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TH2 cytokines regulate gene expression and proinflammatory responses in xenografts
TH2 Cytokines Regulate Gene Expression and Proinflammatory Responses in Xenografts N. Wang, J.M. Lee, M.P. Soares, E. Csizmadia, D. Xu, F.Y. Liew, N. Smith, F.H. Bach, and Y. Lin
I
F A FORM of delayed xenograft rejection (DXR) is prevented in a concordant xenograft, such as hamster hearts transplanted in rats, the graft may encounter a more classical cellular immune response involving T-cell activation and graft infiltration as seen in allogeneic situation.1 We have previously reported that long-term surviving and accommodated hamster hearts in rats were primarily infiltrated with Th2 cells that produced IL-4, IL-10, and IL-13. In contrast, grafts undergoing DXR showed primarily Th1 cell infiltration with associated Th1 cytokines (IL-2, IFN-␥, and TNF-␣).2 In the present study, we investigated the evolution and function of Th2 cells in rats when hamster cardiac xenografts were accommodating. We have identified that antiST2L, a rabbit polyclonal antibody that selectively labels Th2 (ST2L⫹) cells in mice,3 recognizes Th2 cells in rats as well, facilitating identification and separation of these cells. In addition to the previously demonstrated Th2 cell infiltration and expression of Th2 cytokines within accommodated grafts, we detected an increase in the number of Th2 cells in the recipient’s spleen during accommodation. Those Th2 cells upon adoptive transfer facilitated protective gene expression in grafts in vivo and inhibited proinflammatory responses mediated by cytotoxic T lymphocytes (CTL) and lymphokine activated killer (LAK) cells in vitro. MATERIALS AND METHODS Cervical and abdominal heterotopic hamster-to-rat heart transplantation was performed using published techniques.1 Immunosuppressive therapies with Cyclosporin A (CyA; Novartis Pharma, Basel, Switzerland); cobra venom factor (CVF; purity ⱖ 85% by SDS Page; Quidel Co, San Diego, Calif, USA) were described previously.2 Cell surface and intracellular cytokine phenotypes were measured by two- or three-color flow cytometry analysis using a methodology described previously.4 CD4⫹, ST2L⫹ T cells were isolated from rat splenocytes by indirect panning techniques or magnetic beads.1 LAK cells and CTL were generated using published standard techniques.5 Immunohistochemical staining and Western blot analysis were performed to detect gene expression in the graft.2
RESULTS AND DISCUSSION
We found that the structure of the peptide within ST2L in mice3 was highly similar to that within ST2L in rats as described in the literature.6 Based on a study using anti0041-1345/01/$–see front matter PII S0041-1345(00)02249-1
ST2L antibodies, a rabbit polyclonal antibody that selectively labels Th2 (ST2L⫹) cells in mice,3 plus intracellular cytokine analysis, we have demonstrated that anti-ST2L recognizes Th2 cells in rats. These antibodies therefore were used in various studies to understand the role of Th2 cells in xenograft accommodation in a hamster-to-rat heart xenotransplantation model. There was a progressive increase of Th2 cells (CD4⫹, ST2L⫹) in the spleen of CVF ⫹ CyA-treated rats in which hamster hearts were accommodating. These Th2-polarized cells produced predominantly IL-4 and IL-10. In contrast, splenic T cells from untreated rats in which xenografts underwent DXR produced primarily IFN-␥ and TNF-␣. Adoptive transfer of purified Th2 cells (5 ⫻ 107 cells/rat, intravenously) taken from rats carrying an accommodating heart for 20 days into CyA-treated naive rats facilitated expression of protective genes such as heme oxygenase (HO-1) in graft endothelial cells and smooth muscle cells. This was associated with an increased ability of those grafts to resist rejection mediated by adoptively transferred hyperimmune serum. In contrast, Th2-negative cells isolated from same animals did not show such effects. In vitro, Th2 cells activated in vivo were added to cell culture in which xenospecific CTL or rIL-2-induced LAK cells were generated. Th2 cells suppressed the generation of xenospecific CTL and LAK cell activity against hamster target cells. In both cultures, Th2-negative cells isolated from same rats failed to do so. Our findings have provided evidence that T cells undergo Th2 immune deviation during xenograft accommodation. From the Immunobiology Research Center (Y.L., N.W., J.M.L., M.P.S., E.C., F.H.B.), Department of Surgery, Beth Israel Deaconess Medical Center, and Department of Pathology (N.S.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; and Department of Immunology (D.X., F.Y.L.), University of Glasgow, Glasgow, UK. This is paper no. 785 from our laboratories. This work is supported in part by National Institutes of Health Grant RO1HL 58688 (to F.H.B.). F.H.B. is a paid consultant to Novartis Pharma, Basel, Switzerland. Address reprint requests to Yuan Lin, MD, PhD, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
776
Transplantation Proceedings, 33, 776–777 (2001)
TH2 CYTOKINES REGULATE GENE EXPRESSION
777
The Th2 responses appear to aid in xenograft survival by upregulation of protective gene expression in the graft and inhibition of proinflammatory immune responses of the host.
