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Prolonged class II MHC disparate skin allograft survival by treatment with antibodies to the chemokine Mig
Prolonged Class II MHC Disparate Skin Allograft Survival by Treatment with Antibodies to the Chemokine Mig S. Koga, H. Kobayashi, A.C. Novick, H. Toma, and R.L. Fairchild
A
CUTE rejection of allogeneic tissue grafts is initiated by alloantigen–primed T cell recruitment into the graft. Following infiltration of the graft, the T cells are activated by alloantigents to express the immune effector functions mediating destruction of the graft tissue.1,2 The factors recruiting alloantigen-primed T cells into allografts during initiation of the acute rejection process remain unclear. The chemokines are a superfamily of cytokines with chemoattractant properties for leukocyte populations.3,4 The chemokines are organized into four families based on cysteine motifs in the amino terminal end of the protein. The CXC chemokines contain two members, IP–10 (IFN-␥ induced protein-10) and Mig (monokine induced by IFN-␥) with chemoattractant properties for antigen-primed T cells.5,6 Recent studies from this laboratory have indicated the ability to prolong the survival of class II MHC disparate B6.H-2bm12 skin grafts by treating C57BL/6 recipients with a short course of antibodies to Mig.7 The goal of the current study was to test if longer treatment with the Mig antibodies resulted in prolonged B6.H-2bm12 allograft survival and to compare chemokine expression and leukocyte infiltration in these grafts and grafts from control treated recipients experiencing acute rejection. MATERIALS AND METHODS C57BL/6 (H-2b) mice received full thickness skin grafts from syngeneic or class II MHC disparate B6.H-2bm12 donors. Recipients received 0.5 mL aliquots of normal rabbit serum (NRS) or Mig specific antiserum every other day from day 7 to day 21 posttransplantation. In certain experiments, Mig antiserum treatment was continued on a weekly basis after day 21 posttransplantation. At various times after transplantation grafts were retrieved, whole cell RNA was prepared and tested by Northern blot hybridization for expression of various chemokines. Tissue sections were prepared and analyzed for graft infiltrating leukocytes by staining with
hematoxylin and eosin or by immunocytochemistry using antibodies for CD4(GK1.5; rat anti–mouse CD4 mAb), CD8 (53– 67; rat anti-mouse CD8a mAb), or macrophages (MOMA-2; rat antimouse macrophage mAb).
RESULTS
The role of Mig in the rejection of skin allografts was initially tested by giving Mig specific antiserum to C57BL/6 recipients of skin allografts. Because expression of Mig mRNA was not observed in skin allografts until day 9 posttransplantation, this treatment was initiated on day 7 posttransplantation and was continued every other day until day 21 posttransplantation or until the graft was rejected. Treatment with the Mig antiserum had a modest effect on the survival of fully allongeneic (BALB/c 3 C57BL/6) and class I MHC disparate (B6.H–2bm1 3 C57BL/6) skin grafts prolonging survival 3 to 4 days longer than NRS treated recipients. In contrast, survival of the class II MHC disparate combination (B6.H–2bm12 3 C57BL/6) was prolonged from day 14 to 15 in the NRS treated control recipients to day 52 to 55 posttransplant. Associated with the prolonged survival was the absence of infiltrating cells into the B6.H– 2bm12 allografts at day 14 posttransplantation when compared to the NRS treated controls. To test if chronic treatment with the Mig antiserum would prolong the B6.H-2bm12 skin allografts indefinitely, C57BL/6 recipients were treated with the antiserum every From the Urological Institute (S.K., H.K., A.C.N., R.L.F.) and the Department of Immunology (R.L.F.), Cleveland Clinic Foundation, Cleveland, Ohio and Department of Urology (S.K., H.K., H.T.), Tokyo Women’s Medical University, Tokyo, Japan. Supported by USPHS grant AI40459. Address reprint requests to Robert L. Fairchild, PhD, Department of Immunology, NB3-79, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195-0001, USA.
