The influence of therapy with azathioprine and prednisone on the immune system of kidney transplant recipients

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The Influence of Therapy with Azathioprine and Prednisone on the Immune System of Kidney Transplant Recipients R. J. M. TEN BERGE,

P. TH. A. SCHELLEKENS, S. SURACHNO.:‘: J. H. TEN VEEN.” AND J. M. WILMINK”:

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In a group of IS kidney-transplant recipients, the effect of maintenance immunosuppressive therapy with azathioprine and low doses of prednisone on cellular and humoral immunocompetence was studied. These patients had undergone transplantation at least I year prior to the study and had a stable renal function during the last 3 months. A control group consisted of healthy individuals matching those of the patient group in age and sex. In the patients. the number of T and B lymphocytes in peripheral blood did show a significant decrease. Antibody-dependent lymphocyte cytotoxicity (K-cell function) was depressed. Specific antibody responses after primary and secondary immunization in \Y,o were not affected. nor was cellular immune reactivity in vifro. However, primary and secondary delayed-type skin reactivity to antigens was severely depressed. From these data, it seems unlikely that these drugs exert their main immunosuppressive effect through inhibition of antigen recognition or of the proliferative phase of the immune response. Rather. their immunosuppressive action in ,Y\~~~ depends on some other mechanism. probably on an anti-inflammatory effect.

INTRODUCTION The immunosuppressive effects of azathioprine and prednisone have been extensively studied in experimental animals (for review, see (1)). However. the mechanism of action of these drugs on the immune system in human beings has not yet been studied in detail and particularly did not include the necessary control groups. Thus, the data reported so far, appear still inconclusive, probably due to the heterogeneity of the groups of patients with respect to disease and treatment. Furthermore, most investigators have studied only one aspect of the immune response, for example, only the humoral immune response (2) or only secondary responses, without considering primary responses (3). Treatment with azathioprine and prednisone is widely used for a variety of diseases with the aim to achieve immunosuppression. Therefore, it is of great importance to obtain more insight in the nature and the degree of immunosuppression brought about by these drugs. In this study, we have tried to avoid the above-mentioned drawbacks. We selected kidney-transplant recipients who were in good general condition, with a good and stable renal function and who, apart from the immunosuppressive therapy. did not receive any other medication that might possibly influence the immune system. Therefore, in our patient group, any aberrations in the immune system were probably only due to the immunosuppressive treatment. Moreover. 20 0090Copwght All

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IMMUNOSUPPRESSIVE

DRUGS AND IMMUNE

RESPONSE

21

our study included a group of healthy, untreated control individuals, who were matched with the patients as to age and sex. By comparing these groups, we have investigated the effects of the immunosuppressive drugs on cellular and humoral immune responses, both primary and secondary, against well-defined antigens in ~~ivoas well as in vitro. SUBJECTS

From the 15 patients, included in this study, informed consent was obtained. They had received a kidney allograft at least one year ago. All selected patients had a stable renal function for at least the last 3 months before initiation of the study, with a creatinine clearance varying from 63 to 160 ml/min (mean: 101 mYmin.). Their age varied from 27 to 61 years (median: 39 years). During the first 3 months following transplantation, they had received azathioprine, 135 mg/kg, and prednisone 165 mg/kg, (total cumulative doses). Thereafter, they received azathioprine in a mean dose of 2.5 mg/kg/day and prednisone in a mean dose of 30 mg/kg/day; the dose of the latter was gradually tapered off. During the last 6 months or longer prior to the time of this study, they were treated with a daily dose of azathioprine, varying from 2 to 2.5 mg/kg and a daily dose of prednisone, varying from 0.12 to 0.3 mg/kg. All patients were in good health and did not receive other possibly immunosupressive drugs. None of them was splenectomized. The already mentioned control group consisted of 15 healthy individuals, matched with those of the patient group as to age and sex. GENERAL OUTLINE OF STUDY

