Chemokine and toll-like receptor signaling in macrophage mediated islet xenograft rejection

BACKGROUND: Adoptive transfer of antigen-primed T-cell-activated macrophages into NOD-SCID mice within 14 days of foetal porcine pancreatic fragment (FPP) or foetal porcine skin (FPS) transplantation had been shown to cause xenograft rejection. In the present study, it was proposed that signaling between the graft and macrophages promoted specific graft recognition and destruction in this setting. METHODS: Exogenous macrophages isolated from rejecting FPP xenografts were transferred to NOD-SCID FPP recipients and tracked by Ly5.1 surface antigen or via CSFE staining. Monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage inflammatory protein-1beta (MIP-1beta), regulated upon activation, normal T-cell expressed and secreted (RANTES), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), toll-like receptors (TLRs) (1-9) and gene expression in transplanted FPP xenografts was evaluated by real-time polymerase chain reaction. Gene expression of CCR2, CCR5 and TLRs was also analyzed in pooled samples of activated and non-activated macrophages. RESULTS: Exogenous macrophages were shown to track to and reject recently transplanted but not established FPP xenografts. Gene expression for MCP-1, RANTES, MIP-1alpha and MIP-1beta was at least 3-fold greater in recently transplanted compared with established xenografts (P < 0.05), and CCR2 and CCR5 gene expression was 10-fold greater in activated compared non-activated macrophages, suggesting that graft-mediated pro-inflammatory signals were important for macrophage recruitment. Specific graft recognition by macrophages may involve TLR signaling as macrophages exposed to porcine islets had higher levels of TLR gene expression compared with those exposed to allografts regardless of the level of activation. CONCLUSION: Xenografts provide additional activation signals to macrophages that are not seen following allotransplantation. This study identifies chemokines and TLR as important signals in macrophage-mediated recognition and rejection of islet xenografts.     

Proinflammatory cytokines induce NF-kappaB-dependent/NO-independent chemokine gene expression in MIN6 beta cells

BACKGROUND: Interactions between chemokines IP-10, MCP-1, and RANTES and their receptors may mediate graft rejection following islet transplantation. The mechanisms regulating chemokine gene expression in pancreatic islet cells have not been well characterized. We examined the cytokine-induced gene expression profiles for several chemokines in a transformed pancreatic beta-cell line (MIN6) cotreated with an inhibitor of nitric oxide synthase and in a mutated clone of MIN6 made to overexpress a dominant negative inhibitor of NF-kappaB (IkappaBalphaM). METHODS: MIN6 and MIN6-IkappaBalphaM (Bm) cells were cultured in mixtures of IL-1beta and TNF-alpha or IL-1beta, TNF-alpha, and IFN-gamma plus/minus the iNOS inhibitor L-NMMA. RT-PCR and RNase Protection Assay were used to measure mRNA expression for the following chemokines: IP-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES. Enzyme linked immunosorbant assay was used to measure IP-10 and MCP-1 protein release. RESULTS: Cytokine-treated MIN6 and Bm demonstrated increased expression of genes for IP-10 and MCP-1. Expression in MIN6 was first detected at 2 h of incubation and peaked at 6 h. MIN6 demonstrated a more marked increase in chemokine gene expression for both IP-10 and MCP-1 and a more marked increase in IP-10 protein release than did Bm. There was no detectable gene expression for MIP-1alpha, MIP-1beta, or RANTES from MIN6 or Bm. L-NMMA completely blocked NO production from MIN6 and Bm but had no effect on chemokine gene expression in either MIN6 or Bm. CONCLUSIONS: These results suggest that beta cells produce a complement of rejection-relevant chemokines in response to a proinflammatory stimulus and that pathways governing cytokine-induced chemokine gene expression in MIN6 are dependent on NF-kappaB but independent of NO.     

Beneficial effects of targeting CCR5 in allograft recipients

BACKGROUND: The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. METHODS: We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. RESULTS: Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. CONCLUSIONS: We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.     

Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions

To elucidate the possible immunoregulatory role of nitric oxide (NO) in cellular xenograft rejection we performed rat-to-mouse skin xenotransplantation. The rat skin engrafted mice were treated with the inducible NO synthase (iNOS) inhibitors, aminoguanidine (AMG, 200 mg/kg) and NG-nitro-L-arginine methyl ester (L-NAME, 60 mg/kg) every other day until rejection. Skin xenograft survival was monitored and immune cell infiltration and intragraft cytokine and chemokine mRNA expressions were analyzed 7 days after grafting. Compared with the control mice, the AMG- and L-NAME treated mice showed delayed xenograft rejection by approximately 3 days (8.9 +/- 0.7 days vs. 11.7 +/- 1.2 and 12.0 +/- 0.9 days, respectively). Infiltrations of CD11b+, MOMA-2+ cells and neutrophils were significantly reduced in both AMG- and L-NAME treated graft but CD4+ and CD8+ cells were not. The expression of cytokines such as IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma in AMG- and L-NAME treated grafts were significantly decreased (P<0.01), whereas IL-10, TNF-alpha and TGF-beta1 were unchanged or enhanced. Additionally, the expressions of CC-chemokines, such as RANTES and MIP-1alpha, were significantly reduced (P<0.01) whereas the expressions of CXC-chemokines, such as IP-10 and MIG, were unchanged. These results imply that prolonged rat-to-mouse skin xenograft survival by iNOS inhibitors may be due to the selective inhibition of pro-inflammatory cytokines and chemokines and suggest the possible regulatory role of NO in cytokine and chemokine expressions during xenotransplant rejection.     

Involvement of CCR5 signaling in macrophage recruitment to porcine islet xenografts

BACKGROUND: Porcine antigen primed and CD4+ T-cell-activated macrophages are capable of both recognition and rejection of porcine xenografts. However, the specific signaling mechanisms involved remains to be addressed. The aim of this study was to examine the role of chemokine receptor and CD40 signaling in macrophage recruitment and graft destruction. METHODS: Macrophages were isolated from rejecting CCR2, CCR5, CD40 and control C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Macrophages isolated from rejecting NPCC xenografts in CD40 and wildtype C57BL/6 mice demonstrated upregulated expression of macrophage activation markers as well as CCR5 and CCR2 genes, and caused pig islet xenograft destruction 8 days after transfer to NOD-SCID recipients. Graft infiltrating macrophages from rejecting CCR2 mice showed a similar activation phenotype and destroyed NPCC xenografts 10 days after transfer to NOD-SCID mice. Blockade of MCP-1 by anti-MCP-1 mAb did not prolong graft survival in CD4+ T cell reconstituted NPCC recipient NOD-SCID mice. By contrast, the graft infiltrating macrophages from rejecting CCR5 recipients showed impaired macrophage activation when compared to control C57BL/6 recipients, and transfer of these macrophages did not result in xenograft destruction in NOD-SCID recipients until day 16 after transfer. Analysis of graft infiltrating macrophages from these rejecting NOD-SCID mice showed an impaired activation phenotype. CONCLUSION: These results demonstrate that CCR5 is involved in both the activation and recruitment of macrophages to rejecting islet xenografts but other pathways are involved.     

Renal allograft rejection: beta-chemokine involvement in the development of tubulitis

