Increase in Natured Killer Activity in Cyclosporine-Treated Ren~ AUograft Recipients During Rejection

Martin Lefkowicz, D~ane Jorkasky, and Jacki Kornbiuth

ABSTRACT: Natural killer (N~) activity was a~sessed in a prespeetlve fashion i~ 15 renal tramplau~ recipients recoiving single HLA-hapletype matched allografls and ~i~t~ined on cyda~/min~ (CYA) immunosuppression. There was marhed vaeiabili¢y in NK activity pretmnsptautati~ in this popalation; however, a strong corrdasion (r = 0,92, p < 0.01) was f iuud b~tw~ determinations in an i.dividual patient u~on repeated teeing. No rignifeea~t dtp~siou of NK activity eceurred within the first 12 weeks fdlewing tm.splantatiun. Whole Mead CYA ~ did not correlate with NK activity, Although NK activity pd*r to transplantati, u did net tnndict clinical outcome of the allografi, a marked rise in NK activity was *~serud in patknt~ undergoing rejection corapa~M with thee not rate. ring (p < O,OD. A large inomasut in acti*i~y was seen in eight of 11 rejwtion episodes; a similar increase was ~rdy sten in the aksem~ of ~¢jeetiou. Those resedts indicate that NK activity is stimulatd du~ing Ml~gmft ~j¢ction in CYA ~ t e d renal transplant mipients. It m~ains to b~ determised wheth~ this rise iu NK ~nc t#n up~eeents a manifestation ofalhaeaaivity acc~rapanying the rejection pmcas ew whether NK cdh dir~c~ contribute to allogmft deswuaion.

ABBREVIATIONS CYA o/closporitm CTL cytotoxic T lymphocytes NK natural killer LU ]ytic umt(s)

INTRODUCTION The introduction of cyclosporine (CYA) to immunosuppressive te#mens for o r~n transpiantatlon has been ~ssoclated with improved gnfft and patient sur- vivals [1,2]. The early favorable results reported by Caine and co-workers in 1978 [3] wit~ CYA immunosuppression have now been repeatedly confirmed. CYA is a nonmyelotoxic immunosuppressant that exerts a selective [nhibito~ effect on T-lymphocyte helper function with relative sparing of T-lymphocyte suppressor, B cell, macrophage, and gran,alocyte functions [4,5]. The effects o f the drug on natural killer (NK) function are less well characterized.

NK cells are lymphoid ceils that exhibit spontaneous cytotoxiciv/in virto against a variety of real/guam ceils, virus-infected cells, and some normal cells without apparent prior sensitization. In addition, NK cells m'¢ potent producers

From the lmmu~i~o~J, Division, Depa~t~nt of P~shdo~ a~vl the Renal Ekttrdyt¢ Division, Dc- ?arzment of M~idN¢, U.izcrsity of Pezmyl~wni~ $chad of Medici., Philaddphi¢, Pexnsyl~Ma.

Address reprint ml*ests to Dr. Jacki Komblath, Department of Pathalogy, UnF~ersity of Pennsyh'ania School of Mcdlciue, Philad¢lphia, PA 19104°6082.

Recei, wd Octeber 23, 1986; ac~qepted FebrNary 27, 1987.

Humsn Immunolog~ 19,139-E49 (1987) © ELsevier Science Pub~s~n~ Co., Inc., 1987 139 52 V~nde~lt Ave., New York, ~Y 1O0|7 01~-8859187/$3.~

140 M. Lefkowitz ¢t aL

of lymphoklnes, such as interferon and interleukin-2, and their cytotoxic activity is in turn rapidly augmented by these iymphokines [6.7]. Evidence h ~ accu- mulated that NK activity is an important in vivo defense n~echanism against certain types of viral infection and the development of malignancies [8,9], conditions to which the transplant population are predisposed. In the routine system, NK cells appear to be the main effector cells med/atiug mural resistance against non-self in bone marrow transplantation [9,10]. NK cells may also become activated during graft-versus-host disease in man [11]. However, the importance of NK activity in solid organ transplantation and aUograft rejection has yet to be defined. Animal studies have demonstrated that NK cells infiltrate rejecting aliografts soon after transplantation, and appear prior to cytotoxic T lymphocytes (CTL) [12]. Analysis of renal biopsy specimens from patients undergoing , .'jecdon parallels these observations in animals [ 13].

