A longitudinal study on the effects of azathioprine and high doses of prednisone on the immune system of kidney-transplant recipients

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

24, 33-46 (1982)

A Longitudinal Study on the Effects of Azathioprine Doses of Prednisone on the Immune System Kidney-Transplant Recipients R. J. M. TEN BERGE,

P. TH. A. SCHELLEKENS, S. SURACHNO,* J. H. TEN VEEN,* AND J. M. WILMINK*

and High of

T. H. THE,?

Central Laboratory of the Netherlands Red Cross Blood Transfusion Ser\>ice and the Laborator) for Experimental and Clinical Immunology. University of Amsterdam: * Department of Internal Medicine, Division of Renal Transplantation, University Hospital Wilhelmina Gasthuis. Am.sterdarn, and t Department of Internal Medicine, Division of Clinical Immunology. University Hospital, Groningen. The Netherlands

The effects of treatment with azathioprine and high doses of prednisone were studied in five kidney-transplant recipients, from whom blood was obtained prior to and frequently after transplantation during a period of 4 months. Just before initiation of drug therapy, the patients were immunized with hemocyanin and DTP vaccine to elicit primary and secondary humoral immune responses. It was found that the numbers of circulating T and B lymphocytes and also the number of monocytes showed a decrease. In addition, the levels of all serum immunoglobulins except IgM decreased. Primary and secondary antibody responses were normal. In contrast, the proliferative response of lymphocytes in vitro after stimulation with the same antigens was low or even absent. This indicates that, although antigen-reactive cells are apparently depleted from the peripheral blood, they may still be present in other lymphoid compartments. Cytotoxic T-cell function was hardly affected in these patients, but K- and NK-cell activity showed a decrease. From this study we conclude that lymphocytes are redistributed under the influence of therapy with azathioprine and high doses of prednisone, and that this drug regimen has only a small effect on the specific immune reactivity in viva.

INTRODUCTION

Previously, we have studied the effect of treatment with azathioprine, together with low doses of prednisone on the immune system of a group kidney-transplant recipients (1). From that study, we concluded that the immunosuppressive effects were mainly due to the anti-inflammatory action of these drugs. However, the results obtained could have been influenced by host factors, which may have been responsible for the successful outcome of the kidney transplantation, e.g., patients with depressed immune functions before transplantation seem to have a better graft survival (2-5). These problems can be circumvented by performing longitudinal studies, in which the patient can serve as his own control. In that way, it is possible to determine whether changes in measured parameters are indeed induced by the drug therapy. For that reason, and to investigate the effects of high doses of prednisone, we have studied five patients during a period of 4 months from the day of transplantation. The results obtained in these patients are concordant and confirm our original conclusions; moreover, they indicate that a 33 0090-1229/82/070033-14$01.00/O Copyright All rights

0 1982 by Academic hess, Inc. of reproduction in any form reserved.

34

TEN

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ET AL.

drug regimen, consisting of azathioprine and high doses of prednisone, besides their anti-inflammatory action, also has a small effect on the immune system. ABBREVIATIONS

ADL ALS CML ConA DNCB DTH DTP

ELISA FcyR FITC k K cell MLC NK cell PHA PPD PWM SEM

antibody-dependent lymphocytotoxicity antilymphocyte serum cell-mediated lympholysis concanavalin A dinitrochlorobenzene delayed-type hypersensitivity a vaccine containing diphtheria toxoid, tetanus toxoid, and killed polio virus, strains I, II, and III enzyme-linked immunosorbent assay receptor for the Fc fragment of IgG fluorescein isothiocyanate immunoglobulin killer cell mixed-lymphocyte culture natural killer cell phytohemagglutinin purified protein derivative of tuberculin pokeweed mitogen standard error of the mean PATIENTS

