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.     

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.     

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.     

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.     

Early increased chemokine expression and production in murine allogeneic skin grafts is mediated by natural killer cells

BACKGROUND: Increased expression of chemokine mRNA is observed in allogeneic but not syngeneic skin grafts 3-4 days after transplantation. The recipient cells mediating this early inflammatory response in allografts remain unidentified. METHODS: Isogeneic and allogeneic skin grafts were transplanted to euthymic and athymic nude mice. mRNA expression and protein production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and the murine homolog of Gro(alpha), i.e. KC, from graft homogenates retrieved 3-4 days posttransplantation was tested by Northern blot hybridization and ELISA. To deplete NK cells, recipients were treated with antiasialo GM1 (ASGM1) antisera or with anti-NK1.1 mAb before transplantation. RESULTS: Expression of KC, MIP-1alpha, and MIP-1beta mRNA was equivalent in C57BL/6 allogeneic skin grafts and BALB/c isografts at day 2 posttransplant. At day 3 posttransplant, chemokine mRNA levels decreased in isografts but were maintained at high levels in the allografts. Increased early chemokine mRNA was also observed in C57BL/6, but not BALB/c++ grafts on BALB/c athymi(nu/nu) recipients. Treatment of allograft recipients with ASGM1 or with anti-NK1.1 antibody eliminated NK cells from the spleen and allograft infiltrating cell populations and decreased early chemokine mRNA levels in allografts 60-70%. Analyses of allograft homogenates indicated increased levels of KC, MIP-1alpha, and MIP-1beta protein at day 4 posttransplant that were decreased in recipients depleted of NK cells. Early chemokine mRNA levels were equivalent in isogeneic and semiallogeneic F1 grafts. CONCLUSIONS: Early chemokine mRNA expression and protein production in allogeneic skin grafts is amplified by recipient natural killer (NK) cells. These results indicate a novel function for infiltrating NK cells in mediating early increased intra-allograft chemokine production and inflammation during the initiation of acute rejection.     

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.     

Chemokine expression in the obstructed kidney.

Chemokines are chemotactic cytokines that are important mediators of leukocyte extravasation and chemotaxis. Herein, we provide evidence that after 1 day of unilateral ureteral obstruction (UUO), the mouse obstructed kidney (OBK) expresses MCP-1 (monocyte chemoattractant protein-1), RANTES (Regulated on activation normal T-cell expressed and secreted) and IP-10 (interferon-gamma-induced protein-10). In addition, by day 7, MIP-2 (macrophage inflammatory protein-2) expression is elevated in the obstructed kidneys compared to the contralateral control kidneys (CLK). After 7 days of obstruction, RANTES was the most abundant of the four chemokines detected in the OBK. In situ hybridization results indicate that several cellular compartments contribute to the expression of RANTES in the OBK. However, clearly cortical tubules within the OBK contribute substantially to the elevated expression of RANTES. These data support the contention that the cortical tubular epithelium plays a pivotal role in the inflammation associated with experimental hydronephrosis.     

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.     

Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray

BACKGOUND: Interferon (IFN)-gamma and the IFN-gamma-dependent pathway are prominent in vascularized allograft during acute rejection. However, IFN-gamma deficient (IFN-gamma-/-) mice can rapidly reject cardiac allografts. To bring the alternative pathway during allograft rejection into more precise focus, we investigated the gene expression profile in murine cardiac allografts in IFN-gamma-/- mice by means of DNA microarray. MATERIAL AND METHOD: We screened for gene expression changes in murine cardiac allografts of BALB/c H-2d into both wild-type C57BL/6 H-2b (n=3) and IFN-gamma-/- C57BL/6 H-2b(IFN-gamma-/-, n=4) using Affymetrix oligonucleotide arrays to monitor more than 11,000 genes and expressed sequence tag (ESTs). The heart was heterotopically transplanted. Transplanted hearts were harvested on day 5. As a control, isografts (C57BL/6 to C57BL/6) were also harvested on day 5. RESULTS: On day 5, 64 of the 84 genes induced in the allografts in wild-type mice were not up-regulated in IFN-gamma-/- mice. We identified a group of 54 genes that were up-regulated in allografts in IFN-gamma-/- mice. Several chemokine genes, including monocyte chemoattractant protein=1 and macrophage inflammatory protein, were induced in the allografts in both wild-type and IFN-gamma-/- mice. Interestingly, a group of genes, including C10-like chemokine and platelet factor 4, were specifically induced in the IFN-gamma-/- mice. CONCLUSION: DNA microarray analysis reveals a unique pattern of mRNA expression in allografts in IFN-gamma-/- mice as well as a group of genes induced in cardiac allografts in both wild-type and IFN-gamma-/- mice, including monocyte chemoattractant protein-1 and monocyte chemoattractant protein-1.     