4. Maino VC, Suni MA, Ruitenberg JJ: Cytometry 20:127, 1995
REFERENCES
5. Lin Y, Proud G, Taylor RM, et al: Immunology 79:290, 1993
1. Lin Y, Soares MP, Sato K, et al: J Immunol 162:1206, 1999 2. Bach FH, Ferran C, Hechenleitner P, et al: Nat Med 3:196, 1997
3. Xu D, Chan WL, Leung BP, et al: J Exp Med 187:787, 1998
6. Bergers G, Reikerstorfer A, Braselmann S, et al: EMBO J 13:1176, 1994
I
F A FORM of delayed xenograft rejection (DXR) is prevented in a concordant xenograft, such as hamster hearts transplanted in rats, the graft may encounter a more classical cellular immune response involving T-cell activation and graft infiltration as seen in allogeneic situation.1 We have previously reported that long-term surviving and accommodated hamster hearts in rats were primarily infiltrated with Th2 cells that produced IL-4, IL-10, and IL-13. In contrast, grafts undergoing DXR showed primarily Th1 cell infiltration with associated Th1 cytokines (IL-2, IFN-␥, and TNF-␣).2 In the present study, we investigated the evolution and function of Th2 cells in rats when hamster cardiac xenografts were accommodating. We have identified that antiST2L, a rabbit polyclonal antibody that selectively labels Th2 (ST2L⫹) cells in mice,3 recognizes Th2 cells in rats as well, facilitating identification and separation of these cells. In addition to the previously demonstrated Th2 cell infiltration and expression of Th2 cytokines within accommodated grafts, we detected an increase in the number of Th2 cells in the recipient’s spleen during accommodation. Those Th2 cells upon adoptive transfer facilitated protective gene expression in grafts in vivo and inhibited proinflammatory responses mediated by cytotoxic T lymphocytes (CTL) and lymphokine activated killer (LAK) cells in vitro. MATERIALS AND METHODS Cervical and abdominal heterotopic hamster-to-rat heart transplantation was performed using published techniques.1 Immunosuppressive therapies with Cyclosporin A (CyA; Novartis Pharma, Basel, Switzerland); cobra venom factor (CVF; purity ⱖ 85% by SDS Page; Quidel Co, San Diego, Calif, USA) were described previously.2 Cell surface and intracellular cytokine phenotypes were measured by two- or three-color flow cytometry analysis using a methodology described previously.4 CD4⫹, ST2L⫹ T cells were isolated from rat splenocytes by indirect panning techniques or magnetic beads.1 LAK cells and CTL were generated using published standard techniques.5 Immunohistochemical staining and Western blot analysis were performed to detect gene expression in the graft.2
RESULTS AND DISCUSSION
We found that the structure of the peptide within ST2L in mice3 was highly similar to that within ST2L in rats as described in the literature.6 Based on a study using anti0041-1345/01/$–see front matter PII S0041-1345(00)02249-1
ST2L antibodies, a rabbit polyclonal antibody that selectively labels Th2 (ST2L⫹) cells in mice,3 plus intracellular cytokine analysis, we have demonstrated that anti-ST2L recognizes Th2 cells in rats. These antibodies therefore were used in various studies to understand the role of Th2 cells in xenograft accommodation in a hamster-to-rat heart xenotransplantation model. There was a progressive increase of Th2 cells (CD4⫹, ST2L⫹) in the spleen of CVF ⫹ CyA-treated rats in which hamster hearts were accommodating. These Th2-polarized cells produced predominantly IL-4 and IL-10. In contrast, splenic T cells from untreated rats in which xenografts underwent DXR produced primarily IFN-␥ and TNF-␣. Adoptive transfer of purified Th2 cells (5 ⫻ 107 cells/rat, intravenously) taken from rats carrying an accommodating heart for 20 days into CyA-treated naive rats facilitated expression of protective genes such as heme oxygenase (HO-1) in graft endothelial cells and smooth muscle cells. This was associated with an increased ability of those grafts to resist rejection mediated by adoptively transferred hyperimmune serum. In contrast, Th2-negative cells isolated from same animals did not show such effects. In vitro, Th2 cells activated in vivo were added to cell culture in which xenospecific CTL or rIL-2-induced LAK cells were generated. Th2 cells suppressed the generation of xenospecific CTL and LAK cell activity against hamster target cells. In both cultures, Th2-negative cells isolated from same rats failed to do so. Our findings have provided evidence that T cells undergo Th2 immune deviation during xenograft accommodation. From the Immunobiology Research Center (Y.L., N.W., J.M.L., M.P.S., E.C., F.H.B.), Department of Surgery, Beth Israel Deaconess Medical Center, and Department of Pathology (N.S.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; and Department of Immunology (D.X., F.Y.L.), University of Glasgow, Glasgow, UK. This is paper no. 785 from our laboratories. This work is supported in part by National Institutes of Health Grant RO1HL 58688 (to F.H.B.). F.H.B. is a paid consultant to Novartis Pharma, Basel, Switzerland. Address reprint requests to Yuan Lin, MD, PhD, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
776
Transplantation Proceedings, 33, 776–777 (2001)
TH2 CYTOKINES REGULATE GENE EXPRESSION
777
The Th2 responses appear to aid in xenograft survival by upregulation of protective gene expression in the graft and inhibition of proinflammatory immune responses of the host.
4. Maino VC, Suni MA, Ruitenberg JJ: Cytometry 20:127, 1995
REFERENCES
5. Lin Y, Proud G, Taylor RM, et al: Immunology 79:290, 1993
1. Lin Y, Soares MP, Sato K, et al: J Immunol 162:1206, 1999 2. Bach FH, Ferran C, Hechenleitner P, et al: Nat Med 3:196, 1997
3. Xu D, Chan WL, Leung BP, et al: J Exp Med 187:787, 1998
6. Bergers G, Reikerstorfer A, Braselmann S, et al: EMBO J 13:1176, 1994