From all patients included in this study, informed consent was obtained. Clinical data regarding these patients are summarized in Table 1. Starting on the day of transplantation, azathioprine was given at a dose of 200 mg per day during the first 3 days, and thereafter at doses as high as possible with a maximum of 3 mg/kg; depending on renal function, liver function, and the number of leukocytes and thrombocytes in peripheral blood (average dose, 100 to 150 mg daily). Furthermore, each patient received 200 mg prednisone daily in the first week, after which the dose was tapered off within 4 months to 30 mg daily. Moreover, doses of 1000 mg methylprednisolone were given on Days 0,3,7, and 14. When a rejection crisis occurred (i.e., at the time-points indicated in Table l), three pulse doses of 1000 TABLE

I

PATIENTS Patient

As

1 2

27 19

3 4 5

32 26 23

Original

kidney

disease

Chronic pyelonephritis Chronic glomerulonephritis (Alport’s syndrome) Focal glomerulosclerosis Membranous glomerulonephritis Chronic glomerulonephritis

Rejection

crisis 31 34 66 33 -

on Day

IMMUNOSUPPRESSION

IN

KIDNEY-TRANSPLANT

RECIPIENTS

35

mg methylprednisolone on consecutive days were administered; at the same time, the standard scheme of azathioprine and prednisone was resumed, as described for the period immediate post-transplantation. GENERAL

OUTLINE

OF STUDY

Just before transplantation and initiation of drug therapy, blood was collected and each patient was immunized with hemocyanin from a-Helix pomatia to elicit a primary immune response, and with a DTP vaccine containing diphteria toxoid, tetanus toxoid, and killed polio virus to evoke secondary immune responses. During the first month after transplantation, blood was collected twice weekly: during the second month once weekly, and during the third and fourth months once every 2 weeks. A complete evaluation of specific immune reactivity should preferably include tests on cellular immunity in vivo (i.e., DTH skin tests). However, to avoid aporte &entree in these severely immunosuppressed patients, no skin tests were performed. For reasons of clarity, we present the results obtained at those time points, which are most relevant with regard to the level of immunosuppression achieved. Point A represents the day of transplantation on which blood was drawn just before initiation of drug therapy. Point B, about 21 days after transplantation (range 17 to 25 days), was chosen because observed changes in parameters in each of our patients were most pronounced at that point of time. Points C and D represent values some time before (range 3 to 6 days) and after (range 3 to 8 days) antirejection therapy. Because one of the patients did not undergo a rejection crisis, all values at points C and D represent the mean of four patients instead of five. Finally, point E represents the latest point in time at which parameters of each patient were tested, i.e., 4 months after transplantation. MATERIALS

Preparation

AND

METHODS

of Spleen Cell Suspensions

Splenic tissue fragments were minced and squeezed through tantalum gauze. The cell suspension was layered on top of a Ficoll-Isopaque mixture with a density of 1.079 g/ml and centrifuged for 20 min (1OOOg at 22°C) to remove dead cells, cell clumps, tissue fragments, and cells with a density greater than 1.079 g/ml, such as granulocytes and erythrocytes. The mononuclear cells were washed and stored in liquid nitrogen (7). Blood Samples Serum obtained from each individual was frozen and kept at -20°C. Mononuclear cells were isolated from defibrinated blood by means of Ficoll-Isopaque density-gradient centrifugation (6) and preserved in liquid nitrogen (7). To avoid day-to-day variations in the lymphocyte cultures, all cultures on samples of one patient were performed in one experiment on the same day. Crll Numbers The absolute numbers of lymphocytes determined by electronic cell counting

and monocytes in peripheral blood were (Coulter counter) and May-Grttnwald-

36

TEN

BERGE

ET

AL.

Giemsa-stained blood smears. T lymphocytes were determined both by rosette formation with sheep erythrocytes (8) 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 (9). 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. Specific Humoral

Immune

Responses

To determine the capacity to mount a primary antibody response, each individual was immunized subcutaneously with 1.0 mg hemocyanin (hemocyanin of cy-Helix pomatia) (10). Several times thereafter, specific antibodies to this antigen in the IgM, IgG, and IgA classes were measured by indirect ELISA, as described by Weits et al. (11). To study secondary antibody responses, 1 ml of a DTP vaccine containing diphteria toxoid, tetanus toxoid, and killed polio virus, was injected intramuscularly. Again, the antibody responses were measured several times afterward. The total antibody titer against diphteria 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 by a semiquantitative Ouchterlony technique combined with radioautography (12). Antibodies against polio virus were measured by a virus neutralization test. Cellrrlar Immune

