FK 506 is a recently discovered immunosuppressive macrolide1 that has been shown to be significantly more potent than cyclosporine A (CyA) in a variety of in vitro and in vivo assays.2-5 Like CyA, FK 506 inhibits the transcription of early T-cell activation genes6 following binding to a cytoplasmic receptor FK-binding protein (FKBP).7-9
It has recently been demonstrated that transforming growth factor beta (TGF-β) has the ability to regulate immune functions in vitro, 10,11 and has therefore been used as an immunosuppressant in cardiac and islet allotransplantation. 12-14 It is interesting to speculate that one of the pathways by which immunosuppressants like FK 506 may act is by inducing endogenous TGF-β expression.
MATERIALS AND METHODS
A rat liver transplant model was used to study the effect of FK 506 administration on FKBP and TGF-β gene expression. Livers harvested from ACI donors were orthotopically transplanted in Lewis recipients. 15 Following orthotopic liver transplantation (OLTX), recipients were either treated with 1.28 mg/kg body weight of intramuscular FK 506 for 4 days (n = 15) or left untreated (control, n = 12). Three animals each were killed on postoperative days 3, 6, and 9 in the control group and on days 3, 6, 9, and 12 in the FK 506-treated group. Total cellular RNA was extracted and purified by the method of Chomczynski and Sacchi. 16 For Northern analysis, RNA samples were fractionated on 0.7% agarose, 18% formaldehyde gels and transferred to nitrocellulose paper. Hybridization was carried out at 60°C overnight in Church buffer17 and a 32P-labeled complementary DNA probe. The filters were washed twice for 20 minutes at room temperature in 1 × SSC (SSC = 150 mmol/L sodium chloride, 15 mmol/L sodium citrate, pH 7), 0.1% sodium dodecyl sulfate and 0.1 SSC, 0.1% sodium dodecyl sulfate at 65°C. The filter was exposed to Kodak X-omat film for 4 days with an intensifying screen at −70°C.
RESULTS
Fig 1 depicts the autoradiographs for FKBP expression. FKBP gene expression was observed in both untreated control, and FK 506-treated recipients on all the days studied.
Fig 1.
Autoradiograph for FKBP expression. FKBP gene expression was observed in both control (lanes 2–4) and FK 506-treated recipients (lanes 5–8) on all the days studied.
Fig 2 is the autoradiographs for TGF-β expression. No evidence of TGF-β gene expression was observed in untreated control livers. On the other hand, FK 506-treated livers showed an induction of TGF-β gene expression on days 3 and 6, which tapered off on days 9 and 12 following orthotopic transplantation.
Fig 2.
Autoradiograph for TGF-β expression. No evidence of TGF-β gene expression was observed in control livers (lanes 1–3). On the other hand, FK 506-treated livers showed an induction of TGF-β gene expression on days 3 and 6 (lanes 4 and 5), which tapered off on days 9 and 12 (lanes 6 and 7) following OLTx.
DISCUSSION
Our results demonstrated no association between treatment with FK 506 and FKBP gene expression. Both FKBP and its cyclosporine binding counterpart cyclophilin are ubiquitous immunophilins with rotamase activity.18,19 It has recently been reported that inhibition of the rotamase activity of FKB following FK 506 binding cannot explain the biologic effects of FK 506 administration. 18 This would appear to confirm the lack of correlation between FK 506 treatment and FKBP expression observed in our study.
TGF-β gene expression was observed as early as the third postoperative day and persisted on the sixth postoperative day, tapering off on days 9 and 12. No evidence of TGF-β gene expression was noted in untreated control livers. The immunoregulatory properties of TGF-β include inhibition of cytokine production,20 cytotoxic T-cell generation, 21 thymocyte proliferation,22 and T cell/B cell generation. 23 It has peen successfully used to prevent allorejection in murine cardiac and islet transplantation 12-15 and in murine models of autoimmune disease.24,25 It is therefore interesting to speculate that FK 506 may be exerting its immunosuppressant action through the induction of TGF-β.
Acknowledgments
The authors wish to thank Thomas Cunningham for his expert technical experience and Mary Ellen Keener for secretarial services.
References
- 1.Thomson AW. Immunology Today. 1989;10:6. doi: 10.1016/0167-5699(89)90057-1. [DOI] [PubMed] [Google Scholar]
- 2.Shevach EM. Annu Rev Immunol. 1985;3:397. doi: 10.1146/annurev.iy.03.040185.002145. [DOI] [PubMed] [Google Scholar]
- 3.Starzl T, Fung J, Venkataramman R, et al. Lancet. 1989;1:1000. doi: 10.1016/s0140-6736(89)91014-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Handschumacher RE, Harding MW, Rice J, et al. Science. 1984;226:544. doi: 10.1126/science.6238408. [DOI] [PubMed] [Google Scholar]
- 5.Takahashi N, Hayano T, Suzuki M. Nature. 1989;337:473. doi: 10.1038/337473a0. [DOI] [PubMed] [Google Scholar]
- 6.Tocci MJ, Matkovich DA, Collier KA, et al. J Immunol. 1989;143:718. [PubMed] [Google Scholar]
- 7.Harding MW, Galata, Uehling DE, et al. Nature. 1989;341:758. doi: 10.1038/341758a0. [DOI] [PubMed] [Google Scholar]
- 8.Siekierka JJ, Hung SHY, Poe M, et al. Nature. 1989;341:755. doi: 10.1038/341755a0. [DOI] [PubMed] [Google Scholar]
- 9.Standaert RF, Galat A, Verdine GL, et al. Nature. 1990;346:671. doi: 10.1038/346671a0. [DOI] [PubMed] [Google Scholar]
- 10.Wallick SC, Figari IS, Morris RE, et al. J Exp Med. 1990;172:1777. doi: 10.1084/jem.172.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Palladino MA, Morris RE, Starnes HF, et al. Ann NY Acad Sci. 1990;593:181. doi: 10.1111/j.1749-6632.1990.tb16110.x. [DOI] [PubMed] [Google Scholar]
- 12.Morris R, Meiser B, Figari I, et al. Recombinant human transforming growth factor-beta 1 is a potent immunosuppressant in vivo. 7th International Congress of Immunology; Berlin. 1989. [Google Scholar]
- 13.Wu J, Palladino MA, Figari IS, et al. Transplant Proc. 1991;23:238. [PubMed] [Google Scholar]
- 14.Gill RG. Transplant Proc. 1991;23:747. [PubMed] [Google Scholar]
- 15.Rao PN, Walsh TR, Makowka L, et al. Transplantation. 1990;49:1055. doi: 10.1097/00007890-199006000-00006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Chomczynski P, Sacchi N. Anal Biochem. 1987;162:156. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- 17.Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory NY; 1987. [Google Scholar]
- 18.Bierer BE, Mattila PS, Standaert RF, et al. Proc Natl Acad Sci USA. 1990;87:9231. doi: 10.1073/pnas.87.23.9231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Fischer G, Liebold BW, Lang K, et al. Nature. 1989;337:476. doi: 10.1038/337476a0. [DOI] [PubMed] [Google Scholar]
- 20.Espevik T, Figari IS, Shalaby MR, et al. J Exp Med. 1987;166:571. doi: 10.1084/jem.166.2.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Ranges GE, Figari IS, Espevik T, et al. J Exp Med. 1987;166:991. doi: 10.1084/jem.166.4.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Ellingsworth LR, Nakayama D, Segarini P, et al. Cell Immunol. 1988;114:41. doi: 10.1016/0008-8749(88)90253-5. [DOI] [PubMed] [Google Scholar]
- 23.Kehrl JH, Wakefield LM, Roberts AB, et al. J Exp Med. 1986;163:1037. doi: 10.1084/jem.163.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Racke MK, Dhib-Jalbut S, Cannella B, et al. J Immunol. 1991;146:3012. [PubMed] [Google Scholar]
- 25.Kuruvilla AP, Shah R, Hochwald GM, et al. Proc Natl Acad Sci USA. 1991;88:2918. doi: 10.1073/pnas.88.7.2918. [DOI] [PMC free article] [PubMed] [Google Scholar]