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FK506 and SMS 201-995: effect on heterotopic heart transplantation in rats
FK506 and SMS 201-995: Effect on Heterotopic Heart Transplantation in Rats R. Orsenigo, S. Gatti, L. Latham, P. Trezza, and O. Marelli
T
HE NERVOUS and the immune systems are closely related and may communicate in both directions through soluble mediators such as neuropeptides and cytokines. The nervous system may modulate immunological and inflammatory responses as demonstrated by the identification of neuropeptide receptors on leukocytes and by the regulatory activities on the leukocyte functions following the interaction of such neuropeptides with their receptors. Somatostatin (SS) was originally described as a growth hormone release-inhibiting factor synthesized in the hypothalamus. Five distinct SS receptors (SSTR1–5) have been characterized so far, and SSTR2 has been localized on lymphocytes.1–3 The presence of SS-mRNA in lymphoid tissue indicates that lymphocytes are able to synthesize SS, and this may act as a paracrine factor modulating immune responses.4 SS has an inhibitory effect on mitogen-induced lamina propria and peripheral blood lymphocyte proliferation and also decreases human natural killer cell activity. Because of the very short plasma half-life of native SS, analogues such as SMS 201-995 (octreotide acetate) have been developed. SMS 201-995 binds preferentially SSTR2.3 Previous studies in vitro and in vivo have shown evidences of a synergistic action of SMS 201-995 and FK506.5 An immunosuppressive effect of SMS 201-995 was observed on the proliferation of rat spleen cells induced in vitro, either by polyclonal mitogens (PHA) or by alloantigens. With PHA stimulation, SMS 201-995 significantly enhanced the immunosuppressive action of FK506, and the addition of SMS 201-995 in MLR increased the antiproliferative effect of FK506.5 The association of FK506 and SMS 201-995 was tested in vivo in an allogeneic skin graft model using Lewis (Lew) rats as donors and Brown Norway (BN) rats as recipients. BN treated with 0.1 mg/kg FK506 and 0.5 to 10 g/kg SMS 201-995 showed a significant increase in mean skin allograft survival time when compared to either a monotherapy or control group.5 The aim of the present study was to evaluate the same drug combination in an allogeneic vascularized heterotopic heart model using the same rodent combination. MATERIALS AND METHODS Animals and Transplant Procedures Male BN (RT1n) and Lew (RT1l) rats weighing 200 to 300 g were purchased as recipients and donors of heterotopic heart allograft0041-1345/01/$–see front matter PII S0041-1345(00)02140-0
ing, respectively, and maintained in conventional facilities housed one per cage with food and water ad libitum. The Ono and Lindsay microsurgical technique was used.6
Immunosuppression Both FK506 and SMS 201-995 were administered intraperitoneally, diluted in saline solution. Both drugs were commenced at the time of surgery and administered until rejection occurred.
Experimental Design Four groups of rats (n ⫽ 6 in each group) were included in this study. Group A received the vehicle; group B received FK506 0.05 mg/kg/d; group C received FK506 0.02 mg/kg/d; group D received FK506 0.02 mg/kg/d and SMS 201-995 10 g/kg/bid. The graft survival was assessed through daily abdominal palpation. Rejection was diagnosed by the cessation of palpable heart beat, at which time the animals were sacrificed. Graft survival times expressed in days are given as means ⫾ the standard deviation.
Pathologic Studies An autopsy was carried out on all rats. Samples from grafts were fixed in formalin for paraffin embedding and routine H&E staining.
