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Transplllnt Immunology 1993: 1: 14fr150

Effects of combined administration of FK 506 and the purine biosynthesis inhibitors mizoribine or on lymphocyte DNA synthesis and activation molecule expression in human mixed lymphocyte cultures AW Thomson, J Woo, GZ Yao, S Todo, TE Starzl and A Zeevi Transplant Institute and Departments of Surgery and Pathology, University of Pittsburgh Health Science Center, Pittsburgh, Pennsylvania

Abstnct: Our objective was to obtain new information on the in vitro antilymphocytic action of the cytokine synthesis inhibitor FK 506 and the punne biosynthesis inhibitors mycophcnolic acid (MPA; the active mOlctv of RS6l443) and mizoribine (MZB) when used alone or in combination. When added at the initiation of six·day human mixed lymphocyte cultures (MLC). FK 506. MPA or MZB exhibited dose·dependent inhibition 01 T·lymphocvtc DNA synthesis. FK 506. however. was «Xl· fold more potent than MPA. and ((}IXlO·fold more potent than MZB. Combination of FK 506 with either MPA or MZB. each at suboptlonal concentrations. produced no more than additive inhibitory effects on 'H thvmldine Incorporation. Two-colour !low cytometnc analvsls 01 Iymphocvtes revealed that none of the drugs affected cell surface activatIOn molecule expressIOn (CD25 = IL·2R 55 kD lJ·chain. HLA·DR or CD71 = transferrin receptor ITRJ) on allosllmulated CD4' or CD!!' cells harvested at three days 01 culture. Bv day SIX. however. all three agents. at levels which markedly inhibited proliferation. suppressed the expression of activation markers on both CD4' and CD!!' cells. Also at day six. Inhibition 01 activation molecule expression on CD4' cells was achieved with the combination of FK 506 and either MPA or MZB at concentrations which. on their own. were ineffective. These data provide new. additional informallon on the in vitro antilymphocytic action of FK 506. MPA and MZB when used alone and in combinallon.

Introduction superior therapeutic index to CsA both in transplantal1on~ II and autoimmune disease," The ctticacy and clinical pOlential of a variety of new im· Mycophenolic acid (MPA; the active moiety of RS· munosuppressive drugs when used alone or in combinallon () 1443 = mycophenolate mofetil) and mizoribine (MZR) are are currently the subjects of critical evaluation. I.; The macro­ strong inhibitors of enzymes which catalyse the de novo lide anlIbiotic FK 506 exhibits a similar molecular action to synthesis of guanine nucleotides that arc essenlIal for T cell cyclosporin A (CsA)J and interferes with T cell proliferation replication. They thus act later in the cell cycle than FK 506. sttmulated via the T cell receptor (TCR)lCD3 pathway. This By decreasing nucleotide availability. however. MPA and dfect IS achieved by blocking of the transcription of genes MZR may also inhibit signal transduction and the synthesis of encoding interleukin-2 (IL·2) and other cylokines.< FK 506 cell surface proteins and receptors.II).. 15 Both drugs have has recently been shown to be effective in the prevention and recently been shown to inhibit T cell responses in vitro.lb-IM In rescue of human organ allograft rejection and may have a addition. MPA or MZR suppresses transplant rejection and each drug has been reported to act synergistically with CsA to prolong survival of experimental organ allografts. I .... 21 Moreo­ Address for correspondence: A W Thomson. Pillsburllh Transplant Instllute. Depanment of Surllery. W1544 Biomedical Science Tower. ver. MPA and MZR appear to be less toxic than UniversllY 01' PitlsbuTllh. PiIlSbur!lh. PA 15213. USA. to the intestine and bone marrow. Thus. both MPA and MZR Effects 0/ drug combination on human MLR 147 are of prospective clinical value as adjunctive immunosup­ Cultured mononuclear cells were washed and the expres­ pressive agents for use in combination with FK 506. sion of activation antigens was detected as described pre­ viously24 by staining cells with mC-conjugated anti-C025 (IL-2 receptor. a chain). anti-HLA-DR or anti-CD71 (TR) Objectives mouse IgG monoclonal and either phycoerythrin (PE)-conjugated anti-C04 or anti-CDS mouse monoclonal To examine further the effects of FK 506. MP A and MZR antibodies. All antibodies were from DAKO and were used on human T cell activation. we investigated the expression of at a final I : 10 dilution. mc- and PE-conjugated mouse IgO several activation molecules (CD25 = IL-2R 55 kD a-chain. were used as background control. After washing, cells were HLA-DR and C071 = transferrin receptor[TRJ) on allosti­ fixed with 1% wlv paraformaldehyde. followed by analysis in mulated CD4+ and CDS+ lymphocytes cultured in the pres­ a FACSTAR flow cytometer (Becton-Dickinson, Rariton. ence of FK 506, MPA or MZR or when treated with NJ, USA). Five thousand cells were counted for each sample. combinations of FK 506 and either purine nucleotide synthe­ Significances of differences between means were determined sis inhibitor. It was anticipated that the results obtained using Student's t-test. would provide additional. new information on the effect of these drugs on lymphocyte activation and assist in the design of drug combination therapies. Results

