Inhibition of chemokine-glycosaminoglycan interactions in donor tissue reduces mouse allograft vasculopathy and transplant rejection

Abstract

Background: Binding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts.

Methodology/principal findings: Analysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1(f/f)TekCre(+)) heparan sulfate (GAG)-deficient (Ndst1(-/-), p<0.044) and CCR2-deficient (Ccr2(-/-), p<0.04) donor transplants. Donor tissue GAG or CCR2 deficiency markedly reduced inflammation and vasculopathy, whereas recipient deficiencies did not. Treatment with three CMPs was also investigated; Poxviral M-T1 blocks CC chemokine receptor binding, M-T7 blocks C, CC, and CXC GAG binding, and herpesviral M3 binds receptor and GAG binding for all classes. M-T7 reduced intimal hyperplasia in wild type (WT) (Ccr2(+/+), p< or =0.003 and Ccr2(-/-), p</=0.027) aortic allografts, but not in Ndst1(-/-) aortic allografts (p = 0.933). M-T1 and M3 inhibited WT (Ccr2(+/+) and Ndst1(+/+), p< or =0.006) allograft vasculopathy, but did not block vasculopathy in Ccr2(-/-) (p = 0.61). M-T7 treatment alone, even without immunosuppressive drugs, also significantly prolonged survival of renal allograft transplants (p< or =0.001).

Conclusions/significance: Interruption of chemokine-GAG interactions, even in the absence of chemokine-receptor blockade, is a highly effective approach to reduction of allograft rejection, reducing vascular inflammation and prolonging allograft survival. Although chemokines direct both local and systemic cell migration, interruption of inherent chemokine responses in the donor tissue unexpectedly had a greater therapeutic impact on allograft vasculopathy.

Figure 1. Local, but not systemic, GAG or receptor deficiency reduces neointimal hyperplasia in mouse aortic allograft transplants.

Histology cross sections of aortic allograft transplants stained with Masson's trichrome demonstrating reduced inflammatory cell invasion with Ndst1-deficient (Ndst1 ^−/−) (B) and Ccr2-deficient (Ccr2 ^−/−) (D) donor aortic allografts when compared to wild type (WT)(A, B). Bar graphs demonstrate similar, significant reductions in mean neointimal area for Ccr2 ^−/− (D) and Ndst1 ^−/− (E) transplants. Reduced intimal area is detected with donor aortic deficiency, but not with recipient deficiency (F, reverse transplant WT into Ccr2 ^−/− or Ndst1 ^−/− deficient recipient mice). Measurements reported as mean ± S.E. Arrows bracket intimal plaque, arrow heads indicates mononuclear cell infiltrates. Mag 400X.

Figure 2. Viral chemokine modulating proteins (CMPs) significantly reduce allograft neointimal hyperplasia with donor receptor and GAG dependent specificity.

H & E stained cross sections. M-T1 (A) and M-T7 (C) at 0.6 µg, or greater, doses reduced plaque in Ccr2 ^+/+ aortic transplants. M-T1 (B) did not inhibit intimal hyperplasia in Ccr2 ^−/− donor allografts (B) whereas M-T7 did (D). Bar graphs illustrate significant reductions in intimal to medial thickness with M-T1 (E), M3 (F), and M-T7 treatment of Ccr2+/+ donor allografts, whereas M-T1 (E) and M3 (F) are inactive in Ccr2 ^−/− donors. M-T7 did significantly reduce intimal hyperplasia in Ccr2 ^−/− donor allografts (G). Reverse transplant to WT Ccr2 ^+/+ mice did significantly reduce intimal area, but Ccr2 ^−/− recipients show only a trend toward reduction with M-T7 treatment (H). Measurements reported as mean ± S.E. Arrows bracket intimal plaque. Arrowheads point to inflammatory cell invasion. Mag. 200X.

Figure 3. M-T7 CMP loses inhibitory activity in Ndst1^−/− donor allografts.