RESULTS
Control animals (group A), which received only saline solution, rejected the heterotopic hearts with a mean survival time (MST) of 6.5 ⫾ 0.5 days. All three treated groups showed significant improvement in graft survival when compared with the control group. Group B animals receiving FK506 0.05 mg/kg/d showed an unlimited graft survival (⬎100 days). Rats receiving FK 0.02 mg/kg/d with or without the coadministration of SMS 201-995 showed similar MST: 36 ⫾ 0.5 days for group C and 35 ⫾ 0.5 days for group D, respectively. None of the animals died or showed signs of drug-related toxicity. At sacrifice, the grafts harvested by the control group showed a histopathologic picture of severe acute rejection. From the Fujisawa srl (R.O.), Milan, Italy; Laboratorio di Microchirurgia e Chirurgia Sperimentale (S.G., L.L., P.T.), Ospedale Maggiore di Milano IRCCS, Milan, Italy; and Dipartimento di Farmacologia (O.M.), Universita` di Milano, Milan, Italy. Address reprint requests to R. Orsenigo, Fujisawa srl, Corso Sempione 4, 20122 Milan, Italy. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
554
Transplantation Proceedings, 33, 554–555 (2001)
FK506 AND SMS 201-995
The grafts from group B showed a pattern of fibrointimal hyperplasia in spite of a preserved graft function. Group C and D specimens showed a pattern of acute rejection with severe mononuclear myocardial infarction. DISCUSSION
Advances in understanding the cellular and molecular mechanisms that regulate the immune responses, including evidences of interactions between neuropeptides and immunocompetent cells, may offer the potential of new strategies in immunosuppression. SS and its receptor occur in normal and pathological lymphoid tissues, and the stable SS analogue SMS 201-995 acts on the immune response via an inhibiting effect both on polyclonal and oligoclonal T cell-induced proliferation, with a more marked effect on the latter, acting preferentially on the CD28 rather than CD3 activation pathway.5 On the other hand, the immunosuppressant FK506 inhibits T cell activation via CD3 pathway, while the CD28 is unaffected.6 These combined observation led to the hypothesis that SMS 201-995 might act synergistically with FK506 by interfering with different pathways of T cell activation. It was previously demonstrated that SMS 201-995 enhanced the immunosuppressive effect of FK506 at doses that were 100 to 1000 times lower than those that were effective on T lymphocyte activation in vitro, either by mitogens or by alloantigens, confirming the validity of this hypothesis.5 It is important to stress that the SMS 201-995 concentrations that were effective in vitro are in the picomolar range, that is to say, close to the physiological concentrations. Interestingly, SMS 201-995 enhanced the antiproliferative effect of FK506 irrespectively of the doses used when the splenocytes were stimulate by alloantigens, while it was effective only when tested together with the lower dose of FK506 in the case of PHA stimulation.5 The association of FK506 and SMS 201-995 was tested in vivo using an allogenic skin graft model.5 The FK506 regimen selected was the highest dose that did poorly or was not effective in protecting the allograft and that showed no toxicity.7 The SMS 201-995 concentrations selected were the more effective in prolonging graft survival and were without side effects. In the skin model, animals receiving the two drugs in association showed a graft survival time that was significantly improved when compared with each monotherapy group and with the control group, suggesting that a combined therapy might lead to a down-regulation of the immune response without side effects.5 The same associa-
555
tion was therefore tested in another in vivo model, the vascularized heterotopic heart allograft, using the same couple of donor and recipient strains. The two drugs were administered using the same route and using the same criteria for the dose selection.9 In this particular animal model, we found that FK506 at 0.05 mg/kg/d is still therapeutic, being efficacious in preventing acute rejection and leading to an unlimited graft survival time; nevertheless, in the long term this dosage does not avoid the development of chronic rejection. FK506 at a lower dose (0.02 mg/kg/d) seems to delay the onset of the acute rejection in comparison with the control group but does not offer protective long-term effects on the graft survival. The addition of 10 g/kg/bid SMS 201-995 seems not to offer any advantage in this donor/recipient model, but different FK506-SMS combination regimens remain to be evaluated. Padberg and colleagues reported indeed a synergistic effect of 16 g/kg/ 2xd when administered subcutaneously in combination with 1.5 mg/kg/d intramuscular CyA in prolonging MST of heart allografts both vs untreated controls and vs SMS 201-995 or low-dose CyA given alone.9 Other possible strategies are related to the use of long-acting depot formulations of SMS 201-995, which may yield constant levels of SMS 201-995 over several weeks upon single injections.10 ACKNOWLEDGMENT We thank Fujisawa GmbH, Munich, for providing FK506 used in this study.
