Duquesnoy, R

Eiras, G

Makowka, L

Rabinowich, H

Starzl, TE

Todo, S

Zeevi, A

English

PubMed

D-Scholarship@Pitt

Immunosuppressive Effect of FK-506 on In Vitro Lymphocyte Alloactivation: Synergism With Cyclosporine A A. Zeevi, R. DUQuesnoy, G. Eiras, H. Rabinowich, S. Todo, L. Makowka, and T.E. Starzl RECENT STUDIES have described a new potent immunosuppressive drug FK-506 (FK) that induces considerable prolongation of allograft survival.'·) This drug is isolated from Streptomyces tsukubaensis. has a molecular weight of 822 daltons, and is struc-turally different from cyclosporine A (CsA). In vitro investigations have demonstrated a strong inhibitory effect on lymphocyte activa-tion that appears to be mediated by blocking of interleukin 2 (IL-2) release.4 Our studies have extended these findings by showing that FK inhibits secondary proliferation of allo-reactive T lymphocytes generated from mixed lymphocyte reaction (MLR) cultures or pro-pagated from organ transplant biopsy speci-mens with cellular infiltrates.s The in vitro immunosuppressive effect of FK is generally several hundredfold greater than that of CsA. On the other hand, FK does not inhibit sec-ondary proliferation of activated T cells stim-ulated by IL-2. With respect to the possibility that FK could be used together with CsA as an immu-nosuppressive drug regimen to prolong allo-graft survival, we studied in vitro effects of low doses of these drug combinations on lym-phocyte proliferation. The results demon-strate synergism between FK and CsA in their From the Department of Immunopathology. Univer-sity of Pittsburgh. Supported in part by Grant Nos. AI23467 and HL36416 from the Nationa/Institutes of Health and a grant from the American Heart Association, Western Pennsylvania Chapter. Address reprint requests to A. Zeevi. PhD. University of Pittsburgh, Department of Immunopathology, One Children's Place, Room 571I, Pittsburgh, P A 15213-3417. C 1987 by Grune & Stratton, Inc. 0041-1345/87/1905-6oo8$OJOO/0 inhibition of both primary lymphocyte alloac-tivation and secondary proliferation of allo-reactive T cells. This in vitro synergism may have clinical application in that low doses of FK and CsA .:ombinations may be effective in increasing transplant survival. MATERIALS AND METHODS M LR-Induced Proliferation Human peripheral blood lymphocytes were isolated from heparinized blood by Ficoll-Hypaque density gra-dient centrifugation. Undirectional MLR cultures were set up with 105 responder and 105 irradiated (2,000 R) stimulator cells in a volume of 200 ItL of tissue culture medium (TCM) supplemented with 10% human serum and incubated for six days. During the final 20 hours of incubation, each culture was labeled with I ItCi of lH-thymidine. The cultures were harvested and counted in a liquid scintillation counter. Primed Lymphocyte Test Alloreactive lymphocyte cultures were propagated from human heart and liver transplant biopsy specimens in the presence of IL-2 as previously reported.·" Second-ary proliferation of alloreactive T cells was assessed in a three-day primed lymphocyt~ test (PLT) whereby 10' responder cells were incubated with lOS irradiated (2,000 R) stimulator cells. Lymphocyte proliferation was deter-mined by lH-thymidine incorporation as described ear-lier. Drug Sources FK was supplied by the Fujisawa Pharmaceutical Co, Ltd, Osaka, Japan. This drug was received as a crystalline powder, and before use it was dissolved in methanol and further diluted in TCM. CsA was obtained from Sandoz, Inc, Hanover, NJ. It was dissolved in ethanol and further diluted in TCM to various concentrations. Dose Effects of FK and CsA on Lymphocyte Proliferation The inhibitory effects of FK and CsA on MLR reactiv-ity and PL T response of alloreactive lymphOCytes were measured at different concentrations of drugs ranging from 0.06 to 500 ng/mL. The results were expressed as 40 Transplantation Proceedings, Vol XIX, No 5, Suppl 6 (October), 1987: pp 40-44 ~h-RMS -ivation: ne A