- Email: [email protected]
Inhibition of calcineurin activity and protection against cyclosporine A induced cytotoxicity by prednisolone sodium succinate in human peripheral mononuclear cells
Immunopharmacology 48 Ž2000. 87–92 www.elsevier.comrlocaterimmpharm
Short communication
Inhibition of calcineurin activity and protection against cyclosporine A induced cytotoxicity by prednisolone sodium succinate in human peripheral mononuclear cells Sandor Sipka a,) , Kornelia Szanto ´ ´ Szucs ¨ b, Sandor ´ ´ ´ a, Ildiko´ Kovacs ´ a, Gabriella Lakos a , Peter ´ Antal-Szalmas ´ a, Gyula Szegedi a, Pal ´ Gergely b a
3rd Department of Internal Medicine, UniÕersity Medical School of Debrecen, Moricz Zs. ut ´ ´ 22, H-4004 Debrecen, Hungary b Department of Medical Chemistry, UniÕersity Medical School of Debrecen, Debrecen, Hungary Received 19 August 1999; received in revised form 6 January 2000; accepted 6 January 2000
Abstract We have investigated the effects of prednisolone sodium succinate ŽPss. and cyclosporin A ŽCSA., applied alone or concurrently, on the release of arachidonic acid ŽAA. Žcytosolic phospholipase A 2 ŽcPLA 2 . activity. and on the calcineurin ŽCN. activity of human peripheral blood mononuclear cells ŽPBMC.. The cytotoxic damage to the cells treated by the drugs was estimated by the release of lactate dehydrogenase ŽLDH.. We found that Pss Ž10y5 M. could inhibit the CN activity and higher concentrations Ž10y4 M. could decrease the cytotoxic damage caused by CSA Ž10y4 M. during their combined application. CSA had no specific effect on the release of AA from the cells. In the combined clinical use of glucocorticosteroids ŽGCS. and CSA, their additive inhibitory effect on CN activity and the protective membrane influence of GCS against the cytotoxicity of CSA may be beneficial. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Glucocorticosteroid; Cyclosporin A; Calcineurin; Arachidonic acid
1. Introduction Both glucocorticosteroids ŽGCS. and cyclosporin Ž A CSA. are widely used drugs in immunosuppres-
AbbreÕiations: Pss, prednisolone sodium succinate; CSA, cyclosporin A; GCS, glucocorticostreoids; AA, arachidonic acid; CN, calcineurin; cPLA 2 , cytosolic phospholipase A 2 ; PBMC, peripheral blood mononuclear cells; LDH, lactate dehydrogenase; PMA, phorbol myristate acetate ) Corresponding author. Tel.: q36-52-414-969; fax: q36-52414-969. E-mail address: [email protected] ŽS. Sipka..
sive and anti-inflammatory therapeutic protocols. GCS can block the production of various pro-inflammatory mediators, e.g. prostaglandins, leukotrienes, platelet activating factor, histamine and cytokines, which have both mediatory and immunoregulatory functions ŽBarnes, 1996.. In these effects, the inhibition of cytosolic phospholipase A 2 ŽcPLA 2 . plays an important role, as arachidonic acid ŽAA., one of the products of cPLA 2 , is the precursor molecule of prostaglandins, thromboxanes and leukotrienes ŽDennis, 1997; Leslie, 1997.. GCS also have a direct membrane effect and can inhibit the influx of Ca2q ŽButtgereit et al., 1998..
0162-3109r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 0 0 . 0 0 1 8 0 - 6
88
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
In the immunosuppressive effect of CSA, the main element is its intracellular coupling to cyclophilin. The CSA–cyclophilin complex can block the activity of the calmodulinrCa2q-dependent protein phosphatase Žcalcineurin ŽCN.. resulting in a decreased dephosphorylation of the transcriptional factor NF-AT Žnuclear factor of activated T cells., which leads to a reduced production of a series of cytokines ŽIL-2, IL-4, etc.. and cell surface molecules ŽCD40, LCAM, etc.., ŽHo et al., 1996; Ruhlmann ¨ and Nordheim, 1997.. Some clinical and experimental observations have shown advantageous immunosuppressive effects in the combined applications of GCS and CSA ŽSzegedi et al., 1996; Quien et al., 1997; Redington et al., 1998; Kurosawa et al., 1999.. In this study, we show that GCS can inhibit the activity of CN and can additively contribute to the decreased CN activity caused by CSA. In addition, GCS may have some protective membrane effect against the cytotoxicity of CSA during their combined use in vitro.