All patients and control individuals were studied according to an identical scheme. After blood collection, each individual was immunized with hemocyanin from a-Helix pomatia and DNCB’ to elicit a primary immune response, and with diphteria, tetanus toxoid, and polio vaccine to evoke secondary immune responses. After 14 days, blood was drawn again and skin tests were performed against DNCB and a variety of recall antigens (PPD, varidase, mumps, trichophyton and candida). After another 48 hr, skin tests were read. MATERIALS

AND METHODS

Blood Samplrs

Serum obtained from each individual was frozen and kept at -20°C. Mononuclear cells were isolated from de&brinated blood by means of Ficoll-Isopaque density gradient centrifugation (4) and preserved in liquid nitrogen (5). Lymphocyte cultures of one patient and the matched control individual were performed in ’ Abbreviations used: ADL, antibody-dependent lymphocytotoxicity: ALS. antilymphocyte serum: CML, cell-mediated lympholysis; Con A, Concanavalin A; DNCB. dinitrochlorobenzene; DTH, delayed-type hypersensitivity: DTP-vaccine. a vaccine containing diphtheria toxoid, tetanus toxoid. and killed polio virus, strains I, II, and III: E-RFC. lymphocytes forming rosettes with sheep erythrocytes: EAT-RFC, lymphocytes forming rosettes with sensitized human erythrocytes; ELISA, enzyme-linked immunosorbent assay; Fc-yR, receptor for the Fc fragment of IgG: FITC, fluorescein isothiocyanate: Ig, immunoglobulin: K cell, killer cell; KLH, keyhole limpet hemocyanin; MLC. mixed lymphocyte culture; 6MP. 6mercaptopurine: NDMA. paranitrosodimethyl aniline; PHA. phytohemagglutinin: PPD. purified protein derivative of tuberculin: PWM, pokeweed mitogen.

71 --

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one experiment on the same day, thus avoiding phocyte cultures. Cell Numbers

in Peripheral

41~.

day-to-day variations

in the lym-

Blood

The absolute numbers of lymphocytes and monocytes in peripheral blood were determined by electronic cell counting (Coulter counter) and May-GrtinwaldGiemsa-stained blood smears. T lymphocytes were determined both by rosette formation with sheep erythrocytes (6) and by an indirect immunofluorescence test with a heterologous anti-T-cell serum prepared in our institute. B lymphocytes were determined by a direct immunofluorescence test with F(ab’), fragments of a rabbit-IgG anti-human-Ig F(ab’),, labeled with FITC (7). Fc receptors for IgG were determined by rosette formation with human Rh-D erythrocytes, sensitized with IgG-anti-D antibodies (8). Serum Proteins

The total serum-protein concentration and serum-protein spectrum were determined by standard methods. The levels of IgG, IgM, and IgA were determined by a nephelometric technique, and those of IgD and IgE by a radioimmunoassay. The levels of complement components were determined by radial immunodiffusion. Specific

Humoral

Immune

Responses

To determine the capacity to mount a primary antibody response, each individual was immunized subcutaneously with 1.0 mg hemocyanin (cll-hemocyanin of Helix pomatia) (9). Fourteen days later, specific antibodies to this antigen in the IgM, IgG. and IgA classes were measured by indirect ELISA, as described by Weits et al. (10). To study secondary antibody responses, 1 ml of a DTP vaccine, containing diphtheria toxoid, tetanus toxoid, and killed polio virus, was injected intramuscularly. The antibody responses were measured 14 days later. The total antibody titer against diptheria toxoid was measured by ELISA. Total antibodies and those of the IgG class against tetanus toxoid were determined by radioimmunoassay. The IgM- and IgA-anti-tetanus antibodies were estimated through a semiquantitative Ouchterlony technique combined with autoradiography ( 11). Anti-polio virus antibodies were measured by virus neutralization. Cellular

immune

Reactivity

in Vitro

In vitro lymphocyte transformation tests. After thawing, viability of the cell suspensions used for the experiments was determined by trypan blue exclusion. Viability was always higher than 90%. The number of mononuclear cells available was not always sufficient to perform all the in vitro tests mentioned below. Cultures were performed in microtiter plates (12), containing 30,000 lymphocytes per well in 150 ~1 of medium supplemented with 20% pooled, human heatinactivated serum, and standard antibiotics. The following stimulants were used (1) The nonspecific mitogens PHA (Wellcome), final concentration: 50 pg/ml; horse-anti-human ALS, 1:4; Con A (Sigma), final concentration: 120 pg/ml; PWM (Gibco), final concentration: 100 puglml.