NK activity has been examined in renal transplant recipients receiving con- ventional immunosuppression (azathioprine and prednisone). In these studies, NK activity was consistently depressed in patients maim,fined or, this drug reg- imen [14,15]. CYA is now the most commonly used immunosuppressive agent, which is much more selective in its suppressive effects than the combination of azathioprine and prednisone [5]; however, much less is known about the effect of CYA on NK activity either in vitro or in transplant patients receiving CYA.

Studies on the in vitro effects of CYA on NK cell activity have demonstrated that NK cells are relatively resistant to the immunosuppressant effects of CYA at therapeutic concentrations (100--200 rig/rob [16-18], a dosage at which T helper cell function is severely inhibited [19~}. Previous work in this hborato~ has shown an inhibitory effect of CYA on NK function only at high CYA con- centrations (5 t~g/mi) [20]. By virtue of their pharmacologic resistance to CYA, therefore, NK cells may play a more significant role in immune surveillance and possibly allorejection in CYA treated transplant recipients than in patients on other forms of immunosuppresslon.

The purpose of this prospective study was twofold: first, to define the levels of NK activity in CYA treated renal allograft recipients, and second, to determine whether NK activity changes during rejection episodes.

METHODS Patients and controls. Fifteen renal allograft recipients (mean age 32 yr, range 18-53 yr) were studied prospectlveiy from October 1985 to August 1986. All patients received a one HLA haplotype-matched kidney from a living related donor. NK activity was determined twice prior to transpl,t~mtion to establish a reliable baseline and at frequent reguhr intervals after tz~asplantation for 3 months. Whole blood CYA levels were measured with each NK activity determination. CYA levels were assayed using high-performance liquid chromatography [21]. The NK activity of 26 healthy volunteers from the Univetsi%. community was also assessed.

lmmunosuppressive r¢gimen. All transplant recipients were maintained on a com- bination of CYA and prednisone. CYA (14 mgdkg) was administered on the day prior to transplantation, and the dosage was subsequently tapered to maintain whole blood CYA levels between 100 and 200 ng/ml. Methylprednisolone, 1 grn intravenously, was given intraoperatively followed by peednisone (2 mg/kg) on the first postoperative day. Prednisone dosage was subsequendy reduced by 10-20 my/day until a total daily dosage of 30 mg was reached followed by a

NK Activity and AHogrsft Rejection 141

slower dose reduction. The addidon of azathiopr/ne to the immunesuppre,sive re#men excluded the patient from subsequent analysis.

Rejection. Episodes of acute rejection were di~geosed by clinical criteria after exclusion of other causes for deteriorating renal function. Acute rejection was defined as a rise in serum creatiulne coacemrat/on in the absence of an e~cated CYA level (i.e., less than 200 n~/ml), with a l~ck of respome to a reduction in CYA dose. Other causes for deteriorating renal function, such as obstruction and urinary leaks, were ruled nut by radiographic techniques. All but two episodes of rejection were confirmed by renal ~ograf t biopsy utilizing histotogic~ criteria of reiection in the CYA-treated patient as repotted by Sibley et al. [22]. Therapy for rejection consisted o f I gin methyipredulsolone intravenously on 3-5 con- s¢cufive days. CYA doses w¢~ not charted during the com's¢ of ~ t h e ~ y .

NK cytotoxicity assay. Hepadnized samples of peripheral venous blood were ob- tained from all transplant recipients m the same time each morning prior to the administsatioa of a~y immunosuppresslve drugs. Samples were obtained at ran- dom intervals pretransphnmdon. Mononuclear ceils were separated by cemrif- ugadon on Ficoil-Hypaque gradients and suspended in RPMb1640 supplemented with 10% heat inactivated human serum (complete medium). These cells were tested for N K effector function at four effectur-to-tatget ceil ratio: 100:1, 50:1, 25:1, and 12.5:1 as previously described [23]. Brietty, 0. ! ml alklunts of effector cells were placed in the wells of round bottom microdter plates. Target ceils consisted of the erythroleukemia ceil line K562. In most instances, N K fl~'sis of the T-ceil line MOLT-4 was aho determined. Inmmd/ately before use in the assay, 1-2 x 106 target ceils were labeled with 50 ~Ci ofNaz ~C~O4 (~dmm~mn, New E:,gl~,~d Nuclear, Boston, MA) for 1 hr at 37°(;, washed ~ens ive ly and resuspended in complete medium at a concentration o f 3 × 104 cells per nil. One tenth milliliter aliquots o f target ceils were added to the effector ceils in each microtiter well. The plates were spun (200 xg for I mha) and then incubated for 4 hr at 37°C in a 5% CO2 humidified incubator. The plates were centrifuged again (200 xg for 5 mia) and 0.12 m] supematants were removed from each well and counted in a ~mlma counter.