2. Materials and methods 2.1. Preparation of human peripheral blood mononuclear cells (PBMC) and characterisation of cells by flow cytometry PBMC, containing 88–95% lymphocytes and 5– 12% monocytes, were prepared ŽBoyum, 1968. from the heparinized blood of 6 healthy donors Žthree men and three women, age: 21–54 years.. The averages of the various subsets were detected by flow cytometry: CD3q: 69.4%, CD19q: 11.5%, CD56q: 0.8% and CD14q: 8.3% ŽCoulter EPICS XL flow cytometer, Coulter, Hialeah, FL.. 2.2. Determination of cPLA 2 actiÕities by the measurement of AA release The cells Ž10 5rml. were preincubated with w3 HxAA ŽAmersham, UK. in a CO 2 incubator at 378C for 20 h. After extensive washing, the cells were further incubated without any stimuli for 4 h. The amount of released AA reflected the basal activity of cPLA 2 . Activation of cPLA 2 was achieved by the addition of 5 mM of calcium ionophore ŽA
23187, Sigma, St. Louis, MO. and 50 ngrml of phorbol 12-myristate 13-acetate ŽPMA, Sigma, St. Louis, MO. at 378C for 4 h. The amount of released AA reflects the activity of cPLA 2 of stimulated cells ŽLin et al., 1992; Zor et al., 1993.. GCS or CSA, incubated with the cells for 20 h, did not affect the uptake of w3 HxAA and the cPLA 2 assays were therefore comparable in the two cases. 2.3. Determination of CN actiÕity The cell suspensions were washed with phosphate-buffered saline and collected by centrifugation. Cells were sonicated for 2 = 30 s with a Branson sonifier in 2 vol of homogenisation buffer containing 50 mM Tris–HCl ŽpH 7.0., 0.5 M dithiothreitol, 10 mgrml aprotinin, 10 mgrml leupeptin, 10 mgrml trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride ŽPMSF., 5 mM benzamidine and 0.3% Triton X-100. The homogenate was centrifuged for 10 min at 10,000 = g, supernatants were collected and stored in aliquots at y708C prior to the assay. CN activity was measured by the release of 32 Pi from w32 Pxinhibitor-1 ŽYang et al., 1982; Cohen et al., 1988.. The assay mixture Ž30 ml. containing 50 mM Tris–HCl ŽpH 7.0., 0.3 mM dithiothreitol, 0.2 mM CaCl 2r1 mM MnCl 2r0.04 mgrml calmodulin or 2 mM EGTA, appropriate amount of extract Ž0.5–1.5 mgrml protein. and w32 Pxinhibitor-1 Ž12,000–25,000 cpmrreaction mixture. was incubated at 308C for 10 min. All mixtures contained 10 mgrml aprotinin, 10 mgrml leupeptin, 10 mgrml trypsin inhibitor, 1 mM PMSF, 5 mM benzamidine as protease inhibitors and 20 nM okadaic acid as a protein phosphatase 2A inhibitor. The reaction was terminated by the addition of 100 ml of 10% trichloroacetic acid. 32 Pi of the supernatant fraction was determined after centrifugation by Cerenkov counting in a liquid scintillation counter. The activity of CN was calculated as the difference of 32 Pi counts in the presence and absence of Mn2qrCa2qr calmodulin. 2.4. Measurement of lactate dehydrogenase (LDH) actiÕity LDH activities in cell supernatants represent the degree of cell damage ŽKobayashi et al., 1999. and
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
were determined by a kinetic UV test using the Boehringer SYS2 kit in a Hitachi 717 analyser. The results were expressed in Url. 2.5. Statistical analysis The statistical means and S.D. values of data were calculated. The statistical significance of the differences was determined by Student’s paired and unpaired t tests Ž p - 0.01 was considered significant.. At least three independent experiments were performed and all samples were used in triplicates. 3. Results 3.1. Effects of prednisolone sodium succinate (Pss) and CSA on the AA and LDH releases of PBCM First, the effects of 10y7 , 10y5 and 10y4 M concentrations of Pss and CSA applied alone in the cultures of PBMC for 20 h were investigated on the AA and LDH releases. As shown in Table 1, Pss significantly decreased the AA release at 10y5 M concentration in the stimulated cells, and 10y4 M of Pss resulted in an almost complete inhibition Ž5607 " 513, 4352 " 392 dpm vs. 9621 " 945 dpm, p 0.01 and p - 0.001, respectively.. There was practically no cytotoxic effect of Pss at the concentrations applied, as revealed by LDH release. 10y4 M of
89
CSA also reduced the AA release of stimulated PBMC Ž6605 " 693 dpm vs. 9621 " 945 dpm p 0.01.. This effect could be related to the cytotoxic effect of the drug as shown by the release of LDH in the stimulated and non-stimulated cells Ž89.3 " 9.1, 98.2 " 10.4 Url vs. 37.1 " 4.8, 30.2 " 5.1 Url, respectively, p - 0.001.. On the other hand, 10y5 M of CSA exerted only a cytotoxic effect Ž74.5 " 11.3, 75.4 " 9.8 Url vs. 30.2 " 5.1, 37.1 " 4.8 Url, respectively., without any significant influence on the AA release Ž3645 " 477, 8177 " 792 dpm vs. 3857 " 689, 9621 " 945 dpm, respectively.. It was of interest that in the stimulated cells treated with 10y4 M of Pss, the release of LDH was significantly less than in the Pss-free culture Ž24.2 " 3.5 Url vs. 37.1 " 4.8 Url, p - 0.01. implying that this dose of GCS had some membrane protecting effect. When the two drugs were administered concurrently in 10y4 M doses, the inhibition of AA release was almost complete Ž4426 " 319 dpm vs. 9621 " 945 dpm. with a considerable cell damage Ž57.5 " 8.7, 60.2 " 7.2 Url vs. 30.2 " 5.1, 37.1 " 4.8 Url, respectively.. However, these LDH values were significantly lower than those of the single treatment with CSA Ž57.5 " 8.7, 60.2 " 7.2 Url vs. 98.2 " 10.4, 89.3 " 9.1 Url, respectively, p - 0.01.. Our results demonstrated that 10y4 M of Pss had a protective effect against the cytotoxicity of CSA. 10y7 M of Pss could not prevent the cytotoxicity
Table 1 Effect of Pss and CSA on the releases of AA and LDH in human mononuclear cells stimulated and non-stimulated by phorbol ester and Ca2q ionophore The cells were incubated with Pss orrand CSA for 20 h. PBMC were stimulated by 5 mM of A23187 and 50 ngrml of PMA for 4 h. The releases of AA and LDH were assayed as given in Materials and methods. p values were compared with the respective controls. Concentrations ŽM.
0 10y7 10y5 10y4 Pss: 10y4 qCSA: 10y4 Pss: 10y7 qCSA: 10y5 U
p - 0.01. p - 0.001.
UU
AA release Ždpm. Non-stimulated cells
Stimulated cells
Pss
Pss
CSA
3857 " 689 4311 " 445 3618 " 414 4073 " 432 3645 " 477 4013 " 468 3687 " 486 4251 " 452 3843 " 318
CSA
9621 " 945 7704 " 842 9252 " 860 5607 " 513 ) 8177 " 792 4352 " 392 ) ) 6605 " 693 ) 4426 " 319 ) ) 5004 " 576 ) )
LDH release ŽUrl. Non-stimulated cells
Stimulated cells
Pss
Pss
CSA
CSA
30.2 " 5.1 30.6 " 3.9 46.8 " 8.8 33.7 " 3.5 74.5 " 11.3 ) ) 29 " 4.3 98.2 " 10.4 ) ) 57.5 " 8.7 ) )
37.1 " 4.8 34.2 " 4.2 42.7 " 9.1 30.3 " 4.6 75.4 " 9.8 ) ) ) 24.2 " 3.5 89.3 " 9.1) ) 60.2 " 7.2 ) )
75.7 " 7.6 ) )
72.4 " 5.4 ) )
90
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
Fig. 1. Effect of Pss and CSA on the CN activity of human mononuclear cells. PBMC were incubated with various doses of Pss and CSA administered alone or concurrently for 24 h. CN activity was assayed as given in Materials and methods. The data represent the mean of 3–4 independent experiments and the bar shows the standard deviation. p values were compared with the respective control samples.