REFERENCES 1. Yamada Y, Reisine T, Law SF, et al: Mol Endocrinol 6:2136, 1992 2. Elliot E, Metwali A, Blum A, et al: J Immunol 153:1180, 1994 3. Kaupmann K, Bruns C, Raulf F, et al: EMBO J 14:727, 1995 4. Aguila MC, Dees WL, Haensly WE, et al: Proc Natl Acad Sci USA 88:11485, 1991 5. Perego C, Lattuada D, Casnici C, et al: Int J Immunopharmacol 20:479, 1998 6. Sigal NH, Lin CS, Siekierka JJ: Transplant Proc 23:1, 1991 7. Inamura N, Nakahara K, Kino T, et al: Transplantation 45:206, 1988 8. Murase N, Todo S, Lee PH, et al: Transplant Proc 19:71, 1987 9. Padberg WM, Morgalla H, Buhr J, et al: Transplant Proc 23:348, 1991 10. Schuurman HJ, Beckmann N, Briner U, et al: Transplant Proc 28:3272, 1996
T
HE NERVOUS and the immune systems are closely related and may communicate in both directions through soluble mediators such as neuropeptides and cytokines. The nervous system may modulate immunological and inflammatory responses as demonstrated by the identification of neuropeptide receptors on leukocytes and by the regulatory activities on the leukocyte functions following the interaction of such neuropeptides with their receptors. Somatostatin (SS) was originally described as a growth hormone release-inhibiting factor synthesized in the hypothalamus. Five distinct SS receptors (SSTR1–5) have been characterized so far, and SSTR2 has been localized on lymphocytes.1–3 The presence of SS-mRNA in lymphoid tissue indicates that lymphocytes are able to synthesize SS, and this may act as a paracrine factor modulating immune responses.4 SS has an inhibitory effect on mitogen-induced lamina propria and peripheral blood lymphocyte proliferation and also decreases human natural killer cell activity. Because of the very short plasma half-life of native SS, analogues such as SMS 201-995 (octreotide acetate) have been developed. SMS 201-995 binds preferentially SSTR2.3 Previous studies in vitro and in vivo have shown evidences of a synergistic action of SMS 201-995 and FK506.5 An immunosuppressive effect of SMS 201-995 was observed on the proliferation of rat spleen cells induced in vitro, either by polyclonal mitogens (PHA) or by alloantigens. With PHA stimulation, SMS 201-995 significantly enhanced the immunosuppressive action of FK506, and the addition of SMS 201-995 in MLR increased the antiproliferative effect of FK506.5 The association of FK506 and SMS 201-995 was tested in vivo in an allogeneic skin graft model using Lewis (Lew) rats as donors and Brown Norway (BN) rats as recipients. BN treated with 0.1 mg/kg FK506 and 0.5 to 10 g/kg SMS 201-995 showed a significant increase in mean skin allograft survival time when compared to either a monotherapy or control group.5 The aim of the present study was to evaluate the same drug combination in an allogeneic vascularized heterotopic heart model using the same rodent combination. MATERIALS AND METHODS Animals and Transplant Procedures Male BN (RT1n) and Lew (RT1l) rats weighing 200 to 300 g were purchased as recipients and donors of heterotopic heart allograft0041-1345/01/$–see front matter PII S0041-1345(00)02140-0
ing, respectively, and maintained in conventional facilities housed one per cage with food and water ad libitum. The Ono and Lindsay microsurgical technique was used.6
Immunosuppression Both FK506 and SMS 201-995 were administered intraperitoneally, diluted in saline solution. Both drugs were commenced at the time of surgery and administered until rejection occurred.
Experimental Design Four groups of rats (n ⫽ 6 in each group) were included in this study. Group A received the vehicle; group B received FK506 0.05 mg/kg/d; group C received FK506 0.02 mg/kg/d; group D received FK506 0.02 mg/kg/d and SMS 201-995 10 g/kg/bid. The graft survival was assessed through daily abdominal palpation. Rejection was diagnosed by the cessation of palpable heart beat, at which time the animals were sacrificed. Graft survival times expressed in days are given as means ⫾ the standard deviation.
Pathologic Studies An autopsy was carried out on all rats. Samples from grafts were fixed in formalin for paraffin embedding and routine H&E staining.