The percent~,e of specific ~chremium release was calculated according to the formula:

Experimental release - spontaneous release x I00. Maximum release - spontaneous release

Spontaneous release was measured from target cells incubated alone, and max- imum release obtained from target cells incubated with the detergent hexade- cyltrimethyianuaonium bromide (0.1 M). All determinations were performed ha triplicate. N K activity is expressed in terms of lyric uul~ (LU). One LIT is defined as the number of cells per I0 ~ cells mediating 30% specific ~Xchromium release from K562 tmget cells.

Statistical analysis. Standard errors are #yen after all values for N K activity except where noted. Statistical analysis was performed by the Student's t-test for unpaired, and where applicable, for puked data. The WBcoxon i~',mk sum test was used for calculation of s/gnificance for nonparametric data. Dichotomous var~bles were evaluated using Chi-square analysis.

142 M. Lefkowitz et ai.

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FIGURE 1 Recession: repeated NK activity p#etransp!~t. Peripheral blood lympho. cytes from 15 renal transplant patients were tested for NK activity twice before trans- #aatation m establish baseline levels. NK activity was measured in a 4 hr ~'Cr release assay using the NK sensitive target cell line K562. Results are expressed in lyric units, which are calculated as the number of effecmr cells/l0 T mediating 30% lysis of K562. The cnrreladoa coefficient for repeated testing of NK function pretransplant is 0.92.

RESULTS

Baseline N K Activity

A large degree of variabifity in NK activity was apparent in the peripheral Mood of the 15 patients before transplantation (Figure 1). Lyric activity ranged from 23 to 300 LU, with amean (-+ 1SD) o f i 0 6 - 81 LU. When measured on two separate occasions in the same patient, however, a strong correlation (r = 0.92) existed between that measured on the first and second occasions. The time interval between measurements in an individual patient ranged from 1 week to 2 months.

The NK activity of 26 healthy volunteers was also assessed. A similar large range of N K activity was seen in this poptqation (from 16 to 238 LU, mean -+ l SD = 74 -+ 58 LU). There was no significant difference in NK activity between the control group and the pretransplant group (Student's test). These results confirm findings in other laboratories demonstrating no difference in NK activity between healthy controls and patients with end-stage renal failure maintained on hemodialysis [15,24,25].

Post t ransplantat ion N K Activity No significant change in lyric activky was noted within the first 12 weeks after transplantation (Figure 2). Measurements obtained during rejection episodes were excluded from the analTsis. Three patients did not complete the study: a transplant nephrectomy was performed at 8 weeks in one patient due to irreversible re- jection and azathioprine was added to the immunosuppressive regimen in two patients at 6 and 8 weeks because of continued poor ldlogrdt function.

Relat ionship of CYA Level to N K Activity

Figure 3 demonstrates the lack of correlation between whole blood CYA levels and NK activity (r = 0.03, p = 0.87) observed .n all measurements, irrespective of prednisone dosage. In an attempt to mitigate against the potential effects of steroids on NK activity, a similar analysis was performed on lyric activity measured

NK Activity and A ] l o ~ t Reiectlon I~3

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at low prednlsone dosage. Only, a weak negative correht /on (r = - 0 . 2 5 , p = 0.11) was noted b e a t e n C Y A leve! ~ d N K act/vi~ on deterrniaations m ~ e in patients treated with 15 n ~ or less of prednisone (Figare 4). Similar resuks were obtained in patients being ma/nta/ned on only 10 mg of prednisone (dam not shown).

FIGURE 3 Cyclosporine leveJ vs. HK ~tivity. NK acdvRy was c o m p ~ wkh who|e blood CYA levels in all pgoents at ~ | time points ~cter trgnsphnm¢ion. No s/gnifican~ ¢orrela¢/on was found (r = 0.03, p = 0.87).

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144 M. Lefkowitz et al.