The influence of various concentrations of FK 506, MP A or Materials and methods MZB. added at the start of culture. on the uptake JH-TdR in six-day human MLC is shown in Figure I. Each drug ex­ FK 506 (Fujisawa Pharmaceutical Co. Osaka. Japan) and hibited a dose-related. inhibitory effect with maximal inhibi­ MPA (Sigma Chemical Co. St Louis. MO. USA) were tion of DNA synthesis (85-97%) being achieved with 1.2 nM dissolved initially in absolute ethanol. MZB (Asahi Chemical FK 506.0.3 f.l.M MPA and 386 f.l.M MZB. Combinations of Industry Co Ltd. Tokyo. Japan) was dissolved in sterile. moderately effective concentrations of FK 506 and either distilled water before further dilution in cell cuI lUre medium. MPA or MZB produced no more than an additive inhibitory Peripheral blood mononuclear cells were isolated over Ficoll­ dfect on cell proliferation. Isopaque from heparinized samples obtained from healthy The effects of thc three immunosuppressive agents on adult volunteers of both sexes. One-way mixed lymphocyte human T cell surface IL-2R. HLA-DR and TR expression in reaction (MLR) cultures were established in RPMI 1640. three- and six-day MLC were examined by two-colour flow supplemented with 25 mmollL Hepes buffer and 100 Ulml cytometric analysis. Cultures harvested after three days in the gentamicin. with 10% normal human AB serum. Equal presence of FK 506. MPA or MZB showed no significant numbers (5 X 104 well) of responder and irradiated change in the incidence of CD4 + or CD8~ cells co-expressing (2000 Rad) stimulator cells were set up in flat-bottomed any of the activation markers (data not shown). By day six. micro titre plates in a total volume of 0.2 ml. Cells were however. there were significant inhibitory effects of all three cultured for three or six days. [-'H)-thymidine (I f.l.Ci/welll drugs on the incidence of both CD4' and CD8+ cells co­ was added for the final 16 hours of culture. The cells were <.!xpressing each of the markers (Figure 2). At concentrations harvested onto glass fibre discs. ustng a multiple harvester and ()f each drug which gave maXimal inhibition of DNA syntheSIS ['HI-thymidine uptake was estimated using a 1205 Betaplate (Figure I). but not at tenfold lower concentrations (which liquid scintillation counter (Pharmacla LKB Biotechnology were moderately antiproliferativel. FK 506. MPA and MZR Inc .. Piscataway. NJ). Results are expressed as mean counts significantly inhibited (p < (1.025) expressIOn of IL-2R on both per mmute (cpm). CD4' and COil' cells. The extcnt of supprcssion (5~)%)

FIC_!nM) 0.01 0.11 1.2 0.11 0.12 ..A ()III) 0._ 0.03 0.111 IIZII ()III) 1. 31.. ,. ... DRUG CONCENTRATION f1pre 1 The inftuence of vanous concentrallons ot FK 506. MPA and MZB and of drull combinallon on human MLR. Uptake of ·'HTdR was quantitated over the lasl 16 hours of six-day cultures. Results are means::!: I SO oblained (rom three separale expenments. 148 AW Thomson et al.

