Differential expression of beta-chemokines MCP-1 and RANTES and their receptors CCR1, CCR2, CCR5 in acute rejection and chronic allograft nephropathy of human renal allografts

BACKGROUND: The beta-chemokines MCP-1 (CCL2) and RANTES (CCL5) have been shown to play important roles in acute renal transplant rejection (AR) and chronic allograft nephropathy (CAN). The potential relationship of expression of these chemokines, their chemokine receptors CCR1, CCR2, CCR5, and the cell populations of inflammatory infiltrate, histological and clinical diagnoses were investigated in biopsies at the time of AR and compared with biopsies of CAN. METHODS: In 24 renal transplant biopsies with AR (n = 15) and CAN (n = 9), the expression of MCP-1 and RANTES, their receptors CCR1, CCR2, and CCR5 and the infiltration with monocytes/macrophages and T cells were studied. RESULTS: As previously described, chemokine and chemokine receptor expression was found mainly in mononuclear cells infiltrating the interstitium and glomeruli. In the tubulointerstitial area and glomeruli the expression of MCP-1, RANTES, and their receptors correlated with an infiltration by monocytes/macrophages. Biopsies with CAN revealed a lower expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in tubulointerstitial cells, and a significantly lower infiltration with MRP14-positive monocytes/macrophages than biopsies with AR. In AR, MCP-1 and CCR1 showed a lower expression compared to RANTES, CCR2, and CCR5. CONCLUSIONS: The positive correlation between chemokines and chemokine receptors and infiltrating leukocytes during acute rejection, the lower but detectable expression of MCP-1, RANTES, CCR1, CCR2 and CCR5 in CAN, and the differences in the quantity of expression between the different chemokines and chemokine receptors point to a complex regulation of chemokine expression in renal allografts. Since chemokines are not only involved in inflammation but also in tissue regeneration, this could have impact on the development of CAN.     

Early application of Met-RANTES ameliorates chronic allograft nephropathy

BACKGROUND: Initial insults to kidney allografts, characterized by infiltration of mononuclear inflammatory cells, contribute to chronic allograft nephropathy. Chemokines such as RANTES (regulated upon activation, normal T cell expressed) are thought to be responsible for the recruitment and activation of infiltrating cells. The present study investigated whether early application of Met-RANTES, a chemokine receptor antagonist that blocks the effects of RANTES, can protect renal allografts from long-term deterioration. METHODS: Fisher (F344) rat kidneys were orthotopically transplanted into Lewis recipients and treated with cyclosporine A (1.5 mg/kg/day) for the first 10 days following transplantation, together with either Met-RANTES at 40 microg/day, 200 microg/day or vehicle for the first 7 days. Animals were harvested at 2 and 28 weeks after transplantation for histologic, immunohistologic and molecular analysis. RESULTS: Met-RANTES treatment reduced the infiltration of lymphocytes and macrophages in allografts at 2 weeks after transplantation, accompanied by decreased mRNA expression of interleukin (IL)-2, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and RANTES. At post-transplantation week 28, Met-RANTES treatment at high and low doses reduced urinary protein excretion and significantly ameliorated glomerulosclerosis, interstitial fibrosis, tubular atrophy, intimal proliferation of graft arteries and mononuclear cell infiltration. However, creatinine clearance was not influenced by Met-RANTES. Furthermore, Met-RANTES suppressed the mRNA expression of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor-B (PDGF-B). CONCLUSIONS: Blockade of chemokine receptors by Met-RANTES diminishes early infiltration and activation of mononuclear cells in the grafts, and thus reduces the pace of chronic allograft nephropathy.     

A novel small-molecule compound targeting CCR5 and CXCR3 prevents acute and chronic allograft rejection