Ikowka. and T.E. Starzl primary lymphocyte alloac-ndary proliferation of alIo-'his in vitro synergism may ication in that low doses of )inations may be effective in ant survival. LS AND METHODS o/iferation blood lymphocytes were isolated ,d by Ficoll-Hypaque density gra-Undirectional MLR cultures were nder and 105 irradiated (2,000 R) ()Iume of 200 ilL of tissue culture t!mented with 10% human serum lays. During the final 20 hours of Ire was labeled with I /lCi of ures were harvested and counted 'ounter. fe Test eyte cultures were propagated iver transplant biopsy specimens .s previously reported.6•7 Second-cacti~ T cells was assessed in a 'locyte test (PLT) whereby 10· ,bated with lOS irradiated (2,000 I phocyte proliferation was deter-incorporation as described ear-ie Fujisawa Pharmaceutical Co, I rug was received as a crystalline i was dissolved in methanol and CsA was obtained from Sandoz , dissolved in ethanol and furthe; us concentrations. and CsA on Lymphocyte of FK and CsA on MLR reactiv-)f alloreactive lymphocytes were ::oncentrations of drugs ranging L. The results were expressed as uppl6 (October). 1987: pp40-44 IMMUNOSUPPRESSIVE EFFECT OF FK-506 percent inhibition by using the following formula: percent inhibition - I - (cpm with drugs x 100/cpm without drugs). Pretreatment of Alloreactive T Cells With CsA and FK Heart biopsy specimen-propagated alloreactive T cells were cultured with 20 IU /mL of recombinant IL-2 with and without 10 Ilg/mL of CsA or various concentrations of FK (I Ilg/mL, 0.1 Ilg/mL, and 0.01 Ilg/mL). After four days of incubation, the cell cultures were washed and tested for cell viability. The cells were then tested for donor-specific PL T reactivity by incubating 10· cells with !O5 irradiated stimulators for three days. Control cultures were tested for proliferation in the presence of IL-2. In addition, these pretreated cells were tested for their sensitivity to FK and CsA in PL T assays. RESULTS Synergism Between FK and CsA in Inhibition of Lymphocyte Activation Studies on lymphocyte proliferation in pri-mary MLR have previously shown that inhibi-tion was observed at concentrations of FK that were several hundredfold lower than those of CSA.4 An example of the differences between FK and CsA is shown in upper part of Fig 1. It may be seen that significant inhibition of greater than 75% was observed at concentra-tions of 1 ng FKjmL and 500 ng CsAjmL. The bottom part of Fig 1 illustrates that combinations of 0.1 ngjmL of FK and 10 " INHI8iT1()1 C1 N\.R RESPONSE " INHIBITI()I J()() 7S 50 25 0 500 30 Cf MlR 20 RESP()ISE 10 41 C,A 100 10 1 01 DRUG ng/ml 10"g C,A I "'l (,A .1 "'l FK 10"9 (," 1119 (,A .1ngFK .ln9FK DRUG nglml Fig 1. Synergism between FK and CsA inhibition of MLR response. ngjmL or 1 ngjmL of CsA induced consider- whereas at these concentrations each of these able MLR inhibition, whereas at these low drugs alone had no significant effect. concentrations neither drug had any signifi- These findings demonstrate that at very low cant effect. :~-«-concentrations FK and CsA have synergistic Another example of the synergism between inhibitory effects on both primary and second-low doses of FK and CsA is shown in Fig 2, ary T lymphocyte activation. which depicts the results of studies on donor-specific secondary proliferation of alloacti-vated T lymphocytes propagated from liver transplant biopsy material. The upper part of Fig 2 shows that the inhibition of the PLT response of these cells by FK was effective at a 400-fold lower concentration than that of CsA. The lower part of Fig 2 demonstrates that a combination of low doses of FK and CsA exerted significant inhibitory effects, Effect of FK and CsA Treatment of Alloreactive T Cells Studies were designed to determine whether pretreatment of alloactivated T cells with FK or CsA affected subsequent second-ary donor-specific proliferation. Because these drugs have no effect on IL-2-induced proliferation of activated T cells (Table 1), experiments were set up whereby heart biopsy 42 II)() - .... 