caused by 10y5 M of CSA Ž75.7 " 7.6, 72.4 " 5.4 Url vs. 74.5 " 11.3, 75.4 " 9.8 Url, respectively. ŽTable 1.. 3.2. Effect of Pss and CSA on the CN actiÕity of PBMC Non-stimulated PBMC were incubated with Pss orrand CSA for 24 h. We did not test the stimulated cells. The CN activities were determined from the sonicated lysates of these cells. As shown in Fig. 1, CSA dose dependently inhibited the CN activities, as was expected. Unexpectedly, both 10y5 and 10y4 M concentrations of Pss Žnot toxic doses. also significantly inhibited the CN activity, Ž11.2 " 1.0 and 6.6 " 0.6 pmolrmgrprotein vs. 19.1 " 1.8 pmolrmgrprotein, respectively, p - 0.01.. When CSA and Pss were applied concurrently at the same concentrations, the CN activity was further inhibited compared to the effect of drugs used alone. In addition, there was some additivity in the action of these compounds on the CN activity Ž5.2 " 0.5, 0.75 " 0.1, 0.19 " 0.05 pmolrmgrprotein vs. 6.7 " 0.6, 2.3 " 0.2, 0.97 " 0.1 pmolrmgrprotein, respectively.. The inhibitory effect of Pss ŽGCS. on the CN activity of PBMC is a new observation. However, it
has to be emphasised that a much higher concentration of GCS Ž10y4 M. is needed to obtain an inhibition of CN activity similar to that of CSA Ž10y7 M.. In addition, the presence of GCS may result in some additivity in the inhibition of CN caused by CSA.
4. Discussion The clinical application of GCS and CSA represented considerable progress in the therapy of patients with autoimmune diseases, allergy and organ or bone marrow transplantations. However, both GCS and CSA have well known negative side effects, limiting their prolonged administration to patients. GCS can cause cataract, osteoporosis, muscle atrophy or metabolic disorders ŽBarnes, 1996.. In the nephrotoxicity caused by CSA treatment, both the tubular and vascular systems of the kidney can be dose dependently damaged ŽThiru et. al., 1983. based upon the changing of membrane potential ŽVereb et.al., 1990., the inhibition of Ca2q release from intracellular stores ŽMisra et al., 1998. and the increased local production of reactive oxygen species ŽParra et al., 1998.. Therefore, any approach in the
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
clinical application of these compounds which preserves or improves the efficacy of immunosuppression but which decreases the negative side effects could be of great theoretical and practical importance. Our observations have two major points. First, Pss ŽGCS., as well as CSA, can inhibit CN activity and there may be some additivity in the effects of two compounds. However, GCS are less potent inhibitors of CN activity than CSA. Second, the cell membrane protecting effect of GCS against the cytotoxicity of CSA may be very beneficial during their combined therapeutic use. One may suppose that the decreased activity of CN can lead to a reduced production of NF-AT dependent cytokines: IL-2, IL-4, IL-8, IL-13, etc. ŽFirestein and Manning, 1999.. To understand the mechanism whereby GCS can inhibit CN activity, new connections have to be found in the regulation of transcriptional factors ŽNF-AT, NF kappa B, AP1., heat shock proteins, GCS receptors, immunophilins and ion metabolism ŽRuhlmann and ¨ Nordheim, 1997; Buttgereit et al., 1998.. For example, the intracellular production of an inhibitor of k B is known to be increased under the influence of GCS treatment ŽBarnes, 1996., and it can also be a substrate in the CN mediated dephosphorylation processes ŽRuhlmann and Nordheim, 1997.. GCS can ¨ inhibit the CN-dependent activation of the interleukin 2 gene in human T lymphocytes ŽPaliogianni and Boumpas, 1995.. Furthermore, as the inhibition of CN by CSA is rapidly reversible ŽBatiuk et. al., 1997., in a combined therapy GCS may prolong the effect of CSA. However, the main mechanism involved in the GCS induced inhibition of CN may be related to the reduction of Ca2q available for the calmodulin pathway ŽAntoni, 1996. and to the inhibition of Ca2q influx ŽButtgereit et al., 1998.. The reduction of Ca2q influx and the stabilization of the cell membrane ŽButtgereit et al., 1998. can play important roles in the protective effect of GCS against CSA induced cytotoxicity because CSA modulates the intracellular level of Ca2q ŽNicchitta et al., 1985; Misra et al., 1998. and causes depolarization of the cell membrane ŽDamjanovich et al., 1987; Vereb et al., 1990.. CSA had no specific influence on the release of AA from human peripheral mononuclear cells,
91
namely on the cPLA 2 activity. The decreased AA release found in the cells treated with CSA could be related to the cytotoxic damage caused by the drug. In contrast, CSA was found to activate and not to inhibit the PLA 2 activity in hamster renal tubular cells ŽAnderson et al., 1994.. The measurement of LDH activities for testing the degree of cytotoxic damage caused by CSA proved to be a sensitive and useful method. In addition, Pss and CSA did not induce apoptosis in the cultured cells during 24 h, measured by flow cytometry on the basis of propidium iodide uptake Ždata not shown.. Here we presented data on the CN inhibiting effects of CSA and Pss found in non-stimulated human peripheral mononuclear cells. When we measured the CN activities of the cells stimulated by phorbol ester and Ca2q ionophore, a profound decrease Ž87%. was observed in the absence of CSA or GCS Ždata not shown.. Therefore, we preferred to demonstrate the inhibition of CN activities caused by CSA or GCS ŽPss. in non-stimulated cells because these had about six-fold higher activities than the stimulated cells. The mechanism of the markedly decreased CN activities in the stimulated cells is still obscure. One candidate for the blocking of CN activity could be the Cabin 1 molecule, a member of the protein kinase C induced signaling pathway of T cells ŽSun et al., 1998.. In summary, our data show that GCS can additively contribute to the inhibition of CN activity caused by CSA during combined application of the two drugs. Simultaneously, GCS can protect the cell membranes against the cytotoxic side effects of CSA. Acknowledgements The authors thank Mrs. Julia Hunyadi for her expert technical assistance. This work was supported by grants of the National Scientific Research Fund ŽOTKA T023199 and T026541. and Ministry of Education ŽMKM 0784r97. and Ministry of Health ŽETT 315r96 and 093r96.. References Anderson, R., van Rensburg, C.E.J., Myer, M.S., 1994. Alphatocopherol prevents cyclosporin A-mediated activation of