BACKGROUND: Tubulitis is a defining feature of renal allograft rejection. Graft dysfunction may result from damage inflicted on tubular epithelial cells by intratubular cytotoxic T lymphocytes. Graft cells are known to produce chemokines during acute rejection, but it is not known whether changes in expression of specific chemokines can influence the composition of the intratubular lymphocyte population. We examined expression of individual chemokines in biopsy sections showing different pathological rejection grades. METHODS: Sections from Banff-graded transplant biopsies were examined for the presence of beta-chemokines (MCP-1, MIP-1alpha, MIP-1beta, and RANTES) by immunofluorescence and semiquantitative confocal laser scanning microscopy. RESULTS: Beta-chemokines were expressed predominantly at the basolateral surface of tubular epithelial cells. Expression of MCP-1 and MIP-1beta was significantly higher in sections showing grade 2 rather than grade 1 acute rejection. RANTES and MIP-1alpha showed no significant variation in level of expression between rejection grades. CONCLUSIONS: Beta-chemokines are expressed by tubular epithelial cells during acute rejection. Consistent expression of RANTES and MIP-1alpha suggests a general role in recruiting T lymphocytes. However, MCP-1 and MIP-1beta may play a more subtle role in recruitment of specific T-cell subsets, such as Th1 cells, during acute cellular rejection.     

Genetic deletion of chemokine receptor CXCR3 or antibody blockade of its ligand IP-10 modulates posttransplantation graft-site lymphocytic infiltrates and prolongs functional graft survival in pancreatic islet allograft recipients

BACKGROUND: Interaction of chemokine receptor CXCR3 with its ligand IP-10 mediates effector cell trafficking to sites of allograft rejection in murine models of whole organ allotransplantation. We hypothesized that blocking the CXCR3/IP-10 interaction would impair posttransplantation leukocyte trafficking to and delay rejection of pancreatic islet allografts. METHODS: A/J strain murine islets were implanted to the kidney capsule of H-2 disparate, streptozotocin-induced diabetic wild type (WT), CXCR3 deficient (CXCR3(-/-)) or IP-10 antibody-treated WT (alphaIP-10) C57BL/6 recipients. Representative grafts from each group were harvested at day 7. Ribonuclease protection assay was used to determine gene expression for cell markers F4/80 (macrophages), CD8 (type I T cells), CD4 (type II T cells), and CD 19 (natural killer cells), and for chemokines IP-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES. Immunohistochemistry was used to confirm ribonuclease protection assay infiltrate data. Graft-site chemokine gene expression and cellular infiltrate were correlated with time to functional graft rejection. RESULTS: Untreated WT recipients demonstrated heavy graft-site cell infiltrates and increased graft-site gene expression for cell markers F4/80, CD8, CD4, and CD19, and for chemokines RANTES, IP-10, and MIP-1beta at day 7. In comparison with untreated WT, alphaIP-10-treated WT and CXCR3(-/-) recipients demonstrated the same degree of chemokine gene expression but less lymphocytic infiltrate. The mean length of allograft survival was 12.7 +/- 3.1 days in untreated WT versus 20.2 +/- 2.7 days (P <.05) for CXCR3(-/-)- and 19.7 +/- 2.3 days (P <.05) for alphaIP-10-treated WT recipients. CONCLUSIONS: CXCR3 gene deletion or alphaIP-10 antibody therapy modulates posttransplantation lymphocytic graft infiltration and statistically prolongs graft survival in murine islet allograft recipients.     

The role of CC chemokine receptor 5 (CCR5) in islet allograft rejection

Chemokines are important regulators in the development, differentiation, and anatomic location of leukocytes. CC chemokine receptor 5 (CCR5) is expressed preferentially by CD4(+) T helper 1 (Th1) cells. We sought to determine the role of CCR5 in islet allograft rejection in a streptozotocin-induced diabetic mouse model. BALB/c islet allografts transplanted into CCR5(-/-) (C57BL/6) recipients survived significantly longer (mean survival time, 38 +/- 8 days) compared with those transplanted into wild-type control mice (10 +/- 2 days; P < 0.0001). Twenty percent of islet allografts in CCR5(-/-) animals without other treatment survived >90 days. In CCR5(-/-) mice, intragraft mRNA expression of interleukin-4 and -5 was increased, whereas that of interferon-gamma was decreased, corresponding to a Th2 pattern of T-cell activation in the target tissues compared with a Th1 pattern observed in controls. A similar Th2 response pattern was also observed in the periphery (splenocytes responding to donor cells) by enzyme-linked immunosorbent spot assay. We conclude that CCR5 plays an important role in orchestrating the Th1 immune response leading to islet allograft rejection. Targeting this chemokine receptor, therefore, may provide a clinically useful strategy to prevent islet allograft rejection.     

