Donor-derived IP-10 initiates development of acute allograft rejection

Abstract

An allograft is often considered an immunologically inert playing field on which host leukocytes assemble and wreak havoc. However, we demonstrate that graft-specific physiologic responses to early injury initiate and promulgate destruction of vascularized grafts. Serial analysis of allografts showed that intragraft expression of the three chemokine ligands for the CXC chemo-kine receptor CXCR3 was induced in the order of interferon (IFN)-gamma-inducible protein of 10 kD (IP-10, or CXCL10), IFN-inducible T cell alpha-chemoattractant (I-TAC; CXCL11), and then monokine induced by IFN-gamma (Mig, CXCL9). Initial IP-10 production was localized to endothelial cells, and only IP-10 was induced by isografting. Anti-IP-10 monoclonal antibodies prolonged allograft survival, but surprisingly, IP-10-deficient (IP-10(-/-)) mice acutely rejected allografts. However, though allografts from IP-10(+/+) mice were rejected by day 7, hearts from IP-10(-/-) mice survived long term. Compared with IP-10(+/+) donors, use of IP-10(-/-) donors reduced intragraft expression of cytokines, chemokines and their receptors, and associated leukocyte infiltration and graft injury. Hence, tissue-specific generation of a single chemokine in response to initial ischemia/reperfusion can initiate progressive graft infiltration and amplification of multiple effector pathways, and targeting of this proximal chemokine can prevent acute rejection. These data emphasize the pivotal role of donor-derived IP-10 in initiating alloresponses, with implications for tissue engineering to decrease immunogenicity, and demonstrate that chemokine redundancy may not be operative in vivo.

Figure 1

Rationale for targeting of selected CXCR3 ligands in allograft recipients. (a) Northern blot analysis of cardiac expression of the CXCR3 ligands, IP-10, I-TAC, and Mig, after isografting (H2^b→H2^b; 1 d) or allografting (H2^d→H2^b; 1, 3, and 7 d). IP-10 mRNA was induced in both isografts and allografts, whereas I-TAC and Mig mRNA were restricted to allografts and were first seen at 3 d after transplant. (b) Immunohistologic localization of CXCR3 ligands in portions of the same isografts or allografts as shown in panel a. IP-10 production in isografts and allografts was initially confined to cardiac endothelial cells, whereas by day 3, IP-10 was widely expressed by graft endothelial cells and infiltrating leukocytes; I-TAC was focally expressed by endothelial cells and leukocytes, and Mig was confined to leukocytes, especially large inflammatory macrophages. Cryostat sections, hematoxylin counterstain; original magnifications: ×300. For both Northern blot analysis and immunohistologic studies, data are representative of the results from three samples per group per time point.

Figure 1

Rationale for targeting of selected CXCR3 ligands in allograft recipients. (a) Northern blot analysis of cardiac expression of the CXCR3 ligands, IP-10, I-TAC, and Mig, after isografting (H2^b→H2^b; 1 d) or allografting (H2^d→H2^b; 1, 3, and 7 d). IP-10 mRNA was induced in both isografts and allografts, whereas I-TAC and Mig mRNA were restricted to allografts and were first seen at 3 d after transplant. (b) Immunohistologic localization of CXCR3 ligands in portions of the same isografts or allografts as shown in panel a. IP-10 production in isografts and allografts was initially confined to cardiac endothelial cells, whereas by day 3, IP-10 was widely expressed by graft endothelial cells and infiltrating leukocytes; I-TAC was focally expressed by endothelial cells and leukocytes, and Mig was confined to leukocytes, especially large inflammatory macrophages. Cryostat sections, hematoxylin counterstain; original magnifications: ×300. For both Northern blot analysis and immunohistologic studies, data are representative of the results from three samples per group per time point.

Figure 2

Effects of IP-10 targeting on allograft survival. (a) Cardiac allografts across a full MHC disparity were rejected in ∼7 d, regardless of the choice of donor or recipient, but allograft survival was prolonged by use of neutralizing rat anti–mouse IP-10 mAb (*P < 0.01). Comparable prolongation was seen using a hamster anti–mouse IP-10 mAb. (b) IP-10^−/− mice reject cardiac allografts at the same rate as IP-10^+/+ recipients; however, the survival of allografted hearts from IP-10^−/− mice is markedly prolonged compared with hearts from IP-10^+/+ mice. Data from n = 6 transplants/group for each of the 10 groups shown; *P < 0.001.

Figure 2

Effects of IP-10 targeting on allograft survival. (a) Cardiac allografts across a full MHC disparity were rejected in ∼7 d, regardless of the choice of donor or recipient, but allograft survival was prolonged by use of neutralizing rat anti–mouse IP-10 mAb (*P < 0.01). Comparable prolongation was seen using a hamster anti–mouse IP-10 mAb. (b) IP-10^−/− mice reject cardiac allografts at the same rate as IP-10^+/+ recipients; however, the survival of allografted hearts from IP-10^−/− mice is markedly prolonged compared with hearts from IP-10^+/+ mice. Data from n = 6 transplants/group for each of the 10 groups shown; *P < 0.001.