Novel broad-spectrum chemokine inhibitor protects against lung ischemia-reperfusion injury.

BACKGROUND: Functional roles for chemokines have been demonstrated in several models of ischemia-reperfusion injury. The redundancy inherent to chemokine pathways makes administration of neutralizing antibodies to any single chemokine ineffective in ameliorating injury. This study was undertaken to define the pattern of chemokine expression in lung ischemia-reperfusion injury (LIRI), and to determine whether a broad-spectrum chemokine inhibitor, NR58-3.14.3, could confer significant protection against LIRI. METHODS: Left lungs of rats were rendered ischemic for 90 minutes and then reperfused for 4 hours. Chemokine secretion into the alveolar space was quantified by enzyme-linked immunoassay. Treated animals received NR58-3.14.3 prior to reperfusion. Vascular injury was measured by lung permeability index, neutrophil accumulation in lung parenchyma was determined by myeloperoxidase (MPO) activity, and alveolar leukocyte counts were quantified in bronchoalveolar lavage (BAL) effluent. A ribonuclease protection assay evaluated mRNA expression of various chemokines. RESULTS: Lavage effluent in untreated animals demonstrated significant increases in the secretion of cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1). Animals receiving NR58-3.14.3 demonstrated a 37% (p < 0.001) reduction in vascular injury and a marked reduction in lung MPO activity (p < 0.001) and alveolar cell counts (p = 0.005). Chemokine inhibition decreased mRNA expression of a number of early response cytokines when compared with positive control animals, and caused a significant decrease (p < 0.04) in the secretion of TNF-alpha. CONCLUSIONS: These studies demonstrate that chemokines are expressed after lung ischemia and reperfusion, and that broad-spectrum chemokine inhibition ameliorates reperfusion injury. mRNA expression of early response cytokines was modulated, and the secretion of TNF-alpha was decreased.     

Analysis of the Fas system and Bcl-2 in rat liver allograft rejection.

BACKGROUND: Apoptosis is involved in the mechanism of cell death observed in liver allograft rejection. The liver cells are sensitive to Fas-mediated apoptosis; however, little is known about the involvement of the Fas system in liver allograft rejection. We used rat models to investigate the expression of Fas/Fas ligand and apoptosis-related proteins during liver allograft rejection. MATERIALS AND METHODS: DA rats to Lewis, and Lewis to Lewis orthotopic liver transplantation were performed; liver samples were collected on days 1, 3, 5, 7, and 9 postoperatively (each n = 3). Apoptosis was monitored by TUNEL and electron microscopy. The expression of Fas, FasL, bcl-2, and bax was examined at the mRNA level and by means of immunohistochemistry. RESULTS: The TUNEL index in the allografts and isografts on day 7 was 20.1 +/- 1.5 and 7.7 +/- 2.6/1000 cells, respectively. Fas and bax mRNA were constitutively expressed in both of the groups. The expression of Fas ligand mRNA in the allografts which rose on day 5 was 10 times stronger compared to that in the isografts. On the other hand, bcl-2 mRNA was generally expressed in the isografts while it decreased in the allografts. The immunohistochemical analysis also showed an increased reactivity of Fas ligand on day 5 in the allograft, which was observed both in parenchymal and nonparenchymal cells. CONCLUSIONS: These results strongly suggest that Fas/Fas ligand interaction mediates the liver injury during allograft rejection. In addition, other regulatory factors of apoptosis, such as bcl-2, might also be involved in this pathogenesis.     