Induction of macrophage C-C chemokine expression by titanium alloy and bone cement particles

Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1alpha), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1alpha expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1alpha in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1alpha were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1alpha inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty.     

mRNA expression of chemokines in rat kidneys with ureteral obstruction

BACKGROUNDS: It has been demonstrated that leukocyte infiltration, mainly of macrophages and lymphocytes, into obstructed kidneys (OBK) of rats during unilateral ureteral obstruction (UUO). Chemokines (C-C subfamily) may be involved in this mechanisms. Thus, we accessed the gene expression of chemokines in renal cortex of rats with UUO. MATERIALS AND METHODS: Female SD rats were sacrificed at various time points after UUO. mRNA expression of MCP-1, RANTES and MIP-1 alpha was determined by semi-quantitative RT-PCR. RESULTS: Control kidneys (CNK) showed a weak mRNA expression of MCP-1, RANTES and MIP-1 alpha. OBKs showed an increase in MCP-1 at 2 hours of UUO and a significant increase at 4 hours of UUO as compared with CNKs or contralateral unobstructed kidneys (CLK). The mRNA levels of RANTES and MIP-1 alpha were not increased until 72 hours of UUO in CLKs or OBKs. There were slight, but significant, differences of RANTES and MIP-1 alpha expression between OBKs and CNKs at 120 hours of UUO. CONCLUSIONS: We suggest that the early increase in MCP-1 contributes to the leukocyte infiltration and that RANTES and MIP-1 alpha plays a partial role in a late increases.     

Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro.

C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.     

Early and late chemokine production correlates with cellular recruitment in cardiac allograft vasculopathy

BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the leading cause of late mortality in heart transplant recipients. Activated T lymphocytes and macrophages infiltrate the donor heart before vascular intimal thickening develops, but the specific mediators of mononuclear cell recruitment leading to CAV are unknown. Therefore, we sought to define the relationship between chemokine gene expression and production, T lymphocyte and macrophage recruitment, and intimal thickening in a murine model of CAV. METHODS: B10.A or B10.BR strain hearts were transplanted heterotopically into B10.BR mice. Recipients were killed at 1, 4, 7, 14, and 30 days. Donor hearts were assayed for chemokine gene expression with ribonuclease protection and for protein with ELISA. Intragraft cellular infiltration was defined immunohistochemically. Intimal thickening was quantitated morphometrically. RESULTS: Early and late patterns of intragraft chemokine expression associated with distinct cellular infiltration were identified. First, transient MIP-2 and MCP-1/JE production in isografts and allografts correlated with neutrophil and macrophage infiltration. MCP-1/JE production and macrophage infiltration was greater in allografts than isografts. Second, allografts demonstrated sustained lymphotactin, RANTES, and IP-10 expression, beginning at day 4, correlating with persistent macrophage and T lymphocyte infiltration. Intimal thickening became evident at 14 days. Isografts did not display the late pattern of sustained chemokine gene expression, cellular infiltration, or intimal thickening. CONCLUSIONS: Transient, early MIP-2, and MCP-1/JE production in isografts and allografts correlated with neutrophil and macrophage recruitment, and is likely related to ischemia-reperfusion. In allografts, the delayed induction of chemokines specific for macrophages and T lymphocytes correlated with mononuclear cell infiltration and preceded intimal thickening. This study thus demonstrates a dual pattern of chemokine induction correlating with intragraft mononuclear cell recruitment, associated with ischemia-reperfusion and CAV development. Chemokine-directed interventions may interfere with leukocyte trafficking and inhibit CAV development.