Reactivity

in Vitro

In vitro lymphocyte transformation tests. After thawing, the viability of the cell suspensions used for the experiments was determined by trypan blue exclusion, and found to be 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 (13), containing 3 x IO“ lymphocytes per well in 150 ~1; the following stimulants were used: 1. the nonspecific mitogens PHA (Wellcome), final concentration, 50 pg/ml; horse-anti-human ALS, final dilution, 1:34; ConA (Sigma), final concentration, 120 pg/ml; PWM (Gibco), final concentration, 100 pg/ml; 2. the antigen a-Helix pomatia hemocyanin, final concentration 50 pg/rnl (10); 3. an antigen cocktail, consisting of a mixture of 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 (14) ; 4. 3 x lo4 lymphocytes, irradiated at a dose of 2000 rad. To assess the response in MLC, three sets of cultures were always performed, using as stimulating cells peripheral blood lymphocytes from two unrelated individuals as well as spleen cells from the specific kidney donor. Responses to PHA, ALS, and ConA were assessed after 3 days of culture, responses to PWM after 4

1MMUNOSUPPRESSION

IN

KIDNEY-TRANSPLANT

RECIPIENTS

37

days of culture; mixed-lymphocyte cultures were performed for 5 days, and those stimulated by antigens for 6 days. Twenty-four hours before termination of the cultures, [3H]thymidine was added (0.8 &i per well; specific activity, 400 Cii mol). 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 were consistently within the same, normal range. It should be noted that the culture conditions as described were chosen in such a way that a linear relation existed between the number of lymphocytes per culture and the extent of [3H]thymidine incorporation. Cytotoxiciry assays. The effector function of lymphocytes was assessed by different cytotoxicity assays. In the cell-mediated lympholysis (CML) test (19, the cytotoxic capacity of MLC-activated T lymphocytes was tested against both the spleen cells from the original kidney donor and the peripheral blood lymphocytes from two unrelated healthy individuals. In the antibody-dependent lympholysis (ADL) test, the capacity of the K cells in the lymphocyte suspensions to lyse Wr-labeled mouse P815 mastocytoma cells sensitized with an IgG rabbit-antimastocytoma antibody was measured (16). The function of NK cells was assessed in a 4-hr 51Cr-release assay, using K-542’ cells as target cells (17). All cytotoxic assays were performed in triplicate at six different effector cell concentrations and a constant number of target cells. RESULTS

Cell Numbers in Peripheral Blood After transplantation and the initiation of drug therapy, the absolute number of peripheral blood lymphocytes, both T and B lymphocytes, as well as the number of monocytes showed a significant decrease (Fig. 1). Antirejection therapy did not cause any further decline in the lymphocyte counts. In fact, a tendency to return to normal values was consistently observed. However, it can be seen that-after a recovery -the number of monocytes decreased again after the administration of antirejection therapy. Hrrmorai

Immunity

Serum proteins. The serum levels of IgG and IgA showed a significant decrease, reaching a minimal value at about 3 weeks after initiation of drug therapy, which was only partly paralleled by changes in the serum concentration of albumin (Fig. 2). The serum levels of IgD also showed a decrease. The fluctuations in serum levels of IgM paralleled that of serum albumin. In all patients, serum levels of IgE showed an initial increase, followed by an ongoing decrease. In the four patients who underwent a rejection crisis, the effect of antirejection therapy on immunoglobulin levels was less in comparison to that of the same drug regimen immediately after transplantation. Although serum immunoglobulin levels, except IgE, tended to increase as soon as corticosteroid doses were reduced, they did not fully return to pretransplant values, at least during the time period in which we studied these patients.

38

TEN

BERGE

ET AL.

percent

60

FIG. 1. Number of lymphocytes and monocytes in peripheral blood, expressed as a percentage relative to the mean values measured before initiation of drug therapy (mean of five patients). which were all in the normal range. The meaning of the letters A to E is described under General Outline. The number of total lymphocytes, T and B lymphocytes measured at time points B and C are significantly decreased relative to the pretreatment value (time point A). The number of monocytes is significantly decreased at time point B only. P c 0.05 paired Student’s f test, two tailed.