92
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
phospholipase A 2 and inhibition of NaqrKq-adenosine triphosphatase activity in cultured hamster renal tubular cells. Toxicol. Appl. Pharmacol. 125, 176–183. Antoni, F.A., 1996. Mortyn Jones Memorial Lecture — 1995. Calcium checks cyclic AMP corticosteroid feedback in adenohypophysial corticotrophs. J. Neuroendocrinol. 8, 659–672. Barnes, P.J., 1996. Molecular mechanism of steroid action in asthma. J. Allergy Clin. Immunol. 97, 159–168. Batiuk, T.D., Kung, L., Halloran, P.F., 1997. Evidence that calcineurin is rate-limiting for primary human lymphocyte activation. J. Clin. Invest. 100, 1894–1901. Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. J. Clin. Lab. Invest. Suppl. 97, 77–108. Buttgereit, F., Wehling, M., Burgmeister, G-R., 1998. A new hypothesis of modular glucocorticoid actions. Arthritis Rheum. 41, 761–767. Cohen, P., Foulkes, G., Holmes, C.F.B., Nimmo, G.A., Tonks, N.K., 1988. Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 159, 427–433. Damjanovich, S., Aszalos, A., Mulhem, S., Szollosi, ¨ ¨ J., Balazs, ´ M., Tron, L., Fulwyler, M.J., 1987. Cyclosporin depolarizes human lymphocytes. Earliest observed effect on cell metabolism. Eur. J. Immunol. 17, 763–768. Dennis, E.A., 1997. The growing phospholipase A 2 superfamily of signal transduction enzymes. TIBS 22, 1–2. Firestein, G.S., Manning, A.M., 1999. Signal transduction and transcription factors in rheumatic disease. Arthritis Rheum. 42, 609–621. Ho, S., Clipstone, N., Timmermann, L., Northtrop, J., Graef, I., Fiorentino, D., Nourse, J., Crabtree, G.R., 1996. The mechanism of action of cyclosporin A and FK506. Clin. Immunol. Immunopathol. 80, S40–S45. Kobayashi, T., Okamoto, K., Kobata, T., Hasunuma, T., Sumida, T., Nishioka, T., 1999. Tumor necrosis factor alpha regulation of the FAS-mediated apoptosis-signaling pathway in synovial cells. Arthritis Rheum. 42, 519–526. Kurosawa, M., Amano, H., Kanbe, N., Igarashi, J., Nagata, H., Yamashita, T., Kurimoto, F., Miyachi, Y., 1999. Response to cyclosporin and low dose methylprednisolone in aggressive systemic mastocytosis. J. Allergy Clin. Immunol. 103, S412– S420. Leslie, C.C., 1997. Properties and regulation of cytosolic phospholipase A 2 . J. Biol. Chem. 272, 16709–16712. Lin, L-L., Lin, A.Y., Knopf, J.L., 1992. Cytosolic phospholipase A 2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. U. S. A. 89, 6147–6151. Misra, U.K., Gawdi, G., Pizzo, S.V., 1998. Cyclosporin A inhibits
inositol 1,4,5-triphosphate binding to its receptors and release of calcium from intracellular stores in peritoneal macrophages. J. Immunol. 161, 6122–6127. Nicchitta, C.V., Kamoun, M., Williamson, J.R., 1985. Cyclosporin augments receptor-mediated cellular Ca2q fluxes in isolated hepatocytes. J. Biol. Chem. 260, 13613–13618. Paliogianni, F., Boumpas, D.T., 1995. Glucocorticoids regulate calcineurin dependent transactivating pathways for interleukin2 gene transcription in human T lymphocytes. Transplantation 59, 1333–1339. Parra, T., de Arriba, G., Conejo, J.R., Cantero, M., Arribas, I., Rodriguez-Puyol, D., Rodriguez-Puyol, M., Carballo, F., 1998. Cyclosporine increases local glomerular synthesis of reactive oxygen species in rats. Transplantation 66, 1325–1329. Quien, R.M., Kaiser, B.A, Dunn, S.P., Kulinsky, A., Polinsky, M., Baluarte, H.J., Batiuk, T.D., Halloran, P.F., 1997. Calcineurin activity in children with renal transplants receiving cyclosporin. Transplantation 64, 1486–1489. Redington, A.E., Hardinge, F.M., Madden, J., Holgate, S.T., Howarth, P.H., 1998. Cyclosporin A treatment and airways inflammation in corticosteroid-dependent asthma. Allergy 53, 94–98. Ruhlmann, A., Nordheim, A., 1997. Effects of immunosuppres¨ sive drugs CsA and FK506 on intracellular signaling and gene regulation. Immunobiology 198, 192–206. Sun, L., Youn, H.D., Loh, C., Stolow, M., He, W., Liu, J.O., 1998. Cabin 1, a negativ regulator for calcineurin signaling in T lymphocytes. Immunity 8, 703–711. Szegedi, G., Kiss, E., Bodolay, E., Zeher, M., Danko, ´ K., Czirjak, ´ L., Szucs, ¨ G., Szekanecz, Z., Sipka, S., 1996. Polysystemic autoimmune diseases — clinical and basic research made in Hungary. Allergy Clin. Immunol. 8, 90–93. Thiru, S., Maher, E.R., Hamilton, D.V., Evans, D.B., Calne, R.Y., 1983. Tubular changes in renal transplant recipients on cyclosporin. Transplant. Proc. 15, 2846–28451. Vereb, G., Panyi, G., Balazs, L., Matko, ´ M., Matyus, ´ ´ J., Damjanovich, S., 1990. Effect of cyclosporin A on the membrane potential and Ca2q level of human lymphoid cell lines and mouse thymocytes. Biochim. Biophys. Acta 1019, 159–165. Yang, S.D., Tallant, A.N., Cheung, W.Y., 1982. Calcineurin is a calmodulin-dependent protein phosphatase. Biochem. Biophys. Res. Commun. 4, 1419–1423. Zor, U., Ferber, E., Gergely, P., Szucs, ¨ K., Dombradi, ´ V., Goldman, R., 1993. Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate. Biochem. J. 295, 879– 888.