Cytomegalovirus accelerates chronic allograft nephropathy in a rat renal transplant model with associated provocative chemokine profiles

BACKGROUND: Studies have shown that rat cytomegalovirus (RCMV) infection accelerates transplant vascular sclerosis (TVS) in rat heart and small bowel allotransplants. In these models, RCMV-accelerated TVS results from increased graft infiltration of inflammatory cells through up-regulation of chemokine expression. The aim of this study was to determine if RCMV infection accelerates renal transplant chronic allograft nephropathy (CAN), and the role of chemokines in this process. METHODS: F344 kidneys were transplanted into Lewis recipients with and without RCMV infection. To monitor CAN, serum creatinine (Cr) levels were measured starting at 4 weeks posttransplantation. At 7 and 21 days, and at terminal rejection, grafts were examined for histologic changes, inflammatory cell infiltrates, viral load, and chemokine expression profiles. RESULTS: By week 8, serum Cr showed significant elevation (P < .01) in the RCMV-infected group vs uninfected group, and remained significantly elevated through the end of the study. RCMV+ renal allografts had significant inflammatory cell infiltration and increased CAN at postoperative day (POD) 28. The CC chemokines RANTES, MCP-1, and MIP-1alpha, and the CXC chemokine IP-10 were up-regulated in RCMV-infected vs uninfected allografts. IP-10 was significantly up-regulated early in the process, whereas RANTES and MCP-1 were induced at a later time. CONCLUSIONS: RCMV infection accelerates CAN, with associated graft inflammatory infiltrates, which is paralleled by an increase in expression of CC and CXC chemokines. Our findings suggest that the early induction of IP-10 in the infected allografts promotes alterations in T-cell and monocyte migration to the graft, which initiates accelerated inflammatory and fibrotic changes associated with CAN.     

CCR1 chemokines promote the chemotactic recruitment, RANKL development, and motility of osteoclasts and are induced by inflammatory cytokines in osteoblasts.

Chemoattractants that recruit OC precursors to locally inflamed sites of resorption are not well known. A chemokine receptor, CCR1, was expressed in OC precursors and elevated in mature OCs, and its ligands promoted OC precursor recruitment, RANKL development, and OC motility. Cytokines induced OB release of such chemokines, which may therefore significantly contribute to inflammatory bone loss. INTRODUCTION: Chemokines, primarily of two major (CXC, CC) families, are essential signals for the trafficking and localization of circulating hematopoietic cells into tissues. However, little is known about their potential roles in osteoclast (OC) recruitment, development, or function. Previously, we analyzed CXC receptors in murine OC precursors and found high expression of CXCR4 that mediated their stromal-derived factor-1(SDF-1)-induced chemotaxis and collagen invasion. Here, we investigated if CC receptors and ligands, which are elevated in inflammatory and other osteolytic diseases, also play important roles in the recruitment, formation, or activity of murine bone-resorptive OCs. MATERIALS AND METHODS: CC chemokine receptor (CCR) mRNA expression was analyzed during OC formation induced by RANKL in murine RAW 264.7 cells and primary marrow cells. Corresponding CC chemokines were tested for their ability to elicit precursor chemotaxis or OC development, or to influence motility, bone resorption, adhesion, or survival in RANKL-differentiated OCs. Constitutive and inflammatory cytokine-induced release of the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and regulated on activation, normal T-cell expressed and secreted (RANTES) was measured by ELISA for OCs, osteoblasts (OBs), and their precursor cells. RESULTS: CCR1 was expressed in murine marrow cells, the most prominent CCR in RAW cells, and upregulated by RANKL in marrow or RAW cells. Chemokines that bind CCR1 (MIP-1alpha, RANTES, and monocyte chemoattractant protein-3 [MCP-3]) were produced to varying degrees by murine OCs, OBs, and their precursors, and markedly increased by interleukin (IL)-1alpha and TNFalpha in differentiating OBs. RANTES, and especially MIP-1alpha, increased mature OC motility, but did not alter OC resorption activity, adhesion, or survival. All three chemokines stimulated chemotaxis of marrow or RAW cell precursors, leading to the greater formation of OCs (in number and size) after RANKL development of such chemoattracted marrow cells. All three chemokines also directly and dramatically enhanced OC formation in marrow cultures, through a pathway dependent on the presence of RANKL but without altering RANK expression. CONCLUSIONS: Pathological increases in secretion of these chemokines from activated OBs or other cells may potently stimulate the chemotactic recruitment and RANKL formation of bone-resorptive OCs, thereby exacerbating local osteolysis in multiple skeletal diseases.     

