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Interleukin 12 delays allograft rejection: effect mediated via nitric oxide
Interleukin 12 Delays Allograft Rejection: Effect Mediated Via Nitric Oxide N. Verma, X.Y. He, J. Chen, C. Robinson, R. Boyd, G. Tran, and B.M. Hall
I
NTERLEUKIN 12 is a heterodimeric cytokine composed of two subunits, IL-12p40 and IL-12p35. Originally it was identified as natural killer cell-stimulatory factor (NKSF) from EBV-transformed human B lymphoblastoid cell line.1 Later another factor, cytotoxic lymphocyte maturation factor, was isolated from B cell line,2 and upon cloning of the genes for these two, both were found identical3 and were later termed interleukin-12 (IL-12). This cytokine is produced by B cells, macrophages, and antigen-presenting cells and has major effects on natural killer (NK) cells and T lymphocytes.4 Its specific effects include the production of IFN-␥ from NK cells and T cells. It enhances growth of activated NK cells and increases cell-mediated cytotoxicity. The effects on Th1 cells include the activation of Th1 cells and production of IFN-␥. Through its effect on Th1 cells it causes cytotoxic lymphocyte generation, macrophage activation, and antibody isotype switch to complement-fixing antibodies. More recently IL-12 has been reported to have direct effect on B cells and dendritic cells. IL-12 is a key cytokine involved in the regulation of Th1/Th2 balance in both in vivo and in vitro immune responses.4,5 Allograft rejection is thought to be mediated by Th1 cells that are induced by IL-12 produced by antigen presenting cells. Th1 initiates graft rejection by promoting both CTL and delayed-type hypersensitivity responses, which serve as the terminal effector mechanism of allograft rejection.6,7 To further examine the role of IL-12 in alloimmune responses we cloned both the chains of rat IL-12. RT PCR was used to amplify the IL-12p35 subunit gene from dendritic cells of rat bone marrow origin using primers based on homology between mouse and human gene sequences. Similarly rat IL-12p40 gene was amplified using cDNA from the B cells. Both the genes were cloned in the pCEP4 vector and were expressed in CHO-k cell line alone or together to produce rat IL-12 in its various forms, including IL-12p35, IL-12p40, and IL-12p70, the heterodimer. Both genes were sequenced in full. Rat IL-12p35 has not been sequenced before, and the sequence was submitted to NCBI data base. IL-12 BIOASSAY
IL-12 biological assay was performed using lymphocyte proliferation assay. Lymphocytes (5 ⫻ 104 per well) were 0041-1345/01/$–see front matter PII S0041-1345(00)02074-1
cultured in the presence of various forms of IL-12 for 48 hours. Ability of the cytokine to induce proliferation of T cells in vitro was used as a biological assay activity, and only the p70 heterodimer induced proliferation, indicating that either IL-12p40 or IL-12p35 alone is not biological active. One unit was defined as the amount required to support 50% maximal proliferation. EFFECT OF IL-12 ON ALLOGRAFT SURVIVAL
To examine IL-12’s effect on allograft rejection, DA rats were grafted with fully allogeneic PVG neonatal heart grafts and treated daily with various cytokines for 10 days. Paradoxically, IL-12p70 treatment significantly (P ⬍ .001) delayed rejection (23 ⫾ 7.5 days, mean ⫾ SD, (n ⫽ 16) compared to supernatant from nontransfected CHO-k cell line (15 ⫾ 4 days), IL-12p35 (11 ⫾ 3.5 days), and IL-12p40 (14.8 ⫾ 1 days). MECHANISM OF ALLOGRAFT SURVIVAL