IMMUNOSUPPRESSIVE

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13

(2) The antigen a-Helix pomatia hemocyanin, final concentration: 50 pglml(9): (3) An antigen cocktail, consisting of a mixture or the following antigens: PPD. final concentration: 100 &ml; varidase, final concentration: 100 E/ml; mumps, final concentration: 0.08 cfu/ml; trichophyton, final concentration: 2%: candida, final concentration: 1:200 (13); (4) Irradiated allogeneic mononuclear cells in the MLC test, using cells from the same two healthy donors throughout this study. The stimulatory capacity in MLC was measured, using mononuclear cells from the same two donors as responding cells. Reactivity was measured after 24 hr incorporation of [3H]thymidine (0.8 $Zi per well; specific activity 400 Ci/mol). Responses to PHA, ALS, and Con A were assessed after 3 days of culture, responses to PWM after 4 days of culture: mixed lymphocyte cultures were performed for 5 days, and those stimulated by antigens for 6 days. The amounts of the mitogens and antigens that gave an optimal response of the lymphocytes were determined in a series of experiments using different cell numbers, different doses of stimulant, and by varying time periods of incubation. It should be noted that we define culture conditions as optimal if there is a linear relationship between number of cultured cells and the measured reactivity. Since in the literature there are several reports (14- 16) suggesting that the use of a suboptimal dose of stimulant would be a more sensitive assay to demonstrate small defects in lymphocyte reactivity, in all our lymphocyte cultures stimulation with suboptimal doses of mitogens and antigens was performed too. However, it was found that the interindividual variability in responses to suboptimal doses of stimulant was greater than with the use of optimal doses. From these experiments, no conclusions could be drawn on differences in reactivity between patients and control individuals. On each occasion when the assays were performed, cryopreserved lymphocytes from healthy donors were tested in parallel. The results obtained from these control lymphocyte cultures did not fall outside the normal ranges on any occasion. Cytoroxicity assays. The effector function of lymphocytes from immunosuppressed patients was assayed by two different cytotoxicity assays. In the cellmediated lympholysis (CML) test (17), the cytotoxic capacity of MLC-activated T lymphocytes was tested against two different allogeneic donors. In the antibodydependent lympholysis (ADL) test, the capacity of the lymphocyte suspensions to lyse sensitized “‘Cr-labeled mouse-mastocytoma cells, sensitized with an IgG rabbit-anti-mastocytoma antibody (18) was measured. This activity is a function of K cells, i.e., a population of lymphocytes possessing receptors for the Fc fragment of IgG. Crllular immune Reactivity in Vi\w The induction of delayed hypersensitivity was measured by active sensitization to DNCB (19). The procedure was as follows. On Day 0, each individual was sensitized with a patch, containing 1 mg DNCB. After 14 days, sensitization was

tested with two concentrations of DNCB (3 and IO Fg per patch). Forty-eight hours later the test was evaluated as follows: S(‘OV(’ Erythema 1 7 Erythema and induration Erythema, induration, and blistering 3 Erythema, induration. blistering, and ulcer 4 The final DNCB score was calculated as the sum of the scores of each patch. Delayed-type hypersensitivity to recall antigens was determined by skin tests, using five different antigens, viz., PPD (10 Tu/ml), varidase (50 U SK/SK/ml), mumps (20 cfu/ml), trichophyton (0. I%), and candida (1:300): 0.1 ml of each antigen solution was injected intradermally on the volar surface of the forearm. Reactions were read after 48 hr. An induration of 5 mm or more at two perpendicular diameters was considered positive. Statistical

