Duquesnoy, RJ

Starzl, TE

Todo, S

Venkataramanan, R

Woan, M

Yao, GZ

Zeevi, A

English

PubMed

D-Scholarship@Pitt

f~l 1\1.1. lid I:': Iii 1;, I Comparative In Vitro Studies on the Immunosuppressive Activities of Mycophenolic Acid, Bredinin, FK 506, Cyclosporine, and Rapamycin A. Zeevi, M. Woan, G.-Z. Yao, R. Venkataramanan, S. Todo, T.E. Starzl, and R.J. Duquesnoy OVER the past 25 years various immunosuppressive drugs have been screened for clinical usage in con-trolling allograft rejection, autoimmune disease, and ma-lignancy. In vitro methodologies have permitted a rapid assessment of potential new drugs. In this study we have compared the immunosuppressive effects of two purine synthesis inhibitors mycophenolic acid (MPA)I and bred-inin (BR)2 to the known immunosuppressive drugs FK 506, rapamycin (RAPA), and cyclosporine (CyA). A vari-ety of culture systems were used to study the potency of different drugs and their mechanisms of action. Such models included T-cell activation via calcium-dependent (A23187 and phytohemagglutinin [PH A]) and calcium-independent (interleukin-2 [IL-2J and phorbol myristic acetate [PMAJ) pathways. We tested the effect of immu-nosuppressive drugs on freshly prepared peripheral blood lymphocytes (PBL), and transformed T- and non-T-cell lines. In addition, we also studied the combined effects of MPA, BR, and low doses of FK 506 and CyA in our system to determine whether there is any synergistic or additive effect between these drugs. MATERIALS AND METHODS Cells PBL were isolated by Ficoll-Hypaque density gradient centrifu-gation of heparinized blood from healthy donors. Cells were resuspended in RPMI 1640 tissue culture medium (TCM) supple-mented with 25 mmoVL Hepes buffer and 100 UlmL gentamycin and 5% normal human AB serum. Reagents FK 506 (Fujisawa Pharmaceuticals. Osaka. Japan) and CyA (Sandoz. Basel, Switzerland) were dissolved in methanol (1 mgt mL). MPA (Sigma Chemical Co, St Louis, Mo), BR (Sumitomo Chemical Co Ltd, Takarazuka, Japan), and RAPA (Wyeth-Ayerst Research. Rahway, NJ) were dissolved in ethanol. water, and methanol. respectively, before use. Calcium ionophore (A23187), PHA, and PMA were purchased from Sigma. Proliferative Responses of Lymphocytes Mitogen-Induced Proliferation Assays. Mononuclear cells were cultured in TCM at lOS cells/well (Nunc round bottom tissue culture plates) in the presence of A23187 (l ~miFI PHA (1%), or PMA (0.1 ~miF in a volume of 200 "L for 3 days. Mixed Lymphocyte Reaction (MLR assay). One-way MLR cultures were established with equal numbers (5 x 104/well) of responder and irradiated stimulator cells in TCM and incubated for 6 days. PLT Test. Alloreactive T cells (2 x 104/well) were incubated with lIP irradiated (2,000 rad) stimulator cells for 3 days. IL-2 Induced Proliferation. Alloreactive T-cell lines (2 x 104/ well) were incubated with 30 UlmL of recombinant IL-2 for 3 days. [n all assays, proliferation was measured by the degree of 3H-thymidine incorporation during the last 20 hours of incubation. Proliferative Responses of Various Cell Lines T-Iymphoma cell lines (DND41, Molt 13, Peer), EB virus-trans-formed B-Iymphoblastoid cell lines (DT, CK, RK. DN), B-lym. phoma cell line (RPMI-1788), erythroleukemia cell line (K562), and promyelocytic cell line CHL-60) were maintained in TCM supplemented with 10% fetal calf serum (FCS). DND4I. Molt 13, and Peer were kindly provided by Dr C. Milcareck (Molecular Biology, University of Pittsburgh). RPMI-1788 and HL-60 were obtained from American Type Culture Collection (Rockville. Md). K562 was supplied by Dr T. Whiteside (Pittsburgh Canc"f Institute). Proliferation was measured after 72 hours of incubaul 1 by 3H-thymidine incorporation. Drug Inhibition Assays The inhibitory effects of various drugs alone or in combination were measured at different concentrations. The % inhibition was calculated as follows: % inhibition = (I-cpm with drug/cpm without drug) x 100. The drug dose required to obtain 50% inhibition (lC50) was calculated by computer analysis, [BMP, using a nonlinear sigmoi· dal model curve fitting program (SAS. Inc, Cary, NC). RESULTS Effects of Immunosuppressive Drugs on T-Cell Proliferation Table I summarizes the IC50 values of five immunosup-pressive drugs in the proliferation of T cells activated b:, Ca-dependent (A213187 and PHA) and Ca-independem (IL-2 and PMA) pathways. Both FK 506 and RAPA exerted profoundly inhibitory effects (IC50 less than I nmollL) of Ca-dependent T-cell proliferative responses whereas CyA and MPA required loo-fold higher concen-trations. The IC50 of BR was about \04-fold higher than those for FK 506 and RAPA. In contrast to FK 506 and Cy A, RAPA caused profound inhibition of Ca-independent T-cell proliferation (Table 1). From the Division of Transplantation Pathology (A.Z .. M.W., G.