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Rejection

Eleven rejecdo:~ episodes occurred in ten patients at the following times after transplamadon: three at ! week, two at 2 weeks, one as 6 weeks, three at 8 weeks, and two at 12 weeks. There was no significant difference in pretransplant NK activity among individuals subsequently rejecting their grafts and those not rejecting (116 -+ 30 LU rejection vs. 108 -+ 28 LU no rejection, p = 0.86). However, a marked rise in NK activity occurred during rejection. Figure 5 illustrates the change in lyric activity from the preceding cytotoxicity assay in individual patients. Although there was little change in NK function from week to week during periods of stable renal funcdon (mean change -- - 4 -+ 6 LU), lyric activity increased by a mean of 53 -+ 13 LU during reiection (p < 0.01). Following treatment of reiection with high-dose steroids, lyric activity dropped back to the values obtained immediately preceding rejection (mean change from rejection value = - 5 8 -+ 17 LU).

A large increase in NK activity, defined as a rise of greater than 40 LU, occurred during eight of 11 rejection episodes (Table 1). A similar increase in lyric activity was rarely seen (l 1 of 82 measurements) in the absence of reiection (p < 0.01, rejection vs. no rejection, Chi-squa~e analysis). Of interest, the episode of ir- reversible rejection occurred in the individual with the greatest increment ( + 1 ! 8) in lytic activity.

DISCUSSION

NK cells are able to lyse a variety of target cells in vitro without prior sensitization. These target cells include neoplastic cells, virus-infected cells, allogeneic cells, hemopoietic stem cells, and dendritic cells [26-29]. Recent evidence for an in vivo role of NK cells in natural resistance to tumors [9,30], viral infecticm.~ [3I], and bone marrow transplan,~.don [9,10] indicates the potential importance of N K cells in immune sureeilhnce. For example, in a routine model, N K cells mediate resistance to radiation-induced [hymic leukemia, challenge with mela- noma tumor cells and lethal cytomegalovirus infection [9,31]. It is of interest, therefore, to examine N K activity in renal transplant recipients, a population at risk for both malignancy and viral infection.

NK Acdvi~ and Allngraft Rejection I45

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::::::::::::::::::::::::::::: _ _

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FIGURE 5 Change in NK activity during re~al ~ s p h n t rejections. Ci~mzge~ in NK activity in patients undergoing rojecdon web; compared wid~ the change~ in NK ~cdvity in patients not undergoing rejection. Changes in lyric units from the preceding NK ~ a y were calcuk~ed for each patient. In the sbseace of reiactinn, NK activity ~,ied for egfh patient very little from assay to assay (mean ¢h~--e -4*-6 LU). There w~ n significant rise in NK activity during rejection (mean change +53 ± I3 LU) ~p < 0.01). After treatment of rejection, NK levels returned m those nhudned immediately preceding rejection (mean change from rejection value - 5 8 ± 17 LU) (p<0.01).

Previous investigations have consistendy demonsLmted a severe depression o f circulating NK activity in azathioprine and prednisone ~eazed transplant recip- ients [14,15]. In this regard, it should be noted that treamaent of renal transphnt recipiems with prednisone as the so|e/mmunosuppressive agent in daily dosages of 20 mg or less was reported as having no effect on N K activity [32]. Among CYA treated patients, some studies report that N K activity is decreased but less so than in patients on a2atl~ioprine immunosuppression {24,33], while others cannot find any significant depression o f activity [34~. In this prospective study, NK activity in 15 transplant recipients receiving CYA was not significantly altered within the first 12 weeks of transpiantatlnn. Thus, CYA treatment, in sharp contrast m ~zathiopfine adminlstratinn, spares NK activity in renal allograft recipients.

The lack of a significant change in NK funct/on among CYA treated patients is consistent with in vitro data [16--18,20]. Akhoagh conflict exis~ in the lit- erature, all investigators agree that N K cell activity in vitro is relatively resistant to the immunosuppressive effect~ of CYA at therapeutic CYA concentrations (100-200 ng/mD. The maximal decrease in NK activity reported in any study this CYA concentration is 30% [18]. i~ addition, we found no sign;_~icant cor- felat/on between NK activity and levels of CYA in the peripheral blood of transplaut recipients. It is of particular interest that the incidence ofvlral infection in CYA-treated patients is much reduced compared to the incidence/11 azathio- pfine treated renal allngraft recipients [35,36J. No definitive information is yet