Specific antibody responses. The patients were immunized on the day of transplantation, just before the initiation of drug therapy. Secondary humoral responses to diphteria toxoid, tetanus toxoid, and killed polio virus (data not shown), and even the primary humoral immune response to hemocyanin (Fig. 3) appeared to be normal despite the administration of very high doses of immunosuppressive drugs during sensitization. It could be demonstrated that the antibodies produced were of all three major immunoglobulin classes (IgM, IgG, IgA). Moreover, the time course of the responses appeared to be quite normal in comparison to that of healthy control individuals. Cellular immune reactivity in vitro. As shown in Table 2, the in vitro reactivity of lymphocytes to nonspecific mitogens did not show any major fluctuations (P > 0.05). The responder capacity of lymphocytes in the MLC test to stimulator cells from two unrelated donors and to spleen cells from the specific kidney donor was also not affected. In contrast, the reactivity of lymphocytes to soluble antigens in vitro was already low or even absent prior to transplantation, and remained so during the whole time period of this study. However, in three patients we observed a strong reactivity toward antigens in vitro, from about 30 days after transplantation, which became again severely depressed after administration of antirejection therapy, whereas no obvious changes in reactivity to nonspecific mitogens were observed (cellular reactivity in vitro of one of them is shown in Fig. 4). The cytotoxic activity of T lymphocytes as measured in the CML test was hardly affected by the immunosuppressive therapy (Fig. 5). Both K- and NK-cell

IMMUNOSUPPRESSION

IN

KIDNEY-TRANSPLANT

RECIPIENTS

39

activity appeared to be depressed already prior to transplantation in four of the five patients, so the influence of therapy on these cytotoxic functions was difficult to evaluate. However, in the course of time after transplantation, a further decrease seemed to occur in four out of five patients (see Table 3). In the fifth patient, these functions were already maximally depressed prior to transplantation, and remained so during the whole period of this study. DISCUSSION

In the present study, the effect of a drug regimen consisting of azathioprine and high doses of prednisone on the immune system of kidney-transplant recipients was studied. In Table 4, the results of the present study are compared with those of a previous study (l), in which azathioprine was administered together with low doses of prednisone. Both drug regimens cause a depression of the number of peripheral blood lymphocytes, and high doses of prednisone appear to depress the number of circulating monocytes as well. As far as corticosteroids are concerned, several investigators (18, 19) have demonstrated a maximal lympho- and monocytopenia 4 to 6 hr after the administration of a single dose of corticosteroids to healthy volunteers, whereas 24 hr later the cell numbers in peripheral blood had returned back percent lk0

140

albumin

60

FIG. 2. Values of serum albumin and serum immunoglobulins. expressed as a percentage relative to the mean values measured before initiation of drug therapy (mean of five patients), which were all in the normal range. The meaning of the letters A to E is described under General Outline. The values of IgG, IgA, and IgD are significantly decreased at time points B, C, and D, relative to the pretreatment value (time point A). At time point E, the values of IgD, IgA, and IgE are significantly decreased relative to the pretreatment value (time point A). P < 0.05, paired Student’s t test, two tailed.

40

TEN BERGE ET AL.

8 6

IgA

c 0

10

20

30

40

50

60

70

80

90

days

FIG. 3. Primary antibody responses to hemocyanin (expressed as “log titer, the first serum dilution being 1:lOO). The shaded area represents the range of values, obtained after immunization of five healthy volunteers.