-Z.Y., R.J.D.), Department of Pathology, School of Pharmacy (R.V.), and the Division of Transplant Surgery (S.T., T.E.S.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. Supported in part by grants AI-23467 and DK-29961 from the National Institutes of Health and the Pathology Education Re-search Foundation. Address reprint requests to Adriana Zeevi, PhD. University of Pittsburgh Medical Center, Biomedical Science Tower. Room W1551. Pittsburgh, PA 15261. © 1991 by Appleton & Lange 0041-1345191/$3.00/+0 2928 Transplantation Proceedings, Vol 23, No 6 (December), 1991: pp 2928-2930 IN VITRO STUDIES OF IMMUNOSUPPRESSANTS Table 1. Inhibitory Effects of Immunosuppressive Drugs on T-Cell Proliferation IC50 (nmollL) T-Cell Activation Pathways Mitogen FK 506 eyA RAPA MPA SR A23187 0.18 23 0.42 81 NT Ca-dependent PHA 0.72 857 0.87 232 8,919 IL·2 No inhibition 0.61 119 7,637 Ca-independent PMA No inhibition 0.35 81 14,861 The same magnitude of IC50 was observed for RAP A in Ca-dependent and Ca-independent T-cell proliferation. Like RAPA, both MPA and BR inhibit T-cell proliferation induced by Ca-dependent and Ca-independent pathways. However, compared with RAPA, the IC50 were 102_ and lOS-fold higher for MPA and BR, respectively. On the other hand. MP A and BR were inhibitory towards Ca-independent T-cell proliferation at similar IC50 levels as observed for Ca-dependent proliferation. The findings sug-gest that RAP A. MP A. and BR inhibited both T-cell activation pathways at similar IC50 values for each drug, but the required concentrations ofMPA and BR were 102_ and lOs-fold higher than those of RAPA. Inhibition of Alloreactive T-Cell Responses by Immunosuppressive Drugs All four drugs tested (FK 506. CyA. MPA, and BR) inhibited the primary MLR responses and the secondary PLT proliferation of alloreactive T cells (Table 2). The IC50 for FK 506 was 0.1 nmol/L whereas IC50 values for MPA and BR were 102_ and lOs-fold higher than for FK 506. Combinations of low Doses of MPA and SR With FK 506 An additive effect on inhibition of PLT response was demonstrated with the combination of MPA (25 ng/mL) and FK 506 (0.001 to 0.1 ng/mL) (Fig 1). Similar results were achieved with combinations of BR (2,500 ng/mL) and FK 506 (0.001 to 0.1 ng/mL) (Fig 1). In contrast. increased levels of MP A and BR produced no significant additive effects with low doses of FK 506 (data not shown). No evidence was obtained for synergism or antagonism be-tween these drugs in the suppression of PLT proliferation. Table 2. Inhibition of Alloreactlve T-Cell Responses by Immunosuppressive Drugs A1loatimulation Primary (MLR) Secondary (PL T) FK506 0.1 0.17 ICSO (nmoVL) CyA 10 29 MPA 103 163 BR 14,706 28,751 2929 1 • K---------------~ B -... • o 0 OcMI1..,.. __ r-KKKIKKKK~-K___f 0.00 0.01 0.0. 0.01 0.01 0.10 0.11 D.DD '.01 0.0. o.oa .... 0.100.12 ng/ .. - IlOl .. PK5DI Fig 1. Inhibition of PL T response. Combination of low doses of FK 506 (0.01 to 0.1 ng/ml) with (a) MPA (25 ng/ml) and (b) BR (2,500 ng/mL). The results are expressed as % inhibition of PL T response in the presence of FK 506 alone (0) or in combination with MPA or BR (.). Effects of Immunosuppressive Drugs on the Proliferation of Various Transformed Cell Lines Table 3 summarizes the antiproliferative effects of MPA, BR. and RAPA on various T-cell lymphomas, EB virus-transformed B-Iymphoblastoid cell lines. and other tumor cell lines. MP A significantly inhibited all cell lines tested at drug concentrations of 90 to 365 nmollL. Similar inhibition was observed with BR, however, the IC50 dose of BR was 100 to 200-fold higher than those of MPA. In contrast. RAPA exhibited a more restricted spectrum of activity than MP and BR. The sensitivity of tumor cell lines to RAPA varied from sensitive (EB virus-transformed B-cell lines) to