146 M. Lefkowitz et al.

TABLE 1 N K activity in renal allograft recipients

NK (LUg' NK (LU) Patient Rejection (preceding rejectlnn) (during rejection) &LU '~

I N o ~ - - - -

2 N o ~ ~

3 No ~ - - 4 No - - - - - - 5 No - - - - - - 6 I wk 28 28 0 7 1 wk 116 185 +69 8 1 wk 139 238 +99 9 2 wks 256 303 +47

10 6 wks 1 l I 145 + 34 11 8 wks 34 123 +89 12 8 wks 33 93 +60 13 12 wks 125 185 +60 14 12 wks 40 83 +43 ! 5 2 wks 119 74 - 45

8 wks 49 167 + 118

"LU: lyric units, calculated as the number of culls/lO 7 mediating 30% lysis of K562 in a 4 hr SICr-r~ie~e assay. These values are the results obtained in Khe NK assay performed immediately p~oceding rejection. This varied from 1-4 weeks before the reiectiov, episodes. b&LU: change in lyric units; +: increase, -: decrease.

available on the frequency of malignancy in CYA-treated patients uuder the current drug regimens. Intact N K function in patients maintained on CYA may therefore be a contributing factor to the decreased incidence of viral infections in this population.

Recently, N K cells with high lyric activity have been isolated front rat kidneys during the initial stages of ailograft rejection [12,37]. These cells appear prior to CTL and disappear from the graft before peak CTL activity is reached. Fol- lowing the disappearance of N K cells from the allngr~t, peripheral blood N K activity reaches maximum levels. Similar to these observations in the rat, serhl biopsy analysis [13] and fine needle aspiration [38] in azathioprine (AZA)-treated renal transplant patients show that the earliest stages of graft rejection are ac- companied by a significant inttmr of N K cells. However, in these studies, either morphological criteria or the monoclonal antibody Leu 7 was used to identify N K cells. These results need to be confirmed using a more NK-specific antibody. interleukin-2 production by peripheral blood lymphocytes in transplant recipi- ents is also markedly increased during acute rejection episodes [39,40]. The enhancement of N K cell activity by interleukin-2 and interferon, coupled with the ability of N K cells to rapidly produce these iymphokines, suggest a possible role Of N K cells as regulators of the early stages of an immune response.

Most investigators have found no significant association between peripheral blood N K activity and kidney allograft rejection in AZA-treated transplant re- cipients [14,15,41]. One report demonstrated an increase in N K cells in !9 of 20 rejection episodes in AZA-treated renal allograft patients using Leu 7 as a marker for N K cells [42]. However, it has subsequently been shown that the number ofLeu7 + cells does not correlate wellwith N K activity [43A4]. Previous studies of N K activity in CYA-treated patients have not evaluated changes in N K function during rejection. Since N K activity remains intact in patients main- tained on CYA, it may be particularly relevant to grfft rejection in these moawdums.

NK Activity and AUograft Rejection i~7

The results of this prospective study of renal transplant recipiems receiving sia#e HLA haplotTpo-mmched kidney allogr~s and ma/nt~ned on CYA indicate that NK activity is not signfficandy depressed in the fi~t 3 months following transplantation. During Mlograft rejection, however, NK activity in peripheeai blood increases dramatically. The increase in NK activity associated with rejection may reflect a state of alloreacdvity, with heightened prodncdon of ime~Jeukin-2 and interferon. Thus, increased NK function may simp|y represent a manifestation of an activated immune cesponse, Alteroadvely, by virtue of flieir ability m both produce and respond to lymphokines, NK ceils may be direcdy involved in the maturation of the CTL response within the graft and in ~dlog~ft destruction. Further studies are ~n progress to clarify this issue.

ACKHOW[ ~DGMENTS

This work w~s snpporte~ by grant CA 37827 from the Nadonal Cancee Insdtote. M. Lel'kowiez wg~ ~uppormd by MIH tra/nlag gram NRSA 2 T32 AM 07006. The authors would like to thank Ms. Sam Doughs, Ms. Delores Russum, and Ms. Ffiieen Mitt~k for help in specimen collection and pedem foUow-up. We also wish to th~k Ms. Val Remenrer a.qd Ms. Myr~ Pollard for preparation of the m~user/pt.

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148 M. Lefkowitz et al.

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NK Activity and Allograft Rejection 149

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