To study the mechanism of IL-12p70-induced delay in rejection, cytokine mRNA expression was studied in the draining lymph node of these animals and donor hearts. IL-12 induced the iNOS and IFN-␥ mRNA expression in the donor hearts and lymph node, while no differences could be noticed in the IL-2, TNF␣, and the IL-12p35 and IL-12p40 expression. As IL-12p70 treatment has been found to prevent injury in some autoimmune models by induction of nitric oxide, we examined if coadministration of inducible nitric oxide synthetase inhibitor (L-NIL) prevents the effect. Rats given both IL-12p70 and L-NIL rejected in normal tempo (13.7 ⫾ 2.4 days), which was significantly faster than with IL-12p70 alone (P ⬍ .001) and was not different from normal rejection or nontransfected CHO-k supernatant. Further measurement of serum reactive nitrogen intermediate (RNI) as a indicator of nitric oxide production also indicated a higher level of RNI in serum of IL-12p70-treated animals, which was reduced to normal rejection level when IL-12p70 treatment was comFrom the Department of Medicine, Liverpool Hospital and University of New South Wales, Liverpool, NSW, Australia. Address reprint requests to Dr B. Hall, Liverpool Hospital, Department of Medicine, 4th Floor HSB, PO Box 107, Liverpool, New Australia 2170, Australia. E-mail: [email protected]. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
416
Transplantation Proceedings, 33, 416–417 (2001)
IL-12 DELAYS ALLOGRAFT REJECTION
bined with L-NIL, indicating that the IL-12p70-induced delay in rejection is due to the increased nitric oxide production. CONCLUSION
This study demonstrate that IL-12p70 could significantly delay the rejection of allograft in neonatal rat heart graft model, and this effect is mediated by nitric oxide production.
417
REFERENCES 1. Kobayashi M, Fitz L, Ryan M, et al: J Exp Med 170:827, 1989 2. Stern AS, Podlaski FJ, Hulmes JD, et al: Proc Natl Acad Sci USA 87:6808, 1990 3. Wolf SF, Temple PA, Kobayashi M, et al: J Immunol 146:3074, 1991 4. Trinchieri G: Annu Rev Immunol 13:251, 1995 5. Hsieh CS, Mcatonia CS, Tripp SF, et al: Science 260:547, 1993 6. Hall BM: Transplantation 51:1141, 1991 7. Rosenberg AS, Singer A: Annu Rev Immunol 10:333, 1992
I
NTERLEUKIN 12 is a heterodimeric cytokine composed of two subunits, IL-12p40 and IL-12p35. Originally it was identified as natural killer cell-stimulatory factor (NKSF) from EBV-transformed human B lymphoblastoid cell line.1 Later another factor, cytotoxic lymphocyte maturation factor, was isolated from B cell line,2 and upon cloning of the genes for these two, both were found identical3 and were later termed interleukin-12 (IL-12). This cytokine is produced by B cells, macrophages, and antigen-presenting cells and has major effects on natural killer (NK) cells and T lymphocytes.4 Its specific effects include the production of IFN-␥ from NK cells and T cells. It enhances growth of activated NK cells and increases cell-mediated cytotoxicity. The effects on Th1 cells include the activation of Th1 cells and production of IFN-␥. Through its effect on Th1 cells it causes cytotoxic lymphocyte generation, macrophage activation, and antibody isotype switch to complement-fixing antibodies. More recently IL-12 has been reported to have direct effect on B cells and dendritic cells. IL-12 is a key cytokine involved in the regulation of Th1/Th2 balance in both in vivo and in vitro immune responses.4,5 Allograft rejection is thought to be mediated by Th1 cells that are induced by IL-12 produced by antigen presenting cells. Th1 initiates graft rejection by promoting both CTL and delayed-type hypersensitivity responses, which serve as the terminal effector mechanism of allograft rejection.6,7 To further examine the role of IL-12 in alloimmune responses we cloned both the chains of rat IL-12. RT PCR was used to amplify the IL-12p35 subunit gene from dendritic cells of rat bone marrow origin using primers based on homology between mouse and human gene sequences. Similarly rat IL-12p40 gene was amplified using cDNA from the B cells. Both the genes were cloned in the pCEP4 vector and were expressed in CHO-k cell line alone or together to produce rat IL-12 in its various forms, including IL-12p35, IL-12p40, and IL-12p70, the heterodimer. Both genes were sequenced in full. Rat IL-12p35 has not been sequenced before, and the sequence was submitted to NCBI data base. IL-12 BIOASSAY