more resistant (K562, PEER) (Table 3). DISCUSSION These data show that MPA and BR inhibit in a dose-dependent manner the proliferative responses of normal T cells to mitogenic and alloantigenic stimulation. Both Ca-dependent and Ca-independent T-cell activation pathways are equally sensitive to MPA and BR immunosuppression. In contrast, FK 506 and Cy A are most efficient in blocking Ca-dependent T-cell responses.3--5 The drug inhibitory activity seems to be cell cycle dependent. Thus, FK 506 and Cy A act at an early stage of T-cell differentiation by inhibiting the cell division cycle at the GJG t interface6 Table 3. Inhibitory Effects of Immunosuppressive Drugs on the Proliferation of Various Transformed Cell Lines ICSO (nmollL) Cell Culture Origin MPA BR RAPA DND41 TLymphoma 91 11,583 NT MOLT 13 TLymphoma 365 96,525 274 PEER T Lymphoma 275 28,275 No inhibition EB-DT B Lymphoblast 167 15,984 11 EB-CK B Lymphoblast 164 25,135 NT EB·RK B Lymphoblast 274 46,332 NT EB·DN B Lymphoblast 292 42,471 NT RPMI-1788 B Lymphoma 182 19,305 10 K562 Erythroleukemia 304 17,760 No inhibition HL-60 Promyelocytic 365 32,007 273 Note: FK 506 and CyA have no InhibitOlY ellee!. i 1 j j flitl ., r. I: If , ;i ". '1. i ,. :i 2930 while MPA and BR act at a later stage of cell activation pathway by blocking the movement of cells from G 1 to S.7.8 Similarly. RAPA acts at a later stage in cell cycle at some point in G 1.6 MP A and BR also block the proliferation of many lymphoid and nonlymphoid cell lines. The inhibitory ef-fects of these drugs are mediated through inhibition of ionosin monophosphate phosphatase dehydrogenase. an enzyme required for guanine nucleotide synthesis.7.8 Other drugs known as inhibitors of purine synthesis are methotrexate9 and azathioprine. 10 However. the cytostatic effects of these drugs are similar for all cell types tested (lymphoid cells. fibroblasts. and endothelial cells) whereas MPA and BR seem to affect primarily T and B cells.7.8 Since RAPA significantly inhibited the in vitro expansion of EB virus-transformed lymphoblastoid cell lines it is likely that RAPA may have clinical efficacy in treating EBV -associated posttransplant lymphoproliferative dis-ease. No synergism between MPA or BR with FK 506 or Cy A was observed in the inhibition of allogeneic T-cell prolif-eration. At most. an additive effect was seen with low doses of drugs tested in the MLR and PL T test. The additive effects have also been reported for combinations of Cy A and BR in an in vitro model of CD3-activated T-cell proliferationS and in experimental and clinical cadaveric renal allotransplantations."·12 Recently. Todo and col-leagues showed a significant prolongation of renal al-ZEEVI. WOAN. YAO ET AL lografts in dogs treated with low doses of FK S06 and BR (personal communication). Thus. MPA and BR may offer an alternative regimen when used in conjunction with other immunosuppressive drugs in the treatment of al-lograft rejection. REFERENCES I. Franklin TJ. et al: Biochem J 113:515. 1969 2. Mizuno K. Tsugino M. Takada M. et a1: J Antibiot (Tokyo) 24:775. 1974 3. Sawada S. Suzuki C. Kawase Y. et a1: J Immunol139:797, 1987 4. Thomson AW. Woo J. Zeevi Z. et aI: In: Brossard C (cd). Handbook of Immune Response Modifien. Berlin. Lavoisierl Springer-Verlag. 1991. in press 5. Kay JE. Benzie CR. Goodier MR. et a1: Immunology 67:473. 1989 6. Metcalfe SM. Richards FM: Transplantation 49:798. 1990 7. Eugui EM, SjaIquist C, Muller CD, et al: Scand J Immunol 33: 1619, 1991 8. Turka LA, Dayton J, Sinclair G. et aI: J Clin Invest 87:940. 1991 9. Jolivet j, Cowan KH. Curt GA. et aI: N Eng! J Med 309:1094, 1983 10. Herlia P. Murelli L. Scotti M, et a1: Carcinogenesis 9:1011, 1988 II. Osakabe T, Vchida H. Masaki Y. et al: Transplant Proc 21:1598. 1989 12. Amemiya H. Suzuki S. Watanabe H. et a1: Transplant Proc 21:956, 1989 

Comparative in vitro studies on the immunosuppressive activities of mycophenolic acid, bredinin, FK 506, cyclosporine, and rapamycin

Yao, G-Z

Name not available

http://d-scholarship.pitt.edu/4706/1/31735062118736.pdf

Comparative in vitro studies on the immunosuppressive activities of mycophenolic acid, bredinin, FK 506, cyclosporine, and rapamycin

Abstract

Similar works

Full text

Available Versions

D-Scholarship@Pitt

Name not available

Name not available