(A) IL-2R HLA·DR TR

• CD4 • CD4 • CD4 30 t:::I COl I>l COl

w > E en o a.. '"

FK 101 CONCENTRATION (nM)

(B) IL-2R HLA-DR TR

~~------CD4 • CD4 CD4

w > en~ o a..

MPA CONCENTRATION hlM)

(C) IL-2R HLA-DR TR

~.------~ CD4 CD4

w > en~ o a.. '"

MZB CONCENTRATION hlM)

F'tpft 2 The incidence of CD4· and CDS' lymphocytes expressing IL·2R. HLA·DR or TR in (A) FK 506-treated: (8) MPA·treated: and (C) MZB·treated MLC determmed by two· colour tlow cytometnc analysis six days after establishment of cultures. Rcsults are means :: 1 SO obtained from three separate expenments. Effects of drug combination on human MLR 149

IL-2R HLA-DR TR

• CD4 • CD4 • CD4 13 COl lSI COl 13 COl

w > Een o ~ fit

.. ~ ~ ; ;;; d :II d '" '".. ..'" '" If Iu ~ • iu ~ • ... ; ; • '"~ '"~ '"~

Figure 3 The incidence of CD4 + and CD8+ lymphocytes expressing IL-2R. HLA-DR or TR in six-day MLC cultures treated witn concentrations of FK 506 and MZB whicn. on their own. did not affect tne expression of these markers. Results are means =1 SO obtained from three separate expenments. MZB 110M; FK 506 nM.

was similar for both T cell subsets. although MZB was lated via the T cell receptor/CD3 pathway:·2~.26 Following especially effective in suppressing CD8+ IL-2R expression binding of FK 506 to its intracellular. cytosolic receptor (>80%). MZR was also the most effective agent and both FK 506 binding protein (FKBP: predominant member FKBP- MZR and MPA more effective than FK 506 in suppressing 12). the FK 506-FKBP-complex binds to the calcium­ HLA-DR expression. Differences between the effects of activated phosphatase .27 Current thinking is that these drugs. however. were not stastically significant. The FK 506 may block dephosphorylation of the cytosolic compo­