Analysis

Cell numbers in peripheral blood, the levels of total protein, albumin, globulins. and complement were analyzed by the paired Student’s t test. The same test was used to study the antibody response to hemocyanin. Results of in vitro lymphocyte cultures, skin tests, and immunoglobulin levels were analyzed by the Wilcoxon’s signed rank test. All tests were two tailed. RESULTS Cell Numbers

in Peripheral

Blood

Figure 1 shows the absolute number of lymphocytes, T and B lymphocytes. and monocytes in the peripheral blood of patients and control individuals. There appears to be a striking difference between the two groups. The total number of lymphocytes was clearly depressed, T and B lymphocytes being about equally affected. The absolute number of monocytes was not different between the two groups. Humoral

Immunity

Serum proteins. Immunoglobulin levels were slightly lower in the patient group as compared with the control group, reaching significance only for IgG, whereas the serum levels of total protein, albumin, globulin, and complement did not differ between patients and control individuals (data not shown). Specific antibody responses. The primary antibody response against hemocyanin was normal. Both the total antibody titer and also the distribution of the antibodies over the several immunoglobulin classes were comparable in patients and their matched control individuals (Table 1). Anamnestic antibody responses were also normal. The antibody response to diphteria toxoid was about the same for patients and control individuals, as were the antibody responses to polio virus, strains I, II and III (Table 1). It should be mentioned that results of antibody production toward tetanus toxoid are not shown. In fact, during the course of our study, it became evident that a comparison between patients and

IMMUNOSUPPRESSIVE

DRUGS AND IMMUNE l

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mon*c. FIG. I. Absolute numbers of lymphocytes and monocytes in peripheral blood, expressed as a percentage relative to the mean values of the control group. Mean value of total number of lymphocytes in the control group: 1.90. IOYiter. Mean value of number of T lymphocytes in the control group: 1.49. log/liter. Mean value of number of B lymphocytes in the control group: 0.17. 10s/liter. Mean value of number of monocytes in the control group: 0.30.10Ylliter. Horizontal bars indicate the mean for each group. t( tal

no.

lymph.

T

lymph.

B lymph

TABLE I HUMORAL~MMUNE PARAMETERS Patients (Before-after immunization) Antibody response to Hemocyanin kG kM Id


Diphteria toxoid (extinction)

0.54

Polio virus Strain I (W/ml) Strain II (I U/ml) Strain III (IUiml)

7 4 9

Control individuals (before-after immunization)

4.4 4.2 3.9 4.09 178 196 205


P value

5.9 5.3 5.1

NS NS NS

3.71

NS

140 221 197

NS NS NS

Nofe. Mean values of antibody responses to hemocyanin are expressed as titer steps. Each titer step represents a twofold serum dilution. starting with a I:40 serum dilution. NS. not significant.

26

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control individuals was not meaningful, because of the majority of the individuals of the control group had regularly received boosting injections with tetanus toxoid. However. it should be mentioned that the patients did produce antibodies against tetanus toxoid in all three major immunoglobulin classes (IgM. IgG. and IgA). Cellular Immurw ReacTi\,ity in Vitro In \pitro lymphocyte transformation. Table 2 shows the proliferative responses of mononuclear cells from patients compared with their control individuals. There was no significant difference between the two groups with regard to the in vitro response to the nonspecific mitogens PHA, ALS. Con A, and PWM. neither in the paired observation for each patient with his matched control nor in the average values obtained for both groups. A normal response was also found to allogeneic cells in the MLC. The stimulatory capacity of the patients’ cells did not appear to be significantly different from that of control individuals. Also the response ill \*itro against all soluble antigens tested appeared to be unimpaired. Finally. the it! \litro cellular reactivity of lymphocytes to the antigen hemocyanin was similar in patients and control individuals. Cytotoxic activity of the lymphocytes. No significant difference could be observed between patient and control group with regard to cytotoxic activity in TABLE LYMPHOCYTE

