CXCR2 Knockout Research Articles

Role of Endothelial TLR4 for Neutrophil Recruitment into Central Nervous System Microvessels in Systemic Inflammation

Brain inflammation is a frequent consequence of sepsis and septic shock. We imaged leukocyte recruitment in brain postcapillary venules induced by i.p. administration of LPS as a simple model of systemic inflammation. The i.p. injection of LPS (0.5 mg/kg) induced significant leukocyte rolling and adhesion in brain postcapillary venules of wild-type (WT) mice and more than 90% were neutrophils. However, no emigrated neutrophils were detected in brain parenchyma. High levels of TNF-alpha and IL-1beta were detected in the plasma after LPS injection but a different profile (IL-1beta but not TNF-alpha) was detected in the brain. LPS caused no recruitment in TLR4 knockout mice. In chimeric mice with TLR4-expressing resident cells but TLR4-deficient bone marrow-derived circulating cells, neutrophil rolling and adhesion was similar to WT mice. This observation is consistent with a requirement for resident cells in the LPS-induced neutrophil recruitment into brain microvessels. Transgenic mice engineered to express TLR4 exclusively on endothelial cells had a similar level of leukocyte recruitment in brain as WT mice in response to LPS. High dose LPS (10 mg/kg) led to neutrophil infiltration in the brain parenchyma in WT mice. High KC and MIP-2 production was observed from brain parenchyma microglial cells, and CXCR2 knockout mice failed to recruit neutrophils. However, neither neutrophil infiltration nor KC or MIP-2 was observed in endothelial TLR4 transgenic mice in response to this LPS dose. Our results demonstrate that direct endothelial activation is sufficient to mediate leukocyte rolling and adhesion in cerebral microvessels but not sufficient for emigration to brain parenchyma.

Attenuation of acute lung inflammation induced by cigarette smoke in CXCR3 knockout mice

BackgroundCD8+ T cells may participate in cigarette smoke (CS) induced-lung inflammation in mice. CXCL10/IP-10 (IFNγ-inducible protein 10) and CXCL9/Mig (monokine induced by IFN-γ) are up-regulated in CS-induced lung injury and may attract T-cell recruitment to the lung. These chemokines together with CXCL11/ITAC (IFN-inducible T-cell alpha chemoattractant) are ligands for the chemokine receptor CXCR3 which is preferentially expressed chiefly in activated CD8+ T cells. The purpose of this investigation was to study the contribution of CXCR3 to acute lung inflammation induced by CS using CXCR3 knockout (KO) mice.MethodsMice (n = 8 per group) were placed in a closed plastic box connected to a smoke generator and were exposed whole body to the tobacco smoke of five cigarettes four times a day for three days. Lung pathological changes, expression of inflammatory mediators in bronchoalveolar lavage (BAL) fluid and lungs at mRNA and protein levels, and lung infiltration of CD8+ T cells were compared between CXCR3-/- mice and wild type (WT) mice.ResultsCompared with the WT littermates, CXCR3 KO mice showed less CS-induced lung inflammation as evidenced by less infiltration of inflammatory cells in airways and lung tissue, particularly fewer CD8+ T cells, lower levels of IFNγ and CXCR3 ligands (particularly CXCL10).ConclusionOur findings show that CXCR3 is important in promoting CD8+ T cell recruitment and in initiating IFNγ and CXCL10 release following CS exposure. CXCR3 may represent a promising therapeutic target for acute lung inflammation induced by CS.

Acute pulmonary inflammation is inhibited in CXCR3 knockout mice after short-term cigarette smoke exposure

CXCR3, via binding its specific ligand CXCL10, plays an important role in cigarette smoke (CS)-induced pulmonary inflammation. CXCR3 is preferentially expressed in activated T cells (chiefly CD8+ T cells). The purpose of this study was to investigate the role of CXCR3 in CS-induced pulmonary injury using CXCR3 gene-deficient (CXCR3-/-) mice. Differences in the infiltration of inflammatory cells and CD8+ T cells and the expression of inflammatory mediators and chemokines in the bronchoalveolar lavage fluid and lungs at the mRNA and protein levels were compared between CXCR3-/- mice and wild-type (WT) mice at 2 h after 3 d of CS exposure. Compared with their WT counterparts, the CXCR3-/- mice showed alleviated inflammation, as evidenced by fewer inflammatory cells, particularly cytotoxic CD8+ T cells, in bronchoalveolar lavage fluid and lung tissues. At both the mRNA and protein levels, there were significantly lower levels of inflammatory and chemotactic cytokines, including TNF-alpha, interleukin-8, interferon-gamma, transforming growth factor-beta1, and CXCL10 in the CXCR3-/- mice. Our data show that CXCR3 is important in recruiting inflammatory cells (particularly CD8+ T cells) into the airways and lungs, as well as initiating inflammatory and fibrotic cytokines release at 2 h following a short-term CS insult. CXCR3 could be a novel target for the treatment of pulmonary inflammation induced by CS.

Intra‐neural administration of fractalkine attenuates neuropathic pain‐related behaviour

There is increasing evidence that a number of cytokines and their receptors are involved in the processes that lead to the development and maintenance of neuropathic pain states. Here we demonstrate that levels of CX3CR1 (the receptor for the chemokine fractalkine) mRNA in lumbar dorsal root ganglia (DRG) increase 5.8-fold 7 days after sciatic nerve axotomy, and 1.7- and 2.9-fold, 3 and 7 days respectively, after the spared nerve injury (SNI) model of neuropathic pain. In contrast, no significant change in the levels of fractalkine mRNA is apparent in the DRG after axotomy or SNI. The increase in CX3CR1 mRNA is paralleled by a 3.9- and 2.1-fold increase in the number of CX3CR1-positive macrophages in the DRG 7 days after axotomy and SNI, respectively. Expression of CX3CR1 in macrophages is also markedly increased in the sciatic nerve proximal to site of injury, by 25.7-fold after axotomy and 16.2-fold after SNI, 7 days after injury. Intra-neural injection into the sciatic nerve of 400 ng or 100 ng of fractalkine in adult 129OlaHsd mice significantly delayed the development of allodynia for 3 days following SNI. Further, CX3CR1 knockout (KO) mice display an increase in allodynia for three weeks after SNI compared to strain-matched Balb/c controls. Taken together, these results suggest an anti-allodynic role for fractalkine and its receptor in the mouse.

Methamphetamine‐induced neurotoxicity and microglial activation are not mediated by fractalkine receptor signaling

Methamphetamine (METH) damages dopamine (DA) nerve endings by a process that has been linked to microglial activation but the signaling pathways that mediate this response have not yet been delineated. Cardona et al. [Nat. Neurosci. 9 (2006), 917] recently identified the microglial-specific fractalkine receptor (CX3CR1) as an important mediator of MPTP-induced neurodegeneration of DA neurons. Because the CNS damage caused by METH and MPTP is highly selective for the DA neuronal system in mouse models of neurotoxicity, we hypothesized that the CX3CR1 plays a role in METH-induced neurotoxicity and microglial activation. Mice in which the