to normal values. It has been demonstrated that the lymphocytopenia in human beings under treatment with corticosteroids is not due to cell death (20, 211, but probably to a redistribution (22, 23). Because in our patients azathioprine was always combined with prednisone, a contribution of azathioprine to the depression of cell numbers could not be determined. Regarding the serum immunoglobulin levels, it should be noted that the levels of serum albumin showed only minor fluctuations, thus the observed effects on immunoglobulin levels appear to be largely due to effects on the immune system. From several studies, it appears that corticosteroids may affect serum levels of immunoglobulins (24-26), mainly by decreasing its synthesis (25). Azathioprine may have induced this decrease as well, although there are data in the literature suggesting that azathioprine does not cause a change of serum immunoglobulin levels (27, 28). In all our patients, an initial increase in the serum level of IgE was observed. This is probably caused by the corticosteroid treatment, because a similar increase in IgE levels has been described in patients with bronchial

IMMUNOSUPPRESSION

IN KIDNEY-TRANSPLANT

TABLE 2 CELLULAR IMMUNE REACTIVITY

IN

41

RECIPIENTS VITRO

Mean values of five patients at different points of time” Stimulants

A

B

C

D

E

Mean value of five control individuals

PHA ALS ConA PWM Irradiated allogeneic cells Donor spleen cells Hemocyanin Tetanus toxoid Antigen cocktail

Il.56 12.5 4.8 5.9

10.2 9.9 3.8 5.7

15.9 13.7 5.9 6.0

16.8 7.3 6.3

10.5 16. I 4.6 5.2

13.1 14.8 6.8 6.7

8.1

6.0

6.9

7.4

7.2

7.8

10.7 0.2 1.1 1.3

7.0 0.7 2.3 0.8

8.8 -

9.7 0.3 1.8 0.2

10.5 1.2 2.4 0.4

4.9’ 7.4 9.3

” The meaning of the letters A to E is described under General Outline. ’ [3H]Thymidine incorporation (cpm x lOma). ’ Fourteen days after immunization.

asthma, after initiation of corticosteroid treatment (26). However, in our patients other factors may also have played a role, for instance, the antigeneic load of the kidney allograft . From our study, it appears that the levels of all immunoglobulins, except IgM, are affected. Because it is well known that for the switch from production of IgM

6 0 I

tetanus

toxoid

antigen

cocktsil

6. o-3,

rn

n

n

n

23

azathioprine

ll

“9 200 0 0

20

4. Cellular immune reactivity in methylprednisolone. FIG.

40 vitro

60

80

days

of lymphocytes from patient 4. ‘“Pulse doses of 1000 mg

42

TEN BERGE ET AL. "/ 51 0 Cr-release

60 1

__----------=c~ _----Mm

10

e-4

E

0 1.25

2.5 nr.

5 of

effector

cells

x

10 10 -4

FIG. 5. Cell-mediated lympholysis (CML) of lymphocytes from patient 2. Wr release (%) was determined at six different numbers of effector cells and at a constant number (20,000) of target cells. The meaning of the letters A to E is described under General Outline. (The number of lymphocytes available at time point D was not sufficient to perform a CML test.) O-O, CML tested against nonrelated third-party cells: 0- --0, CML tested against donor spleen cells. Patient 2 Nonrelated donor Kidney donor

HLA phenotypes Ai-2/BlS-35 A2-3lB7-w62 AZ/B15-31

to production of other immunoglobulins helper T cells are needed, the effect of this drug regimen in viva appears to be mediated via an (indirect) effect on T helper cells rather than via an effect on the B cells themselves. It appeared that the patients under study could develop virtually normal primary and secondary humoral immune responses in Go. Furthermore, it was observed that they developed a rise of antibody titer during infection with cytomegalo virus or herpes simplex virus (data not shown). In view of the considerable range in magnitude of antibody responses in healthy individuals, a small alteration of these responses in our patients which might be expected with respect to the reduced levels of serum immunoglobulins, could have been missed. Still, it appears that specific humoral immunity is only moderately affected, if at all, by the administration of azathioprine and prednisone, even when given in high doses. major With regard to cellular immunity in vitro, we could not demonstrate effects of azathioprine and high doses of prednisone on the reactivity of lymphocytes in vitro to nonspecific mitogens. Although Fauci and Dale (18) observed an effect of corticosteroids on lymphocyte reactivity to mitogens, that decrease was