Table 1. Alteration in Histopathology of B6.H-2bm12 Skin Grafts by Treating C57BL/6 Recipients With Antibodies to Mig Recipient Treatment
Time of Rejection
Mig Expression
MCP-1 Expression
RANTES Expression
Graft Infiltration by mø
NRS Mig AS d7–21 Mig AS d7–21/weekly to day 49
d14-15 d50-55 d57-68
⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫾ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹ ⫹⫹⫹ ⫹⫹⫹
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(00)02136-9 549
Transplantation Proceedings, 33, 549–550 (2001)
550
other day from day 7 through day 21 posttransplantation and then once a week thereafter. This treatment resulted in prolonged survival of 70% of the skin allografts during the course of the antiserum treatment. Histological analysis of grafts indicated the absence of T cell and macrophage infiltration into the allografts during the course of the treatment. Following cessation of treatment, the grafts were quickly infiltrated with CD4⫹ T cells. Because few macrophages infiltrated B6.H-2bm12 skin allografts on NRS treated C57BL/6 recipients at day 14 posttransplantation, allografts from Mig antiserum treated recipients were heavily infiltrated with macrophages shortly following cessation of antiserum treatment. Associated with these changes in cellular infiltration into the B6.H-2bm12 allografts on chronically treated Mig antiserum recipients, extensive tissue fibrosis was clearly evident. In contrast, allografts from control treated recipients at day 14 posttransplantation had the characteristic tissue necrosis associated with acute rejection. The expression of three chemokines was tested during rejection of H-2.B6bm12 skin allografts following cessation of chronic Mig antiserum treatment. When compared to levels in NRS treated recipients at the time of rejection (eg, day 14 posttransplantation), the levels of Mig RNA during rejection of the allografts in chronically Mig antiserum treated recipients was considerably higher. Correlating with the intense macrophage infiltration was a great increase in the intra-allograft levels of MCP-1. More surprisingly, RANTES mRNA was at low to undetectable levels in the allografts from the control, NRS–treated recipients but was expressed at high levels in the allografts from the chronic
KOGA, KOBAYASHI, NOVICK ET AL
Mig antiserum treated recipients at the time of rejection following cessation of treatment. CONCLUSION
The rejection of B6.H-2bm12 skin allografts is dependent on the intra-allograft production of Mig. Administration of neutralizing Mig antibodies inhibits T cell infiltration into the allograft and acute rejection. Following cessation of chronic Mig antiserum treatment, the allografts undergo a rejection process that is quite distinct from acute rejection. This difference includes an intense infiltration of macrophages, tissue fibrosis, and high expression of several chemokines. It is important to note that these events are dependent on the ability of the T cells to infiltrate the allografts in a Mig dependent manner. The results suggest that prolongation of graft survival by treating the Mig antiserum results in alteration in the function of the alloreactive T cells with changes in the histopathology of rejection as a consequence of this alteration. REFERENCES 1. Hall B: Transplantation 51:1141, 1991 2. Rosenberg AS, Singer A: Annu Rev Immunol 10:333, 1993 3. Rollines BJ: Blood 90:909, 1997 4. Oppenheim JJ, Zachariae COC, Mukaida N, et al: Annu Rev Immunol 9:617, 1991 5. Luster AD, Ravetch JV: J Exp Med 166:1084, 1987 6. Liao F, Rabin RL, Yannelli JR, et al: J Exp Med 183:1301, 1995 7. Koga S, Auerbach MB, Engeman TM, et al: J Immunol 163:4878, 1999
A
CUTE rejection of allogeneic tissue grafts is initiated by alloantigen–primed T cell recruitment into the graft. Following infiltration of the graft, the T cells are activated by alloantigents to express the immune effector functions mediating destruction of the graft tissue.1,2 The factors recruiting alloantigen-primed T cells into allografts during initiation of the acute rejection process remain unclear. The chemokines are a superfamily of cytokines with chemoattractant properties for leukocyte populations.3,4 The chemokines are organized into four families based on cysteine motifs in the amino terminal end of the protein. The CXC chemokines contain two members, IP–10 (IFN-␥ induced protein-10) and Mig (monokine induced by IFN-␥) with chemoattractant properties for antigen-primed T cells.5,6 Recent studies from this laboratory have indicated the ability to prolong the survival of class II MHC disparate B6.H-2bm12 skin grafts by treating C57BL/6 recipients with a short course of antibodies to Mig.7 The goal of the current study was to test if longer treatment with the Mig antibodies resulted in prolonged B6.H-2bm12 allograft survival and to compare chemokine expression and leukocyte infiltration in these grafts and grafts from control treated recipients experiencing acute rejection. MATERIALS AND METHODS C57BL/6 (H-2b) mice received full thickness skin grafts from syngeneic or class II MHC disparate B6.H-2bm12 donors. Recipients received 0.5 mL aliquots of normal rabbit serum (NRS) or Mig specific antiserum every other day from day 7 to day 21 posttransplantation. In certain experiments, Mig antiserum treatment was continued on a weekly basis after day 21 posttransplantation. At various times after transplantation grafts were retrieved, whole cell RNA was prepared and tested by Northern blot hybridization for expression of various chemokines. Tissue sections were prepared and analyzed for graft infiltrating leukocytes by staining with
hematoxylin and eosin or by immunocytochemistry using antibodies for CD4(GK1.5; rat anti–mouse CD4 mAb), CD8 (53– 67; rat anti-mouse CD8a mAb), or macrophages (MOMA-2; rat antimouse macrophage mAb).