BACKGROUND: Chemokines and chemokine receptors are critical in leukocyte recruitment, activation, and differentiation. Among them, CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 3 (CXCR3) have been reported to play important roles in alloimmune responses and may be potential targets for posttransplant immunosuppression. METHODS: Fully major histocompatibility complex (MHC)-mismatched murine cardiac and islet transplant models were used to test the effect in vivo of a novel, small-molecule compound TAK-779 by targeting CCR5 and CXCR3 in acute allograft rejection. An MHC class II mismatched cardiac transplant model was used to evaluate its efficacy in chronic allograft rejection. Intragraft expression of cytokines, chemokines, and chemokine receptors was measured by quantitative real-time polymerase chain reaction and by histological analysis. RESULTS: Treatment of TAK-779 significantly prolonged allograft survival across the MHC barrier in two distinct transplant models. The treatment downregulated local immune activation as observed by the reduced expression of several chemokines, cytokines, and chemokine receptors. Thereby, the recruitment of CD4, CD8, and CD11c cells into transplanted allografts were inhibited. Furthermore, TAK-779 treatment significantly attenuated the development of chronic vasculopathy, fibrosis, and cellular infiltration. CONCLUSIONS: Antagonism of CCR5 and CXCR3 has a substantial therapeutic effect on inhibiting both acute and chronic allograft rejection. CCR5 and CXCR3 are functional in the process of allograft rejection and may be potential targets in clinical transplantation in the future.     

Suppression of cardiac allograft vasculopathy in mice by inhibition of CC-motif chemokine receptor 5

Cardiac allograft vasculopathy (CAV) is the leading cause of late morbidity and mortality in heart-transplant patients. Increasing evidences support the important role of chemokines and their receptors in transplant immunology. Chemokine-chemokine receptor interaction and subsequent recruitment of T-lymphocytes to the graft are early events in the development of chronic rejection of transplanted hearts. In this study, we first inhibited CC-motif chemokine receptor 5 (CCR5) expression by using lentiviral-mediated gene transfer of an anti-CCR5 siRNA, which introduced through CD34(+) hematopoietic stem/progenitor cell transplantation. Stably marked lymphocytes expressing siRNA and consistent downregulation of CCR5 expression were detected. Our results showed that survival was significantly prolonged in CCR5 knock-down mice and donor hearts from siRNA-treated mice developed markedly less CAV. Infiltration of CD4(+) and CD8(+) T-lymphocytes into transplanted hearts was also markedly decreased. These findings suggest that CCR5 plays an important role in CAV development and inhibition of this chemokine could improve long-term survival after cardiac transplantation.     

Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes

The infiltration of leukocytes plays a major role in mediating tubulointerstitial inflammation and fibrosis in chronic renal disease. CC chemokines participate in leukocyte migration and infiltration into inflamed renal tissue. Because CC chemokine-directed leukocyte migration is mediated by target cell expression of a group of CC chemokine receptors, this study examined the expression of CC chemokines and their receptors during initiation of tubulointerstitial fibrosis after unilateral ureteral obstruction in C57BL/6 mice. Obstructed kidneys developed hydronephrosis, tubular cell damage, interstitial inflammation, and fibrosis. From days 2 to 10, a progressive interstitial influx of F4/80+ macrophages and CD3+ lymphocytes occurred (macrophages, 4-fold; lymphocytes, 20-fold at day 10, compared with contralateral control kidneys). In parallel, the number of activated fibroblast-specific protein 1+ fibroblasts and interstitial collagen IV accumulation increased from days 2 to 10. The mRNA expression of CC chemokines (predominantly monocyte chemoattractant protein-1 [MCP-1]/CCL2, RANTES/CCL5) and their receptors CCR1, CCR2, CCR5 increased progressively from days 2 to 10. By in situ hybridization, a prominent interstitial mRNA expression of MCP-1 and RANTES and their receptors CCR2 and CCR5 localized to interstitial mononuclear cell infiltrates. MCP-1 and RANTES expression was also seen in tubular epithelial cells. Fluorescence-activated cell sorter analysis of single-cell suspensions from obstructed kidneys revealed a prominent expression of CCR2 and CCR5 by infiltrating macrophages, whereas most lymphocytes expressed CCR5 only. These data demonstrate an increased expression of MCP-1/CCL2 and RANTES/CCL5 at sites of tubulointerstitial damage and progressive fibrosis during unilateral ureteral obstruction that correlates with simultaneous accumulation of interstitial macrophages and T lymphocytes expressing the respective surface receptors CCR2 and CCR5. The chemokine receptor-mediated leukocyte influx into the tubulointerstitium could offer a new potential target for therapeutic intervention in progressive renal tubulointerstitial fibrosis.     