incidence of CD4 + and CD8 + cells expressing TR was also nent of the nuclear factor of activated T cells (NFAT)2H that is inhibited (p < 0.02) by all three drugs and to a greater extent required for transcription of IL-2 mRNA. than IL-2R and HLA-DR. although as with the latter activa­ Here we have extended our previous observations24•29 that tion markers. at only the highest drug concentration tested. FK 506 inhibits activation molecule expression on OKT3 Combinations of FK 506 and either MPA or MZB at stimulated or alloactivated human Iymphocvtes. Thus. in the concentraUons which. on their own caused only moderate present study. using two-colour immunoftuorescence staining. Inhibition of DNA synthesis and no significant reducuon In we have shown that FK 506 inhibits lL-2R. HLA-DR and TR cell surface marker expression. reduced the incidence of IL- on both CD4 + and CD8' alloactivated T cells. We could not 2R·. HLA-DR'. and TR· cd Is (Figure J; data shown for confirm an earlier report by Kino el al."~ that FK 506 inhibited FK 506-MZB combination only). IL-2R (CD25) more markedly on CDH' than on alloactivated CD4+ cells. The antiproliferative effects of MFA and MZB differ Discussion distinctly from those of FK 506. Both former drugs are purine biosynthesis inhibitors. It has been reported that Immunosup­ In this study. we have shown that the immunosuppressive pressive doses of MPA do not deplete lymphocytes and may agents. FK 506. MPA (the active moiety of RS-61443) and have no effect on DNA synthesis In nonlymphoid tissues. MZB each has the capacity to profoundly inhibit T cell such as gut epithelium or on most bone marrow progenitor proliferatlon and the expression of T cell surface activation cells.'" We have observed that neither MPA nor MZB affects molecules following allostimulauon. In each instance. sig­ Con A-induced T cell cvtokine gene expressIOn ([L-2. IL-4. mfican! suppression of IL-2R (CD25; 55 kD a-chain). HLA­ IFN-"/ or lL-IO) (data not shown). Little previous data arc DR and TR (CD71) on CD4' and CDS' cells was observed available concermng the effects of MPA or MZB on cyto­ at six but not at three days of culture. Moreover. this effect kines. Turka III al. l ? however. recently reported failure of was achieved at drug concentrations which caused at least MZB to inhibit phorbol mynstate acetate XO% inhibition of cell proliferation: lower concentrations. (PMA) + ionomycin-induced human T celllL-2 gene expres­ which produced <70% inhibition of DNA synthesis. did not sion or cell surface 55 kD IL-2R expressIOn. In the latter suppress actlvation marker expression. instance. the anugen was studied after T cells were stimulated Despite these similarities in the effects of the three agents. with PMA + ionomycin + antl-CD28. a much more powerful marked differences In potency were recorded. FK 506 proved stimulus than the alloantigen used in the present study. This highly effective in inhibiting cell proliferation at approx­ difference In strength of stimulus may explain the apparent imately I nM. whilse MPA and MZB were 100- and discrepancy between the two studies. IOOOO-foid less potent. Our data also confirm the distinct Advances in our understanding of events underlying T cell modes of action of FK 506 on the one hand and MPA and activation have helped pmpomt slles of actlon of immunosup­ MZB on the other. FK 506 has been shown to inhtbit pressive drugs and aided in the design of new drug combina­ selectively the activation and proliferation of T cells stimu- tion therapies. [n this study. combination of a suboptimal 150 AW Thomson et al. concentration of FK 506 with either MP A or MZB was found involvement of a G-protein. FEBS Lea 1987; 213: 199-203. to inhibit cell proliferation at approximately the level seen 14 O'Shea JJ. Urdahl KB. Luong HT. Chused TM. Samelson LE. with tenfold higher concentrations of FK 506. The apparent Klausner RD. Aluminium fluoride induces phosphatidylinositol selectively of MP A and MZB for lymphoid T cells in vivo turnover. elevation of cytoplasmic free calcium and phosphoryla­ tion of the T cell antigen receptor in murine T cells. I Immunol suggests that use of FK 506 together with either MP A or 1987: 139: 3463-69. MZB may be useful for the immunosuppressive therapy of IS Wilson 8S. Deanin GG. Standefer JC et al. Depletion of guanine 21 human allograft rejection. Indeed. Sollinger et al. reported nucleotides with mycophenolic acid