IMMUNOSUPPRESSION

IN KIDNEY-TRANSPLANT TABLE

K-

AND

NK-CELL

Day 0

Patient

ACTIVITY

3

EXPRESSED

AS 5’Cr

About 3 weeks after transplantation

RELEASE

(5%)

About 4 months after transpiantation

K cell activity at lo5 effector cells and 4 x 1W target cells” 17 8 3 4 19 8 44 8 21 8

I 2 3 4 5

43

RECIPIENTS

14 3 8 7 10

NK cell activity at 2 x lo5 effector cells and 10“ target cells” 47 29 33 5 6 6 42 52 15 60 31 20 I6 5 8

I ? 3 4 5

I( Mean value of six control individuals in the same experiments: 44% (SEM 7%). * Mean value of six control individuals in the same experiments: 66% (SEM 5%). TABLE

4

Effect of treatment with azathioprine Parameter Cell numbers in peripheral blood Total number of lymphocytes Number of T lymphocytes Number of B lymphocytes Number of monocytes Immunoglobulin kM kc kA

High-dose prednisone” $” 9 L

and

Low-dose prednisone” 1 f Normalized

levels in serum Not affected

Antibody responses Hemocyanin DTP Cellular reactivity Mitogens Antigens MLC CML ADL NK

in vim

Cellular reactivity DNCB Recall antigens

in viw

i

Not affected 1 Normalized

Not affected Not affected

Not affected Not affected

Not affected 1 Not affected Not affected

Not affected Normalized Not affected Not affected

i Not tested Not tested

” Results from the present study, determined in the time interval (A)-(B). ’ Results from a previous study (see Ref. (1)). r Significantly depressed (p < 0.05).

i 1 L

44

TEN

BERGE

ET

AL.

only transient, reaching a minimal value at 4 to 6 hr after intake of corticosteroids. In our study, we did not collect blood samples at such short intervals and, consequently, we may have missed such short-term changes. The responder capacity of lymphocytes in the MLC test did not seem to be influenced by the administration of azathioprine and high doses of prednisone. The interpretation of the effect of these drugs on the reactivity of lymphocytes to soluble antigens in vitro was hampered by the low levels of reactivity already present before transplantation. However, in three patients a pronounced influence of these drugs on the reactivity to soluble antigens could be demonstrated. It should be noted that even after in vivo immunization, the proliferative response in vitro after stimulation with these antigens remained low or absent in all patients. Yet, at the same time, antibody responses in viva to these antigens were quite normal. Therefore. it appears that antigen-reactive T helper lymphocytes remain present, probably elsewhere in the lymphoid system. This hypothesis is consistent with the data of other investigators on the redistribution of lymphocytes under the influence of corticosteroids (22, 23). An additional role of the observed depression of the number of peripheral blood monocytes cannot be ruled out, although the normal reactivity to the mitogens ConA and PWM, for which the presence of monocytes in the cultures is required, argues against this. In a previous publication (l), we have demonstrated a normal reactivity of lymphocytes to soluble antigens in \sitro, in kidney-transplant recipients who were treated with azathioprine and low doses of prednisone. Thus, apparently, antigen-reactive lymphocytes are only depleted from the peripheral circulation under the influence of high doses of prednisone, and reappear when the dose of prednisone is reduced. Our data indicate that the cytotoxic T-cell function toward the HLAincompatible cells from two healthy unrelated individuals was hardly affected by the drug therapy. All patients, included in this study, showed a low or even absent CML reactivity against cells from the kidney donor, already before transplantation; this is probably due to the HLA compatibility. Consequently, our data provide no information on any role of immunosuppressive drugs in the conversion of CML reactivity to CML nonreactivity in the post-transplantation period, as described by other investigators (29, 30). Regarding CML reactivity against unrelated cells, our findings are sustained by the in vitro experiments of Balow et al. (31) who demonstrated that neither the in vitro generation of cytotoxic T lymphocytes against specific alloantigens nor the lytic phase of the cytotoxic reaction could be inhibited by corticosteroids at pharmacological doses. Again, the effect of azathioprine in this regard is unknown. A depression of K- and NK-cell function in kidney-transplant recipients has already been demonstrated by other investigators (32, 33). However, we could not confirm the findings of Lipinski et al. (34) who reported a dissociation of K- and NK-cell activity in kidney-allograft recipients. Our data are also in contrast with the findings of Parrillo and Fauci (35, 36) who observed a relative increase in K-cell activity, but a profound decrease in NK-cell activity after administration of corticosteroids to healthy volunteers. As has been shown in a previous study (I), the K-cell activity remains depressed in most patients who are treated with azathioprine and low doses of prednisone. Recently, we could demonstrate that