Figure 3

Early graft infiltration by host leukocytes is modulated in IP-10^−/− donor hearts, as shown by quantitative immunohistologic analysis of intragraft CXCR3⁺ cells, mAb DX5⁺ NK cells, and CD3⁺ T cells present at days 0, 1, 3, and 7 after transplant. Data (mean ± SD) from counting of 20 consecutive fields/graft and 3 allografts/group. *Significantly different cell numbers in IP-10^+/+ donor hearts versus IP-10^−/− samples (*P < 0.05; **P < 0.01; ***P < 0.005).

Figure 4

Mechanisms underlying the beneficial effects of targeting donor tissue IP-10 expression. (a) Histology showing acute rejection with extensive mononuclear cell infiltration, vascular injury, and myocyte necrosis in grafts from IP-10^+/+ donors versus almost normal histology in grafts from IP-10^−/− donors (day 7 after transplant). Paraffin sections, hematoxylin; original magnifications: ×300. (b) Immunohistologic detection of IP-10, I-TAC, and Mig in cardiac allografts from IP-10^+/+ but not IP-10^−/− donors (day 7 after transplant). Cryostat sections, hematoxylin; original magnifications: ×450. (c) Immunohistologic analysis showed significant reduction in recruitment of intragraft CD45⁺ cells (all leukocytes), CD4 and CD8 T cell subsets, macrophages, and IL-2R⁺ (CD25⁺) cells in allografts from IP-10^−/− versus IP-10^+/+ donors at day 7 after transplantation. Data (mean ± SD) from counting of 20 consecutive fields/graft and 3 grafts/group. *Significantly reduced cell numbers versus controls (*P < 0.05; **P < 0.01; ***P < 0.005).

Figure 4

Mechanisms underlying the beneficial effects of targeting donor tissue IP-10 expression. (a) Histology showing acute rejection with extensive mononuclear cell infiltration, vascular injury, and myocyte necrosis in grafts from IP-10^+/+ donors versus almost normal histology in grafts from IP-10^−/− donors (day 7 after transplant). Paraffin sections, hematoxylin; original magnifications: ×300. (b) Immunohistologic detection of IP-10, I-TAC, and Mig in cardiac allografts from IP-10^+/+ but not IP-10^−/− donors (day 7 after transplant). Cryostat sections, hematoxylin; original magnifications: ×450. (c) Immunohistologic analysis showed significant reduction in recruitment of intragraft CD45⁺ cells (all leukocytes), CD4 and CD8 T cell subsets, macrophages, and IL-2R⁺ (CD25⁺) cells in allografts from IP-10^−/− versus IP-10^+/+ donors at day 7 after transplantation. Data (mean ± SD) from counting of 20 consecutive fields/graft and 3 grafts/group. *Significantly reduced cell numbers versus controls (*P < 0.05; **P < 0.01; ***P < 0.005).

Figure 4

Mechanisms underlying the beneficial effects of targeting donor tissue IP-10 expression. (a) Histology showing acute rejection with extensive mononuclear cell infiltration, vascular injury, and myocyte necrosis in grafts from IP-10^+/+ donors versus almost normal histology in grafts from IP-10^−/− donors (day 7 after transplant). Paraffin sections, hematoxylin; original magnifications: ×300. (b) Immunohistologic detection of IP-10, I-TAC, and Mig in cardiac allografts from IP-10^+/+ but not IP-10^−/− donors (day 7 after transplant). Cryostat sections, hematoxylin; original magnifications: ×450. (c) Immunohistologic analysis showed significant reduction in recruitment of intragraft CD45⁺ cells (all leukocytes), CD4 and CD8 T cell subsets, macrophages, and IL-2R⁺ (CD25⁺) cells in allografts from IP-10^−/− versus IP-10^+/+ donors at day 7 after transplantation. Data (mean ± SD) from counting of 20 consecutive fields/graft and 3 grafts/group. *Significantly reduced cell numbers versus controls (*P < 0.05; **P < 0.01; ***P < 0.005).

Figure 5

Effects of lack of donor-derived IP-10 on intragraft events. (a) RNase protection assays of intragraft cytokine, chemokine, and chemokine receptor mRNA expression, comparing grafts from IP-10^+/+ (lanes 1–3) to IP-10^−/− (lanes 4–6) donors at day 7 after transplant. (b) Quantitative analysis of RNase protection assay data, showing the significant depression of intragraft IFN-γ, chemokine, and CC and CXC chemokine receptor mRNA expression in IP-10^−/− versus IP-10^+/+ donor hearts. Data are from three transplants/group, as shown, and experiments were performed twice with comparable findings. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Figure 5

Effects of lack of donor-derived IP-10 on intragraft events. (a) RNase protection assays of intragraft cytokine, chemokine, and chemokine receptor mRNA expression, comparing grafts from IP-10^+/+ (lanes 1–3) to IP-10^−/− (lanes 4–6) donors at day 7 after transplant. (b) Quantitative analysis of RNase protection assay data, showing the significant depression of intragraft IFN-γ, chemokine, and CC and CXC chemokine receptor mRNA expression in IP-10^−/− versus IP-10^+/+ donor hearts. Data are from three transplants/group, as shown, and experiments were performed twice with comparable findings. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.