IL-12 biological assay was performed using lymphocyte proliferation assay. Lymphocytes (5 ⫻ 104 per well) were 0041-1345/01/$–see front matter PII S0041-1345(00)02074-1
cultured in the presence of various forms of IL-12 for 48 hours. Ability of the cytokine to induce proliferation of T cells in vitro was used as a biological assay activity, and only the p70 heterodimer induced proliferation, indicating that either IL-12p40 or IL-12p35 alone is not biological active. One unit was defined as the amount required to support 50% maximal proliferation. EFFECT OF IL-12 ON ALLOGRAFT SURVIVAL
To examine IL-12’s effect on allograft rejection, DA rats were grafted with fully allogeneic PVG neonatal heart grafts and treated daily with various cytokines for 10 days. Paradoxically, IL-12p70 treatment significantly (P ⬍ .001) delayed rejection (23 ⫾ 7.5 days, mean ⫾ SD, (n ⫽ 16) compared to supernatant from nontransfected CHO-k cell line (15 ⫾ 4 days), IL-12p35 (11 ⫾ 3.5 days), and IL-12p40 (14.8 ⫾ 1 days). MECHANISM OF ALLOGRAFT SURVIVAL
To study the mechanism of IL-12p70-induced delay in rejection, cytokine mRNA expression was studied in the draining lymph node of these animals and donor hearts. IL-12 induced the iNOS and IFN-␥ mRNA expression in the donor hearts and lymph node, while no differences could be noticed in the IL-2, TNF␣, and the IL-12p35 and IL-12p40 expression. As IL-12p70 treatment has been found to prevent injury in some autoimmune models by induction of nitric oxide, we examined if coadministration of inducible nitric oxide synthetase inhibitor (L-NIL) prevents the effect. Rats given both IL-12p70 and L-NIL rejected in normal tempo (13.7 ⫾ 2.4 days), which was significantly faster than with IL-12p70 alone (P ⬍ .001) and was not different from normal rejection or nontransfected CHO-k supernatant. Further measurement of serum reactive nitrogen intermediate (RNI) as a indicator of nitric oxide production also indicated a higher level of RNI in serum of IL-12p70-treated animals, which was reduced to normal rejection level when IL-12p70 treatment was comFrom the Department of Medicine, Liverpool Hospital and University of New South Wales, Liverpool, NSW, Australia. Address reprint requests to Dr B. Hall, Liverpool Hospital, Department of Medicine, 4th Floor HSB, PO Box 107, Liverpool, New Australia 2170, Australia. E-mail: [email protected]. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
416
Transplantation Proceedings, 33, 416–417 (2001)
IL-12 DELAYS ALLOGRAFT REJECTION
bined with L-NIL, indicating that the IL-12p70-induced delay in rejection is due to the increased nitric oxide production. CONCLUSION
This study demonstrate that IL-12p70 could significantly delay the rejection of allograft in neonatal rat heart graft model, and this effect is mediated by nitric oxide production.
417
REFERENCES 1. Kobayashi M, Fitz L, Ryan M, et al: J Exp Med 170:827, 1989 2. Stern AS, Podlaski FJ, Hulmes JD, et al: Proc Natl Acad Sci USA 87:6808, 1990 3. Wolf SF, Temple PA, Kobayashi M, et al: J Immunol 146:3074, 1991 4. Trinchieri G: Annu Rev Immunol 13:251, 1995 5. Hsieh CS, Mcatonia CS, Tripp SF, et al: Science 260:547, 1993 6. Hall BM: Transplantation 51:1141, 1991 7. Rosenberg AS, Singer A: Annu Rev Immunol 10:333, 1992