CXCR3 and CCR5 positive T-cell recruitment in acute human renal allograft rejection

BACKGROUND: Experimental studies suggest that the infiltration of activated T cells into the allograft, the key event in the development of acute renal allograft rejection, depends on the expression of chemokines and their interaction with chemokine receptors expressed on T cells. METHODS: For a more detailed comprehension of the pathogenesis of T-cell recruitment in human acute rejection, the in situ expression of chemokines and chemokine receptors in allografts of 26 patients between day 3 and 9 after renal transplantation was examined in the present prospective study. RESULTS: Immunohistochemical staining showed a significantly increased number of CXCR3 (P<0.01) and CCR5 positive T cells (P<0.01) in the tubulointerstitium of patients with acute allograft rejection according to Banff grade Ia-IIb. Likewise the intrarenal RNA expression of the CXCR3 ligands IP-10 (5.2-fold) and I-TAC (7.2-fold) and the CCR5 ligand RANTES (5.7-fold), was significantly up-regulated in rejecting organs. In situ hybridization revealed that IP-10 but not I-TAC was predominantly expressed by infiltrating leukocytes in the tubulointerstitial area, co-localizing with CXCR3 positive T cells. To a lesser degree expression by tubular cells could be detected, providing a possible explanation for the increased urinary IP-10 excretion we found in patients with rejecting organs. CONCLUSIONS: These data from a prospective, biopsy-controlled study indicate that the local expression of IP-10 and RANTES leads to the directional movement of activated CXCR3 and CCR5 bearing T cells into the renal allograft and mediates acute rejection. Our data provide a rationale that blocking CXCR3 and CCR5 may offer a unique therapeutic approach to prevent episodes of acute rejection in the early phase after renal transplantation.     

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.     

Expression of IL-8 during reperfusion of renal allografts is dependent on ischemic time

BACKGROUND: Ischemia/reperfusion injury is an inherent consequence of solid organ transplantation that increases tissue inflammation and negatively impacts organ transplant function and survival. This study investigated the expression levels of chemokine and chemokine receptor genes in living versus cadaver donor renal allografts before and after reperfusion. METHODS: This study involved 39 renal transplant patients (19 cadaveric and 20 living donor). The ischemia biopsy was taken just before graft declamping and the reperfusion biopsy 30 min after declamping. Whole-cell RNA was isolated and chemokine (IL-8, CCL2/MCP-1, CXCL10/IP-10 and CCL5/RANTES) and chemokine receptor (CCR2 and CCR5) expression was tested by quantitative PCR. RESULTS: Just prior to declamping, ischemic cadaveric donor grafts had higher expression of CXCL10/IP-10 but not IL-8 or CCL2/MCP-1 than living donor grafts. IL-8 expression increased 50% from ischemia to reperfusion in living donor grafts but increased more than 13-fold during reperfusion of cadaver donor grafts. Increased total ischemia time induced greater IL-8 expression during reperfusion. MCP-1 expression also increased during reperfusion of living and cadaver donor grafts but differences were not observed between the two groups of grafts. RANTES, CCR2, and CCR5 expression did not change in ischemic vs. reperfusion biopsies. CONCLUSIONS: The expression of chemokines directing neutrophil and macrophage recruitment increases during reperfusion of living and cadaveric donor renal allografts. Expression levels of IL-8 correlate with the ischemic time imposed on the renal graft. Early tissue injury may be attenuated by strategies antagonizing chemokines directing the recruitment of neutrophils and macrophages into kidney grafts.     

Chemokine and receptor-gene expression during early and late acute rejection episodes in human cardiac allografts

BACKGROUND: The expression levels of several chemokine genes in heart allografts correlate with histologic rejection grade. Potential molecular differences between early and late rejection (grade > or =2) episodes were examined by testing chemokine and receptor-gene expression. METHODS: Expression of inducible protein (IP)-10, monokine induced by IFN-gamma (Mig), interferon inducible-T cell alpha chemoattractant (I-TAC), regulated on activation normal T-cell expressed and secreted(RANTES), and their receptors CXCR3 and CCR5 was tested in 60 endomyocardial biopsies from 24 patients using quantitative (Taqman) real-time polymerase chain reaction (PCR). The biopsies were taken in the first 3 months or from the 9th to the 12th month following transplantation. RESULTS: IP-10, Mig, RANTES, CXCR3, and CCR5 expression levels were increased in the later versus earlier biopsies (P< or =0.01) despite no change in histologic rejection-grade status.CONCLUSION These results demonstrate significantly increased expression of T-cell chemoattractants in heart allografts during later rejection when compared with episodes occurring shortly after transplantation. The findings suggest increased intensity of inflammation in rejection occurring at later times posttransplant that are revealed by molecular analyses of the graft.     