Short communication
Inhibition of calcineurin activity and protection against cyclosporine A induced cytotoxicity by prednisolone sodium succinate in human peripheral mononuclear cells Sandor Sipka a,) , Kornelia Szanto ´ ´ Szucs ¨ b, Sandor ´ ´ ´ a, Ildiko´ Kovacs ´ a, Gabriella Lakos a , Peter ´ Antal-Szalmas ´ a, Gyula Szegedi a, Pal ´ Gergely b a
3rd Department of Internal Medicine, UniÕersity Medical School of Debrecen, Moricz Zs. ut ´ ´ 22, H-4004 Debrecen, Hungary b Department of Medical Chemistry, UniÕersity Medical School of Debrecen, Debrecen, Hungary Received 19 August 1999; received in revised form 6 January 2000; accepted 6 January 2000
Abstract We have investigated the effects of prednisolone sodium succinate ŽPss. and cyclosporin A ŽCSA., applied alone or concurrently, on the release of arachidonic acid ŽAA. Žcytosolic phospholipase A 2 ŽcPLA 2 . activity. and on the calcineurin ŽCN. activity of human peripheral blood mononuclear cells ŽPBMC.. The cytotoxic damage to the cells treated by the drugs was estimated by the release of lactate dehydrogenase ŽLDH.. We found that Pss Ž10y5 M. could inhibit the CN activity and higher concentrations Ž10y4 M. could decrease the cytotoxic damage caused by CSA Ž10y4 M. during their combined application. CSA had no specific effect on the release of AA from the cells. In the combined clinical use of glucocorticosteroids ŽGCS. and CSA, their additive inhibitory effect on CN activity and the protective membrane influence of GCS against the cytotoxicity of CSA may be beneficial. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Glucocorticosteroid; Cyclosporin A; Calcineurin; Arachidonic acid
1. Introduction Both glucocorticosteroids ŽGCS. and cyclosporin Ž A CSA. are widely used drugs in immunosuppres-
AbbreÕiations: Pss, prednisolone sodium succinate; CSA, cyclosporin A; GCS, glucocorticostreoids; AA, arachidonic acid; CN, calcineurin; cPLA 2 , cytosolic phospholipase A 2 ; PBMC, peripheral blood mononuclear cells; LDH, lactate dehydrogenase; PMA, phorbol myristate acetate ) Corresponding author. Tel.: q36-52-414-969; fax: q36-52414-969. E-mail address: [email protected] ŽS. Sipka..
sive and anti-inflammatory therapeutic protocols. GCS can block the production of various pro-inflammatory mediators, e.g. prostaglandins, leukotrienes, platelet activating factor, histamine and cytokines, which have both mediatory and immunoregulatory functions ŽBarnes, 1996.. In these effects, the inhibition of cytosolic phospholipase A 2 ŽcPLA 2 . plays an important role, as arachidonic acid ŽAA., one of the products of cPLA 2 , is the precursor molecule of prostaglandins, thromboxanes and leukotrienes ŽDennis, 1997; Leslie, 1997.. GCS also have a direct membrane effect and can inhibit the influx of Ca2q ŽButtgereit et al., 1998..
0162-3109r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 0 0 . 0 0 1 8 0 - 6
88
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
In the immunosuppressive effect of CSA, the main element is its intracellular coupling to cyclophilin. The CSA–cyclophilin complex can block the activity of the calmodulinrCa2q-dependent protein phosphatase Žcalcineurin ŽCN.. resulting in a decreased dephosphorylation of the transcriptional factor NF-AT Žnuclear factor of activated T cells., which leads to a reduced production of a series of cytokines ŽIL-2, IL-4, etc.. and cell surface molecules ŽCD40, LCAM, etc.., ŽHo et al., 1996; Ruhlmann ¨ and Nordheim, 1997.. Some clinical and experimental observations have shown advantageous immunosuppressive effects in the combined applications of GCS and CSA ŽSzegedi et al., 1996; Quien et al., 1997; Redington et al., 1998; Kurosawa et al., 1999.. In this study, we show that GCS can inhibit the activity of CN and can additively contribute to the decreased CN activity caused by CSA. In addition, GCS may have some protective membrane effect against the cytotoxicity of CSA during their combined use in vitro.
2. Materials and methods 2.1. Preparation of human peripheral blood mononuclear cells (PBMC) and characterisation of cells by flow cytometry PBMC, containing 88–95% lymphocytes and 5– 12% monocytes, were prepared ŽBoyum, 1968. from the heparinized blood of 6 healthy donors Žthree men and three women, age: 21–54 years.. The averages of the various subsets were detected by flow cytometry: CD3q: 69.4%, CD19q: 11.5%, CD56q: 0.8% and CD14q: 8.3% ŽCoulter EPICS XL flow cytometer, Coulter, Hialeah, FL.. 2.2. Determination of cPLA 2 actiÕities by the measurement of AA release The cells Ž10 5rml. were preincubated with w3 HxAA ŽAmersham, UK. in a CO 2 incubator at 378C for 20 h. After extensive washing, the cells were further incubated without any stimuli for 4 h. The amount of released AA reflected the basal activity of cPLA 2 . Activation of cPLA 2 was achieved by the addition of 5 mM of calcium ionophore ŽA