RESULTS
The role of Mig in the rejection of skin allografts was initially tested by giving Mig specific antiserum to C57BL/6 recipients of skin allografts. Because expression of Mig mRNA was not observed in skin allografts until day 9 posttransplantation, this treatment was initiated on day 7 posttransplantation and was continued every other day until day 21 posttransplantation or until the graft was rejected. Treatment with the Mig antiserum had a modest effect on the survival of fully allongeneic (BALB/c 3 C57BL/6) and class I MHC disparate (B6.H–2bm1 3 C57BL/6) skin grafts prolonging survival 3 to 4 days longer than NRS treated recipients. In contrast, survival of the class II MHC disparate combination (B6.H–2bm12 3 C57BL/6) was prolonged from day 14 to 15 in the NRS treated control recipients to day 52 to 55 posttransplant. Associated with the prolonged survival was the absence of infiltrating cells into the B6.H– 2bm12 allografts at day 14 posttransplantation when compared to the NRS treated controls. To test if chronic treatment with the Mig antiserum would prolong the B6.H-2bm12 skin allografts indefinitely, C57BL/6 recipients were treated with the antiserum every From the Urological Institute (S.K., H.K., A.C.N., R.L.F.) and the Department of Immunology (R.L.F.), Cleveland Clinic Foundation, Cleveland, Ohio and Department of Urology (S.K., H.K., H.T.), Tokyo Women’s Medical University, Tokyo, Japan. Supported by USPHS grant AI40459. Address reprint requests to Robert L. Fairchild, PhD, Department of Immunology, NB3-79, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195-0001, USA.
Table 1. Alteration in Histopathology of B6.H-2bm12 Skin Grafts by Treating C57BL/6 Recipients With Antibodies to Mig Recipient Treatment
Time of Rejection
Mig Expression
MCP-1 Expression
RANTES Expression
Graft Infiltration by mø
NRS Mig AS d7–21 Mig AS d7–21/weekly to day 49
d14-15 d50-55 d57-68
⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫾ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹ ⫹⫹⫹ ⫹⫹⫹
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(00)02136-9 549
Transplantation Proceedings, 33, 549–550 (2001)
550
other day from day 7 through day 21 posttransplantation and then once a week thereafter. This treatment resulted in prolonged survival of 70% of the skin allografts during the course of the antiserum treatment. Histological analysis of grafts indicated the absence of T cell and macrophage infiltration into the allografts during the course of the treatment. Following cessation of treatment, the grafts were quickly infiltrated with CD4⫹ T cells. Because few macrophages infiltrated B6.H-2bm12 skin allografts on NRS treated C57BL/6 recipients at day 14 posttransplantation, allografts from Mig antiserum treated recipients were heavily infiltrated with macrophages shortly following cessation of antiserum treatment. Associated with these changes in cellular infiltration into the B6.H-2bm12 allografts on chronically treated Mig antiserum recipients, extensive tissue fibrosis was clearly evident. In contrast, allografts from control treated recipients at day 14 posttransplantation had the characteristic tissue necrosis associated with acute rejection. The expression of three chemokines was tested during rejection of H-2.B6bm12 skin allografts following cessation of chronic Mig antiserum treatment. When compared to levels in NRS treated recipients at the time of rejection (eg, day 14 posttransplantation), the levels of Mig RNA during rejection of the allografts in chronically Mig antiserum treated recipients was considerably higher. Correlating with the intense macrophage infiltration was a great increase in the intra-allograft levels of MCP-1. More surprisingly, RANTES mRNA was at low to undetectable levels in the allografts from the control, NRS–treated recipients but was expressed at high levels in the allografts from the chronic
KOGA, KOBAYASHI, NOVICK ET AL
Mig antiserum treated recipients at the time of rejection following cessation of treatment. CONCLUSION
The rejection of B6.H-2bm12 skin allografts is dependent on the intra-allograft production of Mig. Administration of neutralizing Mig antibodies inhibits T cell infiltration into the allograft and acute rejection. Following cessation of chronic Mig antiserum treatment, the allografts undergo a rejection process that is quite distinct from acute rejection. This difference includes an intense infiltration of macrophages, tissue fibrosis, and high expression of several chemokines. It is important to note that these events are dependent on the ability of the T cells to infiltrate the allografts in a Mig dependent manner. The results suggest that prolongation of graft survival by treating the Mig antiserum results in alteration in the function of the alloreactive T cells with changes in the histopathology of rejection as a consequence of this alteration. REFERENCES 1. Hall B: Transplantation 51:1141, 1991 2. Rosenberg AS, Singer A: Annu Rev Immunol 10:333, 1993 3. Rollines BJ: Blood 90:909, 1997 4. Oppenheim JJ, Zachariae COC, Mukaida N, et al: Annu Rev Immunol 9:617, 1991 5. Luster AD, Ravetch JV: J Exp Med 166:1084, 1987 6. Liao F, Rabin RL, Yannelli JR, et al: J Exp Med 183:1301, 1995 7. Koga S, Auerbach MB, Engeman TM, et al: J Immunol 163:4878, 1999