Molecular mechanism of contractile dysfunction in cardiac allograft rejection.

Alterations in the beta-adrenergic receptor adenylyl cyclase pathway are well known in heart failure. To determine if an alteration in this pathway occurs during the reversible phase of cardiac allograft rejection, we used a rat heterotopic heart transplant model. Lewis rats received either isografts or Lewis Brown Norway allografts. Cardiac grafts and native hearts were explanted 4, 5, or 6 days later. Receptor-mediated modulation of adenylyl cyclase activity was investigated using isoproterenol, forskolin, and the muscarinic and adenosine receptor agonists carbachol and R-N6-(C2-phenyl-isopropyl)-adenosine (R-PIA), respectively. Allografts demonstrated evidence of histological rejection and a significantly impaired response to forskolin and isoproterenol on all days: [table: see text] (% increase in cAMP in response to forskolin or isoproterenol +/- standard error. All results P less than 0.03 except Day 4 forskolin and Day 5 isoproterenol.) No significant difference was noted between isografts and allografts stimulated with carbachol and R-PIA. These data suggest that a primary alteration in adenylyl cyclase activity may be a component of the molecular basis of reversible contractile dysfunction in cardiac allograft 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.     

Alpha chemokines regulate direct lung ischemia-reperfusion injury.

BACKGROUND: Alpha chemokines function predominantly to recruit and activate neutrophils, which are important effectors of acute lung injury. This study evaluated whether blockade of 2 potent alpha chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), is protective against lung ischemia-reperfusion injury in a warm in situ hilar clamp model. METHODS: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received antibodies to MIP-2 or CINC immediately prior to reperfusion. Lung injury was quantitated by vascular permeability to (125)I-radiolabeled bovine serum albumin, lung tissue neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) fluid. CINC and MIP-2 mRNA expression were assessed by northern blot, while ribonuclease protection assays were performed to evaluate mRNA expression for a number of early response cytokines. MIP-2 and CINC protein expression in injured lungs was determined by immunoblotting. RESULTS: Treatment with antibodies to CINC or MIP-2 was associated with significant protection against increases in vascular permeability, MPO content and alveolar leukocyte sequestration in injured lungs. Expression of CINC and MIP-2 mRNA peaked after 2 hours of reperfusion in injured lungs, and protein levels were evident on immunoblotting after 3 hours of reperfusion. Neither CINC nor MIP-2 blockade appeared to modulate cytokine mRNA expression. CONCLUSIONS: CINC and MIP-2 are important mediators involved in direct lung ischemia-reperfusion injury. They appear to function by modulating neutrophil recruitment, but not inflammatory cytokine release.     

FK506 rescues peripheral nerve allografts in acute rejection

This study investigated the ability of the immunosuppressant FK506 to reverse nerve allograft rejection in progress. Eighty-four Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals. Allografts were left untreated for either 7, 10, or 14 days before receiving daily subcutaneous FK506 injections (2 mg/kg). Time-matched control animals received either an isograft, an allograft with continuous FK506, or an allograft with no postoperative FK506 therapy. All animals underwent weekly evaluation of nerve function by walking track analysis. Experimental group animals were sacrificed either immediately prior to initiation of FK506 therapy (days 7, 10, or 14), after 2 weeks of immunosuppressive treatment, or 8 weeks postsurgery. Histomorphometric analysis, consisting of measurements of total number of nerve fibers, neural density, and percent of neural debris, demonstrated a statistically significant increase in regeneration in the isograft group relative to the untreated allograft group within 28 days of transplantation. Grafts harvested from animals receiving 2 weeks of FK506 after 7 or 10 days of rejection were histomorphometrically similar to time-matched isografts. By contrast, grafts from animals receiving 2 weeks of FK506 following 14 days without therapy resembled untreated allografts and demonstrated significant histomorphometric differences from isografts at the corresponding time point. Analysis of walking track data confirmed that relative to untreated allografts, functional recovery was hastened in animals receiving an isograft, or allograft treated with FK506. This study demonstrated that when started within 10 days of graft placement, FK506 could reverse nerve allograft rejection in rats evaluated following 2 weeks of treatment.