IMMUNOSUPPRESSION

IN KIDNEY-TRANSPLANT

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45

lymphocytes from most of these patients had depressed NK-cell activity too. The role of these cells in cellular immunity and, particularly, their possible role in host immune defense against microbial, tumor, and histocompatibility antigens remains to be established. In conclusion, under the influence of therapy with azathioprine and high doses of prednisone, lymphocytes seem to be redistributed. Specific antibody production and the effector function of T lymphocytes in CML are hardly affected. Thus, the specific immune reactivity in man seems difficult to suppress by this drug regimen. In addition to the already described anti-inflammatory effect (l), only a small influence on the immune system could be demonstrated. However, it seems unlikely that these moderate effects contribute considerably to the survival of the kidney graft. ACKNOWLEDGMENTS We thank Ms. F. de Wilde and Ms. M. Hansen for assistance in collecting the blood samples and for measuring some of the humoral parameters. Dr. G. van Steenis and Dr. A. M. Hagenaars (Rijks lnstituut voor de Volksgezondheid, Bilthoven, The Netherlands) kindly performed the antibody determinations against diphteria toxoid and polio virus. We thank Dr. R. T. Krediet for kindly providing blood samples and clinical data from one patient. We are grateful to Dr. C. J. M. Mehef, Dr. T. A. Out, and Dr. W. P. Zeijlemaker for valuable discussions. This investigation was financially supported by the Foundation for Medical Research FUNGO. which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO), The Hague, The Netherlands (Grant 13-29-42).

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5. 6. 7. 8. 9. 10. I I. 12. 13.

Rolley. R. T., Sterioff, S.. Parks, L. C., and Williams, G. M., Transplant. Proc. 9, 81, 1977. Watson, M. A., Briggs, J. D., Diamandopoulos. A. A., Hamilton. D. N. H.. and Dick, H. M.. La~~ret 2, 1323. 1979. Boyurn. A., J. Clirt. Lab. Invest. 21 (Suppl. 97), 77, 1968. Bois, M. J. G. J. du, Schellekens, P. Th. A., Wit, J. J. F. M. de. and Eijsvoogel. V. P.. Scrrnd. ./. Imnrrtnd. 5 (Suppl. 5). 17, 1976. Jondal, M.. Helm. G., and Wigzell. H., J. Exp. Med. 136, 207, 1972. Pernis, B., Forni, L., and Amante, L., In “Progress in Immunology: First International Congress Immunology” (B. Amos, Ed.), pp. 95-106, Academic Press, New York, 1971. Gast, G. C. de, The, T. H., and Snijder, J. A. M. Arta Med. Scund. 194, 303, 1973. Weits. J.. Gast, G. C. de, The, T. H., Esselink, M. T., Deelder, A. M., Petrovic, M., and Mandema, E., Clilr. E-up. Imrntrtwl. 32, 443, 1970. Thorbecke. G. J., Hochwald, G. M., and Williams, C. A., 111“Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 111, pp. 343-357. Academic Press, New York/London, 1971. Oers, M. H. J. van, Pinkster. J.. and Zeijlemaker, W. P., Inf. Arc,h. Allergy Appl. Immunol. 58, 53. 1979.

Leguit, P., Meinesz. A., Huisman, L., and Eijsvoogel, V. P., Clin. Et-p. Immrtrwl. 14, 149, 1973. 15. Zeijlemaker, W. P., Oers, M. H. J. van. and Eijsvoogel. V. P., Scund. J. fmmunol. 5 (Suppl 5). 14.

143. 1976.

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16. Zeijlemaker, mrrnol.

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ET

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