Immunomonitoring of renal transplant recipients in the early posttransplant period by sequential analysis of chemokine and chemokine receptor gene expression in peripheral blood mononuclear cells

INTRODUCTION: We sought to determine whether sequential changes in chemokine ligand/receptor gene expression in the early posttransplant period of human renal allografts can be detected in peripheral blood mononuclear cells (PBMCs) and whether any such changes are predictive of clinical events. METHODS: Blood samples from 106 renal transplant recipients and 29 donor nephrectomy patients were taken preoperatively and daily for 14 days. Within the study period 22 patients had biopsy-proven acute rejection. From each blood sample PBMCs were separated and gene expression levels for chemokines CCL3, CCL4, CCL5, CXCL10, and their receptors CCR1, CCR5, and CXCR3, were determined using real-time quantitative PCR. RESULTS: Different gene expression patterns were seen between the rejector and nonrejector groups with decreases in CCL4 and CCR5 expression on days 6 to 8 and increases in CCR1 expression on days 9 and 10 posttransplant. With CXCL10, decreases in expression were seen in the nonrejector group but increases were seen in the rejector group posttransplant. With data aligned to time of rejection diagnosis, statistically significant increases, that preceded the clinical detection of acute rejection were seen in CCR1 and CXCL10 expression. Both their expression levels returned to pretransplant baseline values after successful antirejection therapy. CONCLUSION: We have demonstrated that changes in chemokine receptor/ligand gene expression by sequential monitoring in PBMCs can be detected in the early posttransplant period. In particular, CCR1 and CXCL10, which showed increased expression prior to rejection and returned to baseline levels with antirejection therapy, may have potential use in immunomonitoring and as predictive factors of rejection prior to its clinical manifestation.     

Interaction between proximal tubular epithelial cells and infiltrating monocytes/T cells in the proteinuric state

We hypothesize an interaction between T cells/monocytes and the tubules in the development of tubulointerstitial injury in chronic proteinuric nephropathy. We established in vitro co-culture systems of proximal tubular epithelial cells (PTEC) and T cells/monocytes to study the contribution of soluble factors and cell-to-cell contact in the development of tubulointerstitial injury. The release of monocyte chemoattractant protein-1 (MCP1 or CCL2), Regulated upon Activation, normal T cell Expressed and Secreted (RANTES or CCL5), soluble intracellular adhesion molecules-1 (sICAM-1), or interleukin-6 (IL-6) was increased in PTEC following apical exposure to human serum albumin (HSA). The release of CCL2, CCL5, or sICAM-1 from PTEC was enhanced by contact of monocytes/T cells on the basolateral surface. Tumor necrosis factor-alpha (TNF-alpha) and IL-1beta are important soluble factors as suggested by the blocking effect of antibodies (Abs) against TNF-alpha or IL-1beta but not against other cytokines. The percentage of CD4+ T cells expressing both chemokine receptors, CCR2 and CCR5, was increased after culturing with supernatant from the apical or basolateral surface of PTEC following apical exposure to HSA. However, only CCR2 was upregulated in CD8+ T cells, whereas CCR5 expression was increased in monocytes. The chemotaxis of CD4+ or CD8+ T cells to supernatant from PTEC upon apical exposure to HSA was reduced with neutralizing Abs against CCL5 and/or CCL2, whereas the chemotaxis of monocytes was only reduced by anti-CCL5 but not by anti-CCL2. In summary, chemokines released by HSA-activated PTEC are amplified by monocytes/T cells. Mediators released by HSA-activated PTEC can differentially modulate the expression of chemokine receptors in monocytes/T cells and hence, alter their chemotaxis towards activated PTEC. These interactions are pivotal in the development of tubulointerstitial injury.     