Bar graphs of intimal/medial thickness ratios measured in aortic transplant cross-sections at 4 weeks follow up post transplant. M-T7 reduced plaque in WT (C57Bl/6, Ndst1 ^+/+) donor (P<0.032), but not in Ndst1 ^−/− donor (P = 0.933) to Balb/c recipient allografts (A). In the reverse transplant of Balb/c to WT (C57Bl/6, Ndst1 ^+/+) M-T7 again reduced plaque (P<0.050), but not in Balb/c to Ndst1 ^−/− transplants (P = 0.588). M-T1 treatment reduced plaque in Ndst1 ^−/− donor aortic transplanted mice at 4 weeks follow up (C), but this trend is non significant (P = 0.222). Measurements reported as mean ± S.E.

Figure 4. M-T7 CMP treatment of mouse renal allograft transplant recipients markedly prolongs survival.

A. M-T7 treatment alone, with no added immunosuppressive treatment, markedly improved survival of mice to 100 days follow up after renal allograft transplant (100% survival) when compared to controls given no treatment (median survival 22.7±8.1days, 0% survival past 30 days, P<0.001). B. M-T7 also significantly reduced macrophage (Mac-1)(P<0.0001) and CD4 (P<0.0001), CD8 (P<0.002) positive T cell invasion as assessed by analysis histology score on immunostained cross sections taken at follow up. Measurements reported as mean ± S.E.

Figure 5. Interference with chemokine-CCR2 interactions reduces inflammatory cell invasion in aortic allografts.

Immunohistochemical staining of aortic allograft transplant cross sections demonstrated increased lymphocyte (A) invasion in WT (Balb/c, Ccr2 ^+/+) donor aortic transplants at 4 weeks. M-T7 reduced T cell invasion in the WT Balb/c donor transplants (B). In the Ccr2 ^−/− transplants T cell invasion was reduced at 4 weeks in saline treated mice (C), without further significant reduction with M-T7 treatment (D). Bar graphs demonstrate reduced T cell invasion (P<0.0001), but not macrophage (P = 0.106) in Ccr2 ^−/− saline treated allografts when compared to saline treated Ccr2 ^+/+ allografts. M-T1, M-T7, and M3 treatment significantly reduced T cell (P<0.001)(E) and macrophage (P<0.029) (F) invasion in the WT (Balb/c, Ccr2 ^+/+) donor aortic allograft transplants, but not in Ccr2 ^−/− donor allografts (P = NS)(E,F). Measurements reported as mean ± S.E. Arrow heads point to brown, positively stained CD3+ T cells. Arrow points to blue stained suture seen in the top left corner of panel D. Mag 1000X.

Figure 6. Interference with chemokine-GAG interactions blocks inflammatory cell invasion in aortic allografts.

Bar graphs illustrate mean cell count per high power field (HPF) in cross sections taken from WT (C57Bl/6, Ndst1 ^+/+) and Ndst1 ^−/− donor aortic allografts at 4 weeks follow up. Macrophage counts are reduced with M-T7 treatment in Ndst1 ^+/+ donor sections in the intimal (A, P<0.003) and adventitial layers (B, P<0.0001). CD3+ T cell counts are also reduced at 4 weeks follow up with M-T7 treatment in WT (C57Bl/6, Ndst1 ^+/+) donor allografts in both intimal (C) and adventitial (D) layers, but only adventitial counts were significantly altered (P<0021). M-T7 treatment did not reduce either macrophage or CD3+ T cell counts in Ndst1 ^−/− donor aortic allografts (A,B,C,D). Measurements reported as mean ± S.E.

Figure 7. CMP treatment reduces early and local cell invasion into mouse peritoneal ascites in response to chemokine injections.

FACS analysis of cell invasion counts at 18 hours into mouse peritoneal fluid in response to either MCP-1 i.p. injection (A,B,D) or RANTES i.p. injection (C). MCP-1 and RANTES both significantly increased cell migration into the peritoneal space (ascites) in WT mice (A, C, D). M-T1 and M- T7 both reduced cell invasion in WT (Balb/c, Ccr2 ^+/+) mice (A), but M-T7 alone was able to reduce cell invasion in Ccr2 ^−/− mice (B). M-T7 also reduced cell migration into ascites in WT (C57Bl/6, Ndst1 ^+/+) mice (P<0.019, D) but not into Ndst1 ^−/− mice (P = 0.331, D). M-T7 given either locally into the peritoneal space (I.P.) or systemically by I.V. tail vein injection was equally effective at reducing cell migration in WT mice (C). Measurements reported as mean ± S.E.