23187, Sigma, St. Louis, MO. and 50 ngrml of phorbol 12-myristate 13-acetate ŽPMA, Sigma, St. Louis, MO. at 378C for 4 h. The amount of released AA reflects the activity of cPLA 2 of stimulated cells ŽLin et al., 1992; Zor et al., 1993.. GCS or CSA, incubated with the cells for 20 h, did not affect the uptake of w3 HxAA and the cPLA 2 assays were therefore comparable in the two cases. 2.3. Determination of CN actiÕity The cell suspensions were washed with phosphate-buffered saline and collected by centrifugation. Cells were sonicated for 2 = 30 s with a Branson sonifier in 2 vol of homogenisation buffer containing 50 mM Tris–HCl ŽpH 7.0., 0.5 M dithiothreitol, 10 mgrml aprotinin, 10 mgrml leupeptin, 10 mgrml trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride ŽPMSF., 5 mM benzamidine and 0.3% Triton X-100. The homogenate was centrifuged for 10 min at 10,000 = g, supernatants were collected and stored in aliquots at y708C prior to the assay. CN activity was measured by the release of 32 Pi from w32 Pxinhibitor-1 ŽYang et al., 1982; Cohen et al., 1988.. The assay mixture Ž30 ml. containing 50 mM Tris–HCl ŽpH 7.0., 0.3 mM dithiothreitol, 0.2 mM CaCl 2r1 mM MnCl 2r0.04 mgrml calmodulin or 2 mM EGTA, appropriate amount of extract Ž0.5–1.5 mgrml protein. and w32 Pxinhibitor-1 Ž12,000–25,000 cpmrreaction mixture. was incubated at 308C for 10 min. All mixtures contained 10 mgrml aprotinin, 10 mgrml leupeptin, 10 mgrml trypsin inhibitor, 1 mM PMSF, 5 mM benzamidine as protease inhibitors and 20 nM okadaic acid as a protein phosphatase 2A inhibitor. The reaction was terminated by the addition of 100 ml of 10% trichloroacetic acid. 32 Pi of the supernatant fraction was determined after centrifugation by Cerenkov counting in a liquid scintillation counter. The activity of CN was calculated as the difference of 32 Pi counts in the presence and absence of Mn2qrCa2qr calmodulin. 2.4. Measurement of lactate dehydrogenase (LDH) actiÕity LDH activities in cell supernatants represent the degree of cell damage ŽKobayashi et al., 1999. and
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
were determined by a kinetic UV test using the Boehringer SYS2 kit in a Hitachi 717 analyser. The results were expressed in Url. 2.5. Statistical analysis The statistical means and S.D. values of data were calculated. The statistical significance of the differences was determined by Student’s paired and unpaired t tests Ž p - 0.01 was considered significant.. At least three independent experiments were performed and all samples were used in triplicates. 3. Results 3.1. Effects of prednisolone sodium succinate (Pss) and CSA on the AA and LDH releases of PBCM First, the effects of 10y7 , 10y5 and 10y4 M concentrations of Pss and CSA applied alone in the cultures of PBMC for 20 h were investigated on the AA and LDH releases. As shown in Table 1, Pss significantly decreased the AA release at 10y5 M concentration in the stimulated cells, and 10y4 M of Pss resulted in an almost complete inhibition Ž5607 " 513, 4352 " 392 dpm vs. 9621 " 945 dpm, p 0.01 and p - 0.001, respectively.. There was practically no cytotoxic effect of Pss at the concentrations applied, as revealed by LDH release. 10y4 M of
89
CSA also reduced the AA release of stimulated PBMC Ž6605 " 693 dpm vs. 9621 " 945 dpm p 0.01.. This effect could be related to the cytotoxic effect of the drug as shown by the release of LDH in the stimulated and non-stimulated cells Ž89.3 " 9.1, 98.2 " 10.4 Url vs. 37.1 " 4.8, 30.2 " 5.1 Url, respectively, p - 0.001.. On the other hand, 10y5 M of CSA exerted only a cytotoxic effect Ž74.5 " 11.3, 75.4 " 9.8 Url vs. 30.2 " 5.1, 37.1 " 4.8 Url, respectively., without any significant influence on the AA release Ž3645 " 477, 8177 " 792 dpm vs. 3857 " 689, 9621 " 945 dpm, respectively.. It was of interest that in the stimulated cells treated with 10y4 M of Pss, the release of LDH was significantly less than in the Pss-free culture Ž24.2 " 3.5 Url vs. 37.1 " 4.8 Url, p - 0.01. implying that this dose of GCS had some membrane protecting effect. When the two drugs were administered concurrently in 10y4 M doses, the inhibition of AA release was almost complete Ž4426 " 319 dpm vs. 9621 " 945 dpm. with a considerable cell damage Ž57.5 " 8.7, 60.2 " 7.2 Url vs. 30.2 " 5.1, 37.1 " 4.8 Url, respectively.. However, these LDH values were significantly lower than those of the single treatment with CSA Ž57.5 " 8.7, 60.2 " 7.2 Url vs. 98.2 " 10.4, 89.3 " 9.1 Url, respectively, p - 0.01.. Our results demonstrated that 10y4 M of Pss had a protective effect against the cytotoxicity of CSA. 10y7 M of Pss could not prevent the cytotoxicity
Table 1 Effect of Pss and CSA on the releases of AA and LDH in human mononuclear cells stimulated and non-stimulated by phorbol ester and Ca2q ionophore The cells were incubated with Pss orrand CSA for 20 h. PBMC were stimulated by 5 mM of A23187 and 50 ngrml of PMA for 4 h. The releases of AA and LDH were assayed as given in Materials and methods. p values were compared with the respective controls. Concentrations ŽM.
0 10y7 10y5 10y4 Pss: 10y4 qCSA: 10y4 Pss: 10y7 qCSA: 10y5 U
p - 0.01. p - 0.001.
UU
AA release Ždpm. Non-stimulated cells
Stimulated cells
Pss
Pss
CSA
3857 " 689 4311 " 445 3618 " 414 4073 " 432 3645 " 477 4013 " 468 3687 " 486 4251 " 452 3843 " 318
CSA
9621 " 945 7704 " 842 9252 " 860 5607 " 513 ) 8177 " 792 4352 " 392 ) ) 6605 " 693 ) 4426 " 319 ) ) 5004 " 576 ) )
LDH release ŽUrl. Non-stimulated cells
Stimulated cells
Pss
Pss
CSA
CSA
30.2 " 5.1 30.6 " 3.9 46.8 " 8.8 33.7 " 3.5 74.5 " 11.3 ) ) 29 " 4.3 98.2 " 10.4 ) ) 57.5 " 8.7 ) )
37.1 " 4.8 34.2 " 4.2 42.7 " 9.1 30.3 " 4.6 75.4 " 9.8 ) ) ) 24.2 " 3.5 89.3 " 9.1) ) 60.2 " 7.2 ) )
75.7 " 7.6 ) )
72.4 " 5.4 ) )
90
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
Fig. 1. Effect of Pss and CSA on the CN activity of human mononuclear cells. PBMC were incubated with various doses of Pss and CSA administered alone or concurrently for 24 h. CN activity was assayed as given in Materials and methods. The data represent the mean of 3–4 independent experiments and the bar shows the standard deviation. p values were compared with the respective control samples.
caused by 10y5 M of CSA Ž75.7 " 7.6, 72.4 " 5.4 Url vs. 74.5 " 11.3, 75.4 " 9.8 Url, respectively. ŽTable 1.. 3.2. Effect of Pss and CSA on the CN actiÕity of PBMC Non-stimulated PBMC were incubated with Pss orrand CSA for 24 h. We did not test the stimulated cells. The CN activities were determined from the sonicated lysates of these cells. As shown in Fig. 1, CSA dose dependently inhibited the CN activities, as was expected. Unexpectedly, both 10y5 and 10y4 M concentrations of Pss Žnot toxic doses. also significantly inhibited the CN activity, Ž11.2 " 1.0 and 6.6 " 0.6 pmolrmgrprotein vs. 19.1 " 1.8 pmolrmgrprotein, respectively, p - 0.01.. When CSA and Pss were applied concurrently at the same concentrations, the CN activity was further inhibited compared to the effect of drugs used alone. In addition, there was some additivity in the action of these compounds on the CN activity Ž5.2 " 0.5, 0.75 " 0.1, 0.19 " 0.05 pmolrmgrprotein vs. 6.7 " 0.6, 2.3 " 0.2, 0.97 " 0.1 pmolrmgrprotein, respectively.. The inhibitory effect of Pss ŽGCS. on the CN activity of PBMC is a new observation. However, it
has to be emphasised that a much higher concentration of GCS Ž10y4 M. is needed to obtain an inhibition of CN activity similar to that of CSA Ž10y7 M.. In addition, the presence of GCS may result in some additivity in the inhibition of CN caused by CSA.
4. Discussion The clinical application of GCS and CSA represented considerable progress in the therapy of patients with autoimmune diseases, allergy and organ or bone marrow transplantations. However, both GCS and CSA have well known negative side effects, limiting their prolonged administration to patients. GCS can cause cataract, osteoporosis, muscle atrophy or metabolic disorders ŽBarnes, 1996.. In the nephrotoxicity caused by CSA treatment, both the tubular and vascular systems of the kidney can be dose dependently damaged ŽThiru et. al., 1983. based upon the changing of membrane potential ŽVereb et.al., 1990., the inhibition of Ca2q release from intracellular stores ŽMisra et al., 1998. and the increased local production of reactive oxygen species ŽParra et al., 1998.. Therefore, any approach in the
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
clinical application of these compounds which preserves or improves the efficacy of immunosuppression but which decreases the negative side effects could be of great theoretical and practical importance. Our observations have two major points. First, Pss ŽGCS., as well as CSA, can inhibit CN activity and there may be some additivity in the effects of two compounds. However, GCS are less potent inhibitors of CN activity than CSA. Second, the cell membrane protecting effect of GCS against the cytotoxicity of CSA may be very beneficial during their combined therapeutic use. One may suppose that the decreased activity of CN can lead to a reduced production of NF-AT dependent cytokines: IL-2, IL-4, IL-8, IL-13, etc. ŽFirestein and Manning, 1999.. To understand the mechanism whereby GCS can inhibit CN activity, new connections have to be found in the regulation of transcriptional factors ŽNF-AT, NF kappa B, AP1., heat shock proteins, GCS receptors, immunophilins and ion metabolism ŽRuhlmann and ¨ Nordheim, 1997; Buttgereit et al., 1998.. For example, the intracellular production of an inhibitor of k B is known to be increased under the influence of GCS treatment ŽBarnes, 1996., and it can also be a substrate in the CN mediated dephosphorylation processes ŽRuhlmann and Nordheim, 1997.. GCS can ¨ inhibit the CN-dependent activation of the interleukin 2 gene in human T lymphocytes ŽPaliogianni and Boumpas, 1995.. Furthermore, as the inhibition of CN by CSA is rapidly reversible ŽBatiuk et. al., 1997., in a combined therapy GCS may prolong the effect of CSA. However, the main mechanism involved in the GCS induced inhibition of CN may be related to the reduction of Ca2q available for the calmodulin pathway ŽAntoni, 1996. and to the inhibition of Ca2q influx ŽButtgereit et al., 1998.. The reduction of Ca2q influx and the stabilization of the cell membrane ŽButtgereit et al., 1998. can play important roles in the protective effect of GCS against CSA induced cytotoxicity because CSA modulates the intracellular level of Ca2q ŽNicchitta et al., 1985; Misra et al., 1998. and causes depolarization of the cell membrane ŽDamjanovich et al., 1987; Vereb et al., 1990.. CSA had no specific influence on the release of AA from human peripheral mononuclear cells,
91
namely on the cPLA 2 activity. The decreased AA release found in the cells treated with CSA could be related to the cytotoxic damage caused by the drug. In contrast, CSA was found to activate and not to inhibit the PLA 2 activity in hamster renal tubular cells ŽAnderson et al., 1994.. The measurement of LDH activities for testing the degree of cytotoxic damage caused by CSA proved to be a sensitive and useful method. In addition, Pss and CSA did not induce apoptosis in the cultured cells during 24 h, measured by flow cytometry on the basis of propidium iodide uptake Ždata not shown.. Here we presented data on the CN inhibiting effects of CSA and Pss found in non-stimulated human peripheral mononuclear cells. When we measured the CN activities of the cells stimulated by phorbol ester and Ca2q ionophore, a profound decrease Ž87%. was observed in the absence of CSA or GCS Ždata not shown.. Therefore, we preferred to demonstrate the inhibition of CN activities caused by CSA or GCS ŽPss. in non-stimulated cells because these had about six-fold higher activities than the stimulated cells. The mechanism of the markedly decreased CN activities in the stimulated cells is still obscure. One candidate for the blocking of CN activity could be the Cabin 1 molecule, a member of the protein kinase C induced signaling pathway of T cells ŽSun et al., 1998.. In summary, our data show that GCS can additively contribute to the inhibition of CN activity caused by CSA during combined application of the two drugs. Simultaneously, GCS can protect the cell membranes against the cytotoxic side effects of CSA. Acknowledgements The authors thank Mrs. Julia Hunyadi for her expert technical assistance. This work was supported by grants of the National Scientific Research Fund ŽOTKA T023199 and T026541. and Ministry of Education ŽMKM 0784r97. and Ministry of Health ŽETT 315r96 and 093r96.. References Anderson, R., van Rensburg, C.E.J., Myer, M.S., 1994. Alphatocopherol prevents cyclosporin A-mediated activation of
92
S. Sipka et al.r Immunopharmacology 48 (2000) 87–92
phospholipase A 2 and inhibition of NaqrKq-adenosine triphosphatase activity in cultured hamster renal tubular cells. Toxicol. Appl. Pharmacol. 125, 176–183. Antoni, F.A., 1996. Mortyn Jones Memorial Lecture — 1995. Calcium checks cyclic AMP corticosteroid feedback in adenohypophysial corticotrophs. J. Neuroendocrinol. 8, 659–672. Barnes, P.J., 1996. Molecular mechanism of steroid action in asthma. J. Allergy Clin. Immunol. 97, 159–168. Batiuk, T.D., Kung, L., Halloran, P.F., 1997. Evidence that calcineurin is rate-limiting for primary human lymphocyte activation. J. Clin. Invest. 100, 1894–1901. Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. J. Clin. Lab. Invest. Suppl. 97, 77–108. Buttgereit, F., Wehling, M., Burgmeister, G-R., 1998. A new hypothesis of modular glucocorticoid actions. Arthritis Rheum. 41, 761–767. Cohen, P., Foulkes, G., Holmes, C.F.B., Nimmo, G.A., Tonks, N.K., 1988. Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 159, 427–433. Damjanovich, S., Aszalos, A., Mulhem, S., Szollosi, ¨ ¨ J., Balazs, ´ M., Tron, L., Fulwyler, M.J., 1987. Cyclosporin depolarizes human lymphocytes. Earliest observed effect on cell metabolism. Eur. J. Immunol. 17, 763–768. Dennis, E.A., 1997. The growing phospholipase A 2 superfamily of signal transduction enzymes. TIBS 22, 1–2. Firestein, G.S., Manning, A.M., 1999. Signal transduction and transcription factors in rheumatic disease. Arthritis Rheum. 42, 609–621. Ho, S., Clipstone, N., Timmermann, L., Northtrop, J., Graef, I., Fiorentino, D., Nourse, J., Crabtree, G.R., 1996. The mechanism of action of cyclosporin A and FK506. Clin. Immunol. Immunopathol. 80, S40–S45. Kobayashi, T., Okamoto, K., Kobata, T., Hasunuma, T., Sumida, T., Nishioka, T., 1999. Tumor necrosis factor alpha regulation of the FAS-mediated apoptosis-signaling pathway in synovial cells. Arthritis Rheum. 42, 519–526. Kurosawa, M., Amano, H., Kanbe, N., Igarashi, J., Nagata, H., Yamashita, T., Kurimoto, F., Miyachi, Y., 1999. Response to cyclosporin and low dose methylprednisolone in aggressive systemic mastocytosis. J. Allergy Clin. Immunol. 103, S412– S420. Leslie, C.C., 1997. Properties and regulation of cytosolic phospholipase A 2 . J. Biol. Chem. 272, 16709–16712. Lin, L-L., Lin, A.Y., Knopf, J.L., 1992. Cytosolic phospholipase A 2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. U. S. A. 89, 6147–6151. Misra, U.K., Gawdi, G., Pizzo, S.V., 1998. Cyclosporin A inhibits
inositol 1,4,5-triphosphate binding to its receptors and release of calcium from intracellular stores in peritoneal macrophages. J. Immunol. 161, 6122–6127. Nicchitta, C.V., Kamoun, M., Williamson, J.R., 1985. Cyclosporin augments receptor-mediated cellular Ca2q fluxes in isolated hepatocytes. J. Biol. Chem. 260, 13613–13618. Paliogianni, F., Boumpas, D.T., 1995. Glucocorticoids regulate calcineurin dependent transactivating pathways for interleukin2 gene transcription in human T lymphocytes. Transplantation 59, 1333–1339. Parra, T., de Arriba, G., Conejo, J.R., Cantero, M., Arribas, I., Rodriguez-Puyol, D., Rodriguez-Puyol, M., Carballo, F., 1998. Cyclosporine increases local glomerular synthesis of reactive oxygen species in rats. Transplantation 66, 1325–1329. Quien, R.M., Kaiser, B.A, Dunn, S.P., Kulinsky, A., Polinsky, M., Baluarte, H.J., Batiuk, T.D., Halloran, P.F., 1997. Calcineurin activity in children with renal transplants receiving cyclosporin. Transplantation 64, 1486–1489. Redington, A.E., Hardinge, F.M., Madden, J., Holgate, S.T., Howarth, P.H., 1998. Cyclosporin A treatment and airways inflammation in corticosteroid-dependent asthma. Allergy 53, 94–98. Ruhlmann, A., Nordheim, A., 1997. Effects of immunosuppres¨ sive drugs CsA and FK506 on intracellular signaling and gene regulation. Immunobiology 198, 192–206. Sun, L., Youn, H.D., Loh, C., Stolow, M., He, W., Liu, J.O., 1998. Cabin 1, a negativ regulator for calcineurin signaling in T lymphocytes. Immunity 8, 703–711. Szegedi, G., Kiss, E., Bodolay, E., Zeher, M., Danko, ´ K., Czirjak, ´ L., Szucs, ¨ G., Szekanecz, Z., Sipka, S., 1996. Polysystemic autoimmune diseases — clinical and basic research made in Hungary. Allergy Clin. Immunol. 8, 90–93. Thiru, S., Maher, E.R., Hamilton, D.V., Evans, D.B., Calne, R.Y., 1983. Tubular changes in renal transplant recipients on cyclosporin. Transplant. Proc. 15, 2846–28451. Vereb, G., Panyi, G., Balazs, L., Matko, ´ M., Matyus, ´ ´ J., Damjanovich, S., 1990. Effect of cyclosporin A on the membrane potential and Ca2q level of human lymphoid cell lines and mouse thymocytes. Biochim. Biophys. Acta 1019, 159–165. Yang, S.D., Tallant, A.N., Cheung, W.Y., 1982. Calcineurin is a calmodulin-dependent protein phosphatase. Biochem. Biophys. Res. Commun. 4, 1419–1423. Zor, U., Ferber, E., Gergely, P., Szucs, ¨ K., Dombradi, ´ V., Goldman, R., 1993. Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate. Biochem. J. 295, 879– 888.