Lack of association between the −403G/A promoter polymorphism in the human CCL5/RANTES chemokine gene in liver transplant outcome

Chemokines play a major role in the inflammatory and immune responses that mediate allograft outcome. CCL5/RANTES expansion chemokine is potent eosinophil, monocyte, basophils and lymphocyte chemoattractant and has recently been studied in transplantation with discrepant results, but systemic concentrations have been correlated to liver graft survival and incidence of rejection. Recent studies revealed that a functional mutation at -403 in the promoter may have a significance for inflammatory and infectious immune responses. Our objective was to investigate CCL5/RANTES promoter polymorphism in rejection and graft survival in liver transplant. We examined the CCL5/RANTES polymorphism in a series of 218 liver transplants and 101 healthy Caucasian subjects. CCL5/RANTES genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). After comparing recipients (with acute rejection episodes versus without rejection) with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CCL5/RANTES dimorphism in any comparison (P > 0.05). Indeed, 5 years allograft survival was 61.3% in recipients with the GG genotype against 58.8% in recipients with the GA and AA genotypes. These differences were also not statistically significant. In conclusion, human CCL5/RANTES gene promoter polymorphism does not seem to influence acute rejection development and allograft survival in liver recipients.     

Bacterial CpG-DNA aggravates immune complex glomerulonephritis: role of TLR9-mediated expression of chemokines and chemokine receptors

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.     

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.     

Carbon monoxide–releasing molecule attenuates allograft airway rejection

Acute rejection after lung transplantation is the main risk factor for the development of bronchiolitis obliterans (BO). Carbon monoxide (CO) can provide anti‐inflammatory effects and may serve to limit tissue injury in airway transplant. Here, we tested the ability of carbon monoxide releasing molecule‐2 (CORM‐2) to prevent airway rejection. Tracheal grafts from BALB/c or C57BL/6 were transplanted to C57BL/6 recipients. Experimental groups were treated with multiple doses of CORM‐2. Histopathological evaluation of luminal obliteration was blindly reviewed. Immunohistochemistry and real‐time RT‐PCR analyses were performed. Allografts treated with CORM‐2 revealed a striking reduction of thickening in epithelial and subepithelial airway layers (P < 0.01) at day 7 in orthotopic trachea transplantation model compared with allografts treated with vehicle. In heterotopic trachea transplantation model, CORM‐2 treated allografts showed a reduction of luminal obliteration (P < 0.01) at days 14 and 21. There was also a concordant decrease in CD3⁺ lymphocytes and macrophages in CORM‐2 treated allografts. IFN‐γ, IL‐2 and IL17A mRNA expressions were reduced dramatically by systemic administration of CORM‐2. These data implicate CORM‐2‐derived CO has an important protective function in experimental BO, and may represent a target for the therapeutic intervention of chronic lung allograft rejection.     

Donor CCR5 Delta32 polymorphism and outcome following cardiac transplantation

BACKGROUND: Chemokines regulate the recruitment and trafficking of leukocytes during an immune response. Animal models have shown correlations between chemokine production and leukocyte infiltration during allograft rejection. Also, antagonism of chemokine receptors in transplant models has produced prolonged graft survival. Individuals homozygous for a 32 base pair deletion in the CC chemokine receptor 5 (CCR5) gene have an inactive receptor. Renal transplant recipients homozygous for the deletion have been shown to survive significantly longer than those heterozygous or homozygous for the wild type allele. CCR5 ligands are upregulated during allograft rejection aiding infiltration of leukocytes. We investigated the influence of CCR5Delta32 polymorphism on outcome following human cardiac transplantation. METHODS: Recipients and corresponding donors were genotyped for CCR5Delta32 polymorphism using polymerase chain reactions. RESULTS: We found no correlation between recipient genotype and outcome following transplantation. However, there was a significant correlation between donor genotype and mortality in patients transplanted for a nonischemic condition (DD = n/a, ID = 4%, II = 25%, P = .0014). CONCLUSIONS: The induction of CCR5 expression in endomyocardial biopsy tissue is known to correlate with leukocyte graft infiltration. We suggest that donor CCR5 may be more important for leukocyte trafficking during rejection than recipient CCR5 expression. The CCR5 gene is highly conserved, and due to the small population available for this study, more work is required from other centers.     