7S ~ INHIBITI()< Cf PLT 50 obpm~p£ 25 o~----------~--------~ 500 II)() 50 10 25 12 Ob DRUG "g/ml 40 30 " fkefBfqf~ Cf PL r 20 obpm~pb 10 10 ng C.... 1 ng C~KKK .06"g FK 10 ng CsA 1"9 (SA 06 "g FK 06 ng FK DRUG "g/ml Fig 2. Synergism between FK and CsA inhibition of the PL T response of liver biopsy specimen-propagated lymphocytes. material-grown lymphocytes were expanded with IL-2 in the presence of FK or CsA at various concentrations. After four days in culture, these expanded alloreactive T cell cultures were washed and then restimulated with donor cells in three-day PLT assays. The Table 1. Lack of Inhibitory Effect of FK and CsA on IL-2-lnduced Proliferation of Alloreactive T Cell Clone DB29 Dose IL-2 Response Drug (sLg/mL) (cpm) Nothing None 235 IL-2 None 5.296 IL-2 + FR 0.1 5.085 IL-2 + CsA 5,718 The MLR-generated alloreactive T cell clone 0829 is specific for a DQwl-associated cellular determinant. ZEEVI ET AL results shown in Table 2 demonstrate that pretreatment with FK at 1 J.lg/mL caused significant inhibition of PL T stimulation four days later but had no effect on IL-2-induced proliferation. On the other hand. pretreat-ment with CsA or lower doses of FK had no significant effect on the subsequent PL T response. Effect of FK and CsA Pretreatment on Drug Sensitivity of Alloreactive T Cells The PL T response of pretreated cells was also tested for its susceptibility to inhibition by CsA (Fig 3). Pretreatment of IL-2-expanded heart transplant biopsy specimen-grown lymphocytes with 10 J.lg/mL CsA yielded cells whose PL T reactivity had essen-tially the same CsA sensitivity as control cells that were cultured without drugs. On the other hand, pretreatment with FK at 0.1 J.lg/mL yielded cell cultures whose PLT reac-tivity was considerably more sensitive to CsA, and strong inhibition was observed at concen-trations as low as 0.05 J.lg/mL of CsA. It may be seen in Fig 3 that pretreatment with FK at 0.01 J.lg/mL also yielded cells with increased CsA sensitivity of their PLT responses. Further studies were conducted to deter-mine whether pretreatment with CsA or FK leads to an increased sensitivity of alloacti-vated T cells to FK (Table 3). Heart trans-plant biopsy specimen-grown lymphocytes expanded with IL-2 in the presence of 10 J.lg/mL CsA showed similar FK sensitivity of their donor-specific PL T response as control Table 2. Effect of Pretreatment With FK or CsA on the IL-2 Responsiveness and Donor-Specific Reactivity of Lymphocytes Propagated From Heart Biopsy Material Pretreatment Dose Proliferation Responses (cpm) Drug (,,/mL) Background IL-2 PLT Control None 534 22,056 66;447 FK 1 455 30,834 8,126 FK 0.1 449 29,806 81,242 FK 0.01 436 30,638 102.045 CsA 10 351 15,535 57,889 ZEEVI ET AL fable 2 demonstrate that 17K at I J,Lg/mL caused II of PL T stimulation four 110 effect on IL-2-induced he other hand, pretreat-'l)Wer doses of FK had no lfi the subsequent PL T A Pretreatment on Drug 'active T Cells ,e of pretreated cells was usceptibility to inhibition Pretreatment of IL-2-;lsplant biopsy specimen-) with 10 J,Lg/mL CsA PL T reactivity had essen-sensitivity as control cells j without drugs. On the 'atment with FK at 0.1 cultures whose PL T reac-Jly more sensitive to CsA, 1 was observed at concen-15 J,Lg/mL of CsA. It may pretreatment with FK at Ided cells with increased c:ir PL T responses. ere conducted to deter-:ltment with CsA or FK d sensitivity of alloacti-(Table 3). Heart trans-len-grown lymphocytes in the presence of 10 ~imilar FK sensitivity of >L T response as control tment With FK or CsA on the Donor-Specific Reactivity of J From Heart Biopsy Material Proliferation Responses (cpml ground Il-2 PLT ;34 22.056 66.447 >55 30.834 8.126 >49 29.806 81.242 .36 30.638 102.045 :51 15.535 57.889 IMMUNOSUPPRESSIVE EFFECT OF FK-506 43 100 ! 