suppresses IgE-receptor­ recently on the efficacy and safety of MPA (RS-61443) in mediated degranulation in rat basophilic leukemia cells. J Im­ combination with low doses of CsA and prednisone in clinical muno11989: 143: 2S~S. renal transplantation. Furthermore. like FK 506. neither 16 Eugui EM. Almquist SJ. Muller CD. Allison AC. Lymphocyte­ MP A nor MZB has been reported to affect the bone marrow selective cytostatic and immunosuppressive effects of mycophe­ at immunosuppressive doses.1 6.1 7 a possible advantage over nolic acid in vitro: role of deoxyguanosine nucleotide depletion. azathioprine in minimizing the toxicity of drug combination 5amd J Immunoll991; 33: 161-73. therapy. 17 Turka LA. Dayton J. Sinclair G. Thompson CB. Mitchell BS. Guanine ribonucleotide depletion inhibits T cell activation. Mech­ anism of action of the mizoribine. ) Ciin Acknowledgements Invest 1991: 87: 940-48. Supported in part by grant OK 29961 from the National 18 Zeevi A_ Woan M. Yao GZ el al. Comparative in vilro studies on Institutes of Health. We thank Or SC Wang for helpful advice the immunosuppressive activities of mycophenolic acid. bredinin. and Ms Shelly Conklin for typing the manuscript. FK 506. cyclosporine and rapamycin. TranspUmI Proc 1991; 13: 2928-30. 19 Amemiya H. Suzuki S. Niiva S. Watanabe H. Kotake T. Svner­ References gistic effect of cyclosporin~ and mizoribine on survival of dog renal allografts. Transplantalion IlJ8R: 46: 768-71. I Morris RE. Immunopharmacology of new xenobiotic immuno­ 20 Gregory CR. Gourley 1M. Cain GR el al. Effects of combination suppressIVe molecules. Semm Nephroll992: 4: 304-14. cyclosporine/mizoribine on canine renal al­ 2 Thomson A W. The spectrum of action of new immunosup­ lograft recipients. Transplantation IlJM: 45: 856-59. pressive agents. Gill Exp Immlllloll992: 89: 170-73. 21 Platz KP. Sollinger HW. Hullelt OA. Eckhoff DE. Eugui EM. 3 Schreiher SL. Crahtree GR. The mechanism of action of cyclo­ Allison AC. RS-61443 - a new. potent immunosuppressive agent. sporin A and FK 506. Immllllol Todav 1992: 13: 136-42. Tran.fplantalion 1991: 51: 27-31. 4 Tocci MJ. Matkovich DA. Collier KA 1'1 al. The immunosuppres­ 22 Sollinller HW. Dcierhoi MH. Belzer FO. Diethelm AG. Kauff­ sant FK SOn selective Iv inhibits expression of early T cell activa­ man RS. 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S<'anti) Imnlllllol IlJ'I(): 31: 2lJ7-3(14. . . II StaTZI TE. Fun!! J. V.:nkataramanan R. Todo S. Demetris AJ. Jain 25 Kino r. Halanaka H. Miyata S ~I al. FK 506. a novel im­ A. FK SOn for liver. kllinev and pancreas transplantation. l.ancel munosuppressant isolated from a Streptomyces. ll: immunosup­ IlJlIlJ: ii: IIMM~I{MI-4. pressive effect of FK 5(lfI in ~'ltrO. ). AntibiOl (TokwlI IlJ1I7: 40: 'I Thomson A W. Starzl TE. FK SOn and autOImmunity. Perspective 125fHl5. and prospects. Alllmmmllnltv 1l}'/2: 11: 303-13. 26 Sawada S. Suzuki G. Kawase Y. Takaku F. Novel immunosup­ II) Downward J. (imves JA. Warne PH. Rayter S. Cantrell DA. pressive agent. FK 5{lfI. 111 \'itro effects on the cloned T .:ell Stimulation ot 1'21'" upon r "dl activation. Nalllrt! I ')l}f): 346: aCllvallon. ) Imnlllllol I'IM7: 139: 17'17-l!03. 71'1-23. 27 Liu J. Farmer JD. Lane WS. Friedman J. Weissman I. Schreiher II Kiguchi K. C"llart ER. lIemmlO!!-Chuhh C. Huberman E. Cdl SL. Cakineurin is a common target of cyciophilin-cyclospoTln A differentiation and altered IMP-dehydrogenase expression in­ and FKBP-FK 5(lfI complexes. Cell 1991: 66: 1107-15. duced in human r-I"mphohlastoid leukemia cells hy mycophe­ 211 Flanagan WM. Corthesy B. Bram RJ. Crabtree GR. Nuclear nolic acid and tlazolunn. I:'.tp C,'II RI.'.f IlJ'JI): 187: 47-53. assocmtion of aT-cell transcTlption factor hlocked by FK S{lfI and 12 Ledbelter JA.Parsons M. Martin PJ. Hansen JA. Rabinovitch PS. cyc1osporin A. Nlllure 1991: 351: K03~)7. June Clf. Antlhodv hindlO!! to ('05 (Tp67) and Tp44 cell surface 2lJ Woo J. Zeevi A. Yao GZ. Strednak J. Todo S. Thomson AW. molecuh:s: dfects un c"chc nucleotllies. cytoplasmiC free calcium Effects of FK 5{lfI. mycophenohc acid and bredinin on OKT3-. and cAMP-mediated ,uppressllln. J Immllllol 1'IlIn: 137: "MA- and alloanll!len-induced activallon molecule expression on .1299-305. cultured <,'04' and COli' human lymphocytes. Transplant I'roc U Mustelin T. (iTP dependence 01 the transduction of mitollenic 1991: 13: 2939-41). ,ignals throu!!h the T.l .:omplex 10 r lymphocyte IOdicates the