CCR and CC chemokine expression in relation to Flt3 ligand-induced renal dendritic cell mobilization

BACKGROUND: We investigated the expression and function of CC chemokine receptors (CCR) on highly-purified kidney and blood dendritic cells isolated from mice in which dendritic cells were mobilized with fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: CCR and CC chemokine expression were determined by RNase protection assay or flow cytometry, and dendritic cell migratory responses assayed using Transwell chambers. Chemokine production in renal tissue was detected by immunofluorescence staining. Trafficking of fluorochrome-labeled dendritic cells was monitored in vivo. RESULTS: Freshly-isolated renal dendritic cells expressed mRNA for CCR1, 2, 5, and 7 and CCR1 and 5 protein. They did not migrate to inducible chemokines--CCL3 [macrophage inflammatory protein (MIP)-1alpha], CCL5 [regulated upon activation, normal T cell expressed and secreted (RANTES)], or CCL20 (MIP-3alpha). Following lipopolysaccharide (LPS) stimulation, the dendritic cells down-regulated CCR1, 2, and 5 expression, up-regulated or sustained signals for CCR7, and migrated to the constitutively expressed ligands CCL19 (MIP-3beta) and CCL21 (secondary lymphoid tissue chemokine). Normal kidneys expressed weak message for CCL2, 3, and 4, with stronger signals for CCL5 and 19. Intrarenal CCL5 production was enhanced by Flt3L administration, in association with marked increases in interstitial CD45+ mononuclear cells. Mobilized blood dendritic cells migrated to CCR2 and CCR5 ligands and trafficked to renal intertubular sites following adoptive (intravenous) transfer. Their migration to the CCR5 ligand MIP-1beta (CCL4) and homing to kidneys of Flt3L-treated recipients were inhibited by CCR5 antagonism. CONCLUSION: These data implicate specific CCR and their ligands in regulation of the dendritic cell constituency of the kidney. CCR5 antagonism inhibits their directed migration and intrarenal accumulation.     

Role of donor-derived monocyte chemoattractant protein-1 in murine islet transplantation

Monocyte chemoattractant protein-1 (CCL2/MCP-1) is a proinflammatory chemokine produced by several cell types, including pancreatic islets. High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes; however, the causal relationship is unknown. The constitutive and inducible expression of chemokines and their receptors by pancreatic islets in vitro were investigated, specifically the role of donor-derived CCL2 in marginal mass murine islet transplantation. The results showed that inflammatory cytokine stimulation of islets induced de novo expression of CCL2, CCL5/RANTES, CXCL9/MIG, and CXCL10/IP-10 and increased expression of CXCL2/macrophage-inflammatory protein-2. CCL2 mRNA and protein were highly expressed within 2 d in cultures. Transplantation of islets with high levels of CCL2 into syngeneic recipients led to a significantly greater influx of CCR2(+) cells and higher expression of monocyte/macrophage-associated inflammatory cytokines compared with low CCL2-donor islets. The level of pretransplantation CCL2 inversely correlated (P < 0.0001) with isograft function. In contrast, in CCR2-/- recipients, this correlation was not present. A direct toxic effect of CCL2 on islets was excluded by assessing cell viability and insulin release in vitro. In conclusion, CCL2 secreted by islets plays an important role in the immediate islet graft function. Strategies to decrease islet-derived CCL2 release may increase the success of islet transplantation.     

Role of CXCR3 and CCR5 in allograft rejection

Chemokines are known to participate in allograft rejection by mediating leukocyte trafficking. Despite redundancy in chemokine family, several chemokine-chemokine receptor interactions have proven critical in alloimmune responses. We sought to determine the effect of combined blockade of CXCR3 and CCR5, two critical chemokine receptors, in acute rejection. METHODS: Heterotopic heart transplantation was performed using BALB/c to B6/129 mice deficient in CCR5. Following transplantation these mice were treated with goat anti-CXCR3 serum every other day. In the control group, BALB/c hearts were transplanted in wild type B6/129 recipients and treated with goat serum alone. No immunosuppression was given to either group. Recipient mice were then assessed daily for allograft function by abdominal palpation, and graft survival was confirmed by laparotomy. RESULTS: The donor hearts in the control group were rejected at 6 +/- 1 days posttransplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival versus control; all allografts survived to 24 days. In addition, there was a decrease in graft infiltrating CD4 and CD8 lymphocytes in the experimental group at 24 days. CONCLUSION: Combined CXCR3 and CCR5 blockade is effective in prolonging allograft survival in a fully MHC mismatched murine model. Combined chemokine blockade holds promise in control of acute rejection in organ transplantation.