80 7. INHIBITION OF PLT 60 FK 0.1 ug/ml RESPONSE 40 20 0 ~--------~--------~~=======---K----------K .5 .1 .05 .01 .005 CsA ug/ml Fig 3. Increased CsA sensitivity of heart biopsy specimen-grown lymphocytes pretreated with FK. cell cultures grown without drugs. On the other hand, pretreatment with low doses of FK yielded cells that showed increased sensi-tivity to FK, although the effect was not as impressive as the enhanced sensitivity to CsA shown in Fig 3. DISCUSSION These findings demonstrate synergism between the immune suppressive effects of low doses of FK and CsA in two types of in vitro assays. The first deals with a simulta-neous incubation of these drugs during pri-mary or secondary alloactivation of human T lymphocytes. The other assay shows syner-gism through sequential exposure of alloreac-tive lymphocytes to FK followed by CsA. The synergism between FK and CsA suggests that low-dose combinations of these drugs may be effective in prolonging allograft survival. Indeed, our experience with cardiac allografts in rats have shown a strong immunosuppres-sive effect of combinations of FK and CsA.2 An advantage of using combinations of low doses of FK and CsA to prolong transplant survival is that there would be a reduction of undesirable side effects of these drugs. For instance, the nephrotoxicity of CsA in trans-Table 3. Inhibition of PL T Reactivity of Pretreated Heart Biopsy Specimen-Grown Lymphocyte Cultures in the Presence of Various Doses of FK FK Percent Inhibition of PI. T Response HB Cultures· (jtg/mLI Control CsA (10 I'g/mLl FK (0.1 jtg/mLI FK (0.01 I'g/mll 0.1 95 94 95 95 0.01 93 93 93 94 0.001 86 89 90 87 0.00025 0 0 63 21 0.00012 0 0 51 13 -Lymphocytes propagated from heart biopsy (HB) material were cultured with IL-2 in the presence of drugs (CsA and FK) for four days befae the PL T inhibition test. 44 plant patients could be markedly reduced if lower doses are used in combination. perhaps with low doses of FK. Little is understood about the mechanism of the synergism between FK and CsA. For both drugs. the inhibitory effect of lympho-cyte proliferation is mediated by blockage of IL-2 release (Kino et al4 and unpublished data). On the other hand. neither drug causes significant inhibition of IL-2-induced prolif-eration. The presence of IL-2 receptors on activated T cells is required for this prolifera-tion. The appearance of IL-2 receptors on MLR-activated cells has been shown to be inhibited by FK.4 This would mean that such ZEEVI ET AL cells would be less responsive to the stimulat-ing effects of lL-2. However. our current studies do not support this notion because FK pretreatment of alloreactive T cells expanded with lL-2 did not render these cells less responsive to subsequent IL-2 stimulation, although their PL T responsiveness was impaired. Recent studies at our institution have shown that FK pretreatment of lympho-cytes increases the binding of CSA.8 These findings suggest that the synergism between FK and CsA might be related to lymphocyte membrane binding-of these drugs. Studies are currently in progress to obtain a better under-standing of the mechanism of this synergism. REFERENCES 1. Ochiai T. Nakajima K. Kagata M. et al: Transplant Proc 19:1284, 1987 2. Lee P, Murase N, Todos, et al: Surg Res Comm 1987 (in press) 3. Todo S, Makowka L, Starzl T: Surg Res Comm 1987 (in press) 4. Kino T. Goto T. Auki H. et al: (submitted) 5. Zeevi A, Duquesnoy R, Eiras G. et al: Surg Res Comm 1987 (in press) 6. Zeevi A. Fung 1. Zerbe TR, et al: Transplantation 41:620,1986 7. Fung 1, Zeevi A, Starzl TE, et al: Hum Immunol 16:182.1986 8. Sanghvi A, Warty V, Zeevi A, et al: Transplant Proc (this issue) . 

Immunosuppressive effect of FK-506 on in vitro lymphocyte alloactivation: synergism with cyclosporine A.

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Immunosuppressive effect of FK-506 on in vitro lymphocyte alloactivation: Synergism with cyclosporine A

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Immunosuppressive effect of FK-506 on in vitro lymphocyte alloactivation: synergism with cyclosporine A.

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