Mitogens None (Day 3) PHA ALS Con A PWM MLC Stimulator Stimulator Responder Responder

capacity” capacity capacity” capacity

Soluble antigens Antigen cocktail Hemocyanin

2

TRANSFORMATION

TESTS IN

VITRO

Patients

Control individual

Number tests

0.16 + 0.09* 13.3 + 6.9 26.1 f 8.4 13.0 + 5.3 8.1 2 4.9

0.20 i 0. IO 13.1 t 5.1 25.1 +- 13.7 11.2 2 5.8 1.7 -t 4.1

Ii II II 11 II

5.6 8.3 7.1 7.5

t + 2 +

3.5 4.6 5.2 4.2

5.3 + 6.4 3.1 + 6.2

4.9 6.8 8.7 8.5

+- 3.3 + 3.3 c 5.6 t 5.4

9.3 f 9.9 4.9 t 3.3

7 8 II II II II

Notes. “Values represent r’H]thymidine incorporation. expressed as mean counts per minute 2 SD x 10-s. 0 Stimulator capacity is expressed as the response of lymphocytes from two healthy donors to 2000rad irradiated cells from patients and control individuals. b Responder capacity is expressed as the response of lymphocytes from patients and control individuals to irradiated lymphocytes from two healthy donors.

IMMUNOSUPPRESSIVE

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RESPONSE

CML, i.e., the capacity to mount a cytotoxic T-lymphocyte response after stimulation in mixed lymphocyte culture against unrelated donor cells (data not shown). As can be seen from Fig. 2, the cytotoxic capacity of K cells, as measured in the ADL test, was severely impaired in 11 out of 14 kidney-transplant recipients. The same patients had a significantly decreased percentage of Fey-receptor-bearing cells (P value: 0.01, data not shown). C&L&W immunity in viro. The results of the skin tests after sensitization with DNCB are shown in Fig. 3. This figure clearly shows that there is a severe depression of in I?\w reactivity to this antigen. The same holds true for the in \-irv reactivity to recall antigens, as is shown in Fig. 4. It appeared that 4 out of 15 patients showed a failure to react to both primary and any of the secondary antigens, whereas none of the control individuals was anergic to all antigens. DISCUSSION

In this study we have shown that in patients with a well-functioning kidney allograft, who are under treatment with azathioprine and a low dose of prednisone, the numbers of both T and B lymphocytes in peripheral blood are significantly depressed. Both primary and secondary humoral immune responses in viva are not affected. The same holds for the proliferative capacity of lymphocytes in l&-o. Also the cytotoxic activity of T lymphocytes against lymphocytes of an unrelated donor, as measured in the CML test, remains normal. However, serum IgG levels show a significant decrease: the K-cell activity, as measured in the ADL test, is markedly diminished; and also the primary and secondary cellular immune responses in viva, as measured by skin tests, are depressed. Whether the decrease in number of circulating lymphocytes is due to cell death, or to a redistribution out of the circulation into other body compartments, has not been investigated in this study. Fauci and Dale (20) have convincingly demonstrated that normal human lymphocytes, circulating in the peripheral blood, are not lysed by corticosteroids, but are rather redistributed. Whether the low doses

6 c

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2.5 number-

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FIG.2. Antibody-dependent lympholysis(ADL). ADL effector-cellconcentrations and with a constant number

10 cf ivito,-

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was determined (40,000) of mouse

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at six different target cells.

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0

2

1

3

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6 DNCB-scur-e

Foci. 3. Primary

DTH

response

after

sensitization

with

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of prednisone, investigated in this study, can cause such a redistribution is not certain. Probably, the lymphocytopenia in our patients is also due to the influence of azathioprine. The mechanism by which azathioprine induces lymphocytopenia in man has still to be investigated. With respect to the number of peripheral blood T lymphoctyes, it should be noted that we have determined these values both by E-rosette formation (E-RFC) and by a heterologous anti-T-cell serum to exclude a possible interference of the drugs with the ability of lymphocytes to form E rosettes. We could not demonstrate any difference in the results obtained from either of these two methods. Regarding the primary humoral response, our data, indicating a normal antibody response, are in agreement with the findings of Rowley et al. (2) and Pabico et ~11.(21). However, other investigators (22, 23) found an impaired primary humoral response in patients who received 6-MP or azathioprine. As to the secondary humoral response, the observed normal responses are in agreement with those of Denman et al. (3) and Levin et al. (22), who studied patients treated with azathioprine and 6-MP. respectively. However, we could not confirm the findings of Maibach and Epstein (24) and Rowley et nl. (2), who found that therapy with azathioprine, and with both azathioprine and prednisone, respectively, abrogated the secondary humoral response, suggesting an effect of these drugs on m

0 FIG.

1 4. Secondary

2 DTH

3 no. response

of

patientsncontrols

4 positive

to soluble

5 skiirl antigens.

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immunological memory. The discrepancies between our results and those of others may be due to differences in experimental conditions, e.g., the nature of the antigens and their route of administration. From the antibody responses to tetanus toxoid, it appeared that our patients did produce antibodies against this antigen in all three major immunoglobulin classes (IgM, IgG, and IgA). Thus, the switch from IgM to IgG does not seem to be affected by the immunosuppressive treatment. The minor decrease in the level of serum IgG, which appeared to exist in the patients, was apparently not paralleled by decreased levels of antibodies, even those of the IgG class. According to several investigators (2, 23, 24), there seems to be a more rapid decline in antibody titer in patients receiving azathioprine compared with healthy controls. However, these aspects were not considered in this study. The finding of a normal in vitro reactivity of lymphocytes to mitogens, antigens, and alloantigens is in agreement with that of Denman et al. (3) and Heine et al. (25), in that at least those lymphocytes, remaining in the circulation, can mount a normal proliferative response in vitro. Some other investigators (26, 27) found a decreased lymphocyte reactivity that may well be due to the underlying diseases from which their patients suffered. It should be mentioned that the lymphocyte cultures were only performed in medium, supplemented with 20% pooled human serum, and not in the presence of autologous serum. The latter might reveal any influence of immunosuppressive agents present in the blood. However, we have not performed such experiments for the following reasons. First, in general, not all human sera are capable of supporting the growth of human lymphocytes in vitro, and can even have an inhibitory effect on lymphocyte cultures. Therefore, any inhibitory effect of autologous patient serum or plasma cannot readily be interpreted. Second, the blood plasma comprises only a relatively small proportion of the total body fluids. and the half-time for immunosuppressive drugs in plasma is rather short. Moreover, these drugs may be converted outside the blood stream into certain metabolites which actually mediate the immunosuppressive effect. For these reasons, results obtained from such experiments would be very difficult to interpret. Immunosuppressive therapy. as administered to our patients, did not seem to affect the development of cytotoxic T lymphocytes, as measured in the CML test. On the other hand, we found a strongly decreased ADL activity that may be due to both a decreased number of circulating K cells (also indicated by a significantly reduced proportion of EAy-RFCs) and a defect in the function of the remaining K cells. A decrease of ADL activity in patients receiving immunosuppressive drugs has already been demonstrated by several investigators (14. 28, 29). From their studies, it appears that a decrease in ADL activity can be caused by both azathioprine and prednisone when used as single drugs. Both primary and secondary cellular immune responses in i’ir~, as measured by skin tests, were significantly depressed in our patients as compared with the control group, whereas the in vitro reactivity to the same antigens remained normal. Our observation of depressed primary cellular reactivity is in agreement with the studies of Levin et al. (22) (patients received 6-MP) and Swanson and Schwartz (23) (patients received azathioprine). who found a depressed skin reac-

30

I t.I\i BtKGt:

E I AI

tivity to. respectively, the antigens Pastrurrlla tularrtzsis and KLH. That Maibach and Epstein (24)~-studying healthy volunteers-found a normal skin reactivity after sensitization with DNCB and NDMA may be due to the low dose of azathioprine, administered in that study. As to the depression of secondary cellular immune responses in r,i\‘o. the results obtained from previous examinations on human beings are contradictory. Investigators, who studied patients receiving azathioprine as a sole drug, generally found a normal secondary cellular reactivity in \li\Tct (3. 22. 27). Hersh rr (11. (30) and Denman et ul. (3) reported a depressed skinwindow reaction in patients under treatment with azathioprine. This last finding suggests that azathioprine impairs the inflammatory response, as is also suggested by several studies performed on experimental animals (3 l-34). This anti-inflammatory effect of azathioprine might, at least in part. explain our skin-test results. Moreover, it is very likely that the well-known anti-inflammatory action of prednisone is responsible for the depressed expression of DTH as well. Furthermore, it should be noted that the significance of this finding in kidney-transplant recipients is somewhat hampered due to the fact that patients, who show depressed delayed hypersensitivity reactions (so-called low responders), have a better lyear-graft survival than those with a normal skin reactivity to DNBC and recall antigens (so-called high responders) (35-38). Thus, it may be assumed that some of our patients had already depressed delayed hypersensitivity reactions prior to transplantation. However. the fact that without exception all our patients show depressed skin tests strongly suggest an effect of immunosuppressive therapy as well. From our study, it is not possible to attribute the observed effects to either one of the two drugs. To clarify this point, it will be necessary to investigate patients who receive azathioprine only, as well as patients receiving low doses of prednisone as single drug. In conclusion, our findings concerning primary and secondary humoral responses in rive and lymphocyte reactivity in \*itro indicate that the combination of azathioprine and prednisone in this group of patients does not affect the sensitization phase nor the proliferative phase of the immune response. Moreover, the normal CML test suggests an intact effector function of the T cells. Thus, judging from the parameters used in this study, a specific immunological defect at the lymphocyte level does not seem to be responsible for the depressed cellular immune reactivity in \yi\w. Indeed, the moderately decreased number of circulating T lymphocytes may lead to this impaired cellular reactivity in \,irv: however, the anti-inflammatory action of these drugs has to be considered as well. Because of the role of the K cell in the immune system is as yet unresolved, the significance of the decreased ADL activity remains unknown and will require further study. ACKNOWLEDGMENTS We thank Ms. F. de Wilde for her valuable help in collecting the blood samples. Dr. G. van Steenis and Dr. A. M. Hagenaars (Rijks Instituut voor de Volksgezondheid, Bilthoven. The Netherlands) kindly performed the antibody determinations against diphtheria toxoid and polio virus. We are grateful to Dr. C. J. M. Melief. Dr. T. A. Out, and Dr. W. P. Zeijlemaker for valuable discussions. The statistical analysis has been performed by Mr. M. F. M. Janssen.

1MMUNOSUPPRESSIVE

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REFERENCES 1. Bach. J. F., “The mode of action of immunosuppressive agents”. North-Holland, I975. 2. Rowley. M. J., Mackay, I. R., and McKenzie, 1. F. C.. Lancer ii, 708. 1969. 3. Denman. E. J.. Denman, A. M.. Greenwood. B. M., Gall. D.. and Heath. R. B.. Dis.

29,

220.

Amsterdam.

Ann.

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32 36. 37.

11-N Watson. Lawc~r

ii,

Kerman.

R. H..

posium Netherlands 38.

Rolley.

Received

M. A.. 1323.

on

Floyd.

Immunological (Abstract).

R. T..

January

Briggs, 1979.

J. 0.. M..

t.1

Diamandopoulos.

Buren.

Al

A. A..

C. ‘I‘. van.

Monitoring

of

and the

Kahan, Transplant

Hamilton.

I). N. H..

B. D..

“Secondary

Recipient.”

and

Dick.

International

H. M.. Sym-

Noordwijkerhout,

The

1980.

Sterioff.

S.. Parks.

15.

accepted

1981:

HERGt

L. C..

April

and

6. 1981

Williams.

G. M.,

7’run.sp/tr~1r.

Pr~c,.

9, Xl,

1977.