of B and T lymphocytes from human peripheral blood. Clin Immuno1 Immunopathol 3:584-597, 1975 Trinder P: Rapid determination of salicylate in biological fluids. Biochem J 57:301-303, 1965 Forsdyke DR: Serum factors affecting the incorporation of (*H) thymidine by lymphocytes stimulated by antigen. I. Serum concentration. Immunology 25583-595, 1973