RESULTS
Control animals (group A), which received only saline solution, rejected the heterotopic hearts with a mean survival time (MST) of 6.5 ⫾ 0.5 days. All three treated groups showed significant improvement in graft survival when compared with the control group. Group B animals receiving FK506 0.05 mg/kg/d showed an unlimited graft survival (⬎100 days). Rats receiving FK 0.02 mg/kg/d with or without the coadministration of SMS 201-995 showed similar MST: 36 ⫾ 0.5 days for group C and 35 ⫾ 0.5 days for group D, respectively. None of the animals died or showed signs of drug-related toxicity. At sacrifice, the grafts harvested by the control group showed a histopathologic picture of severe acute rejection. From the Fujisawa srl (R.O.), Milan, Italy; Laboratorio di Microchirurgia e Chirurgia Sperimentale (S.G., L.L., P.T.), Ospedale Maggiore di Milano IRCCS, Milan, Italy; and Dipartimento di Farmacologia (O.M.), Universita` di Milano, Milan, Italy. Address reprint requests to R. Orsenigo, Fujisawa srl, Corso Sempione 4, 20122 Milan, Italy. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
554
Transplantation Proceedings, 33, 554–555 (2001)
FK506 AND SMS 201-995
The grafts from group B showed a pattern of fibrointimal hyperplasia in spite of a preserved graft function. Group C and D specimens showed a pattern of acute rejection with severe mononuclear myocardial infarction. DISCUSSION
Advances in understanding the cellular and molecular mechanisms that regulate the immune responses, including evidences of interactions between neuropeptides and immunocompetent cells, may offer the potential of new strategies in immunosuppression. SS and its receptor occur in normal and pathological lymphoid tissues, and the stable SS analogue SMS 201-995 acts on the immune response via an inhibiting effect both on polyclonal and oligoclonal T cell-induced proliferation, with a more marked effect on the latter, acting preferentially on the CD28 rather than CD3 activation pathway.5 On the other hand, the immunosuppressant FK506 inhibits T cell activation via CD3 pathway, while the CD28 is unaffected.6 These combined observation led to the hypothesis that SMS 201-995 might act synergistically with FK506 by interfering with different pathways of T cell activation. It was previously demonstrated that SMS 201-995 enhanced the immunosuppressive effect of FK506 at doses that were 100 to 1000 times lower than those that were effective on T lymphocyte activation in vitro, either by mitogens or by alloantigens, confirming the validity of this hypothesis.5 It is important to stress that the SMS 201-995 concentrations that were effective in vitro are in the picomolar range, that is to say, close to the physiological concentrations. Interestingly, SMS 201-995 enhanced the antiproliferative effect of FK506 irrespectively of the doses used when the splenocytes were stimulate by alloantigens, while it was effective only when tested together with the lower dose of FK506 in the case of PHA stimulation.5 The association of FK506 and SMS 201-995 was tested in vivo using an allogenic skin graft model.5 The FK506 regimen selected was the highest dose that did poorly or was not effective in protecting the allograft and that showed no toxicity.7 The SMS 201-995 concentrations selected were the more effective in prolonging graft survival and were without side effects. In the skin model, animals receiving the two drugs in association showed a graft survival time that was significantly improved when compared with each monotherapy group and with the control group, suggesting that a combined therapy might lead to a down-regulation of the immune response without side effects.5 The same associa-
555
tion was therefore tested in another in vivo model, the vascularized heterotopic heart allograft, using the same couple of donor and recipient strains. The two drugs were administered using the same route and using the same criteria for the dose selection.9 In this particular animal model, we found that FK506 at 0.05 mg/kg/d is still therapeutic, being efficacious in preventing acute rejection and leading to an unlimited graft survival time; nevertheless, in the long term this dosage does not avoid the development of chronic rejection. FK506 at a lower dose (0.02 mg/kg/d) seems to delay the onset of the acute rejection in comparison with the control group but does not offer protective long-term effects on the graft survival. The addition of 10 g/kg/bid SMS 201-995 seems not to offer any advantage in this donor/recipient model, but different FK506-SMS combination regimens remain to be evaluated. Padberg and colleagues reported indeed a synergistic effect of 16 g/kg/ 2xd when administered subcutaneously in combination with 1.5 mg/kg/d intramuscular CyA in prolonging MST of heart allografts both vs untreated controls and vs SMS 201-995 or low-dose CyA given alone.9 Other possible strategies are related to the use of long-acting depot formulations of SMS 201-995, which may yield constant levels of SMS 201-995 over several weeks upon single injections.10 ACKNOWLEDGMENT We thank Fujisawa GmbH, Munich, for providing FK506 used in this study.
REFERENCES 1. Yamada Y, Reisine T, Law SF, et al: Mol Endocrinol 6:2136, 1992 2. Elliot E, Metwali A, Blum A, et al: J Immunol 153:1180, 1994 3. Kaupmann K, Bruns C, Raulf F, et al: EMBO J 14:727, 1995 4. Aguila MC, Dees WL, Haensly WE, et al: Proc Natl Acad Sci USA 88:11485, 1991 5. Perego C, Lattuada D, Casnici C, et al: Int J Immunopharmacol 20:479, 1998 6. Sigal NH, Lin CS, Siekierka JJ: Transplant Proc 23:1, 1991 7. Inamura N, Nakahara K, Kino T, et al: Transplantation 45:206, 1988 8. Murase N, Todo S, Lee PH, et al: Transplant Proc 19:71, 1987 9. Padberg WM, Morgalla H, Buhr J, et al: Transplant Proc 23:348, 1991 10. Schuurman HJ, Beckmann N, Briner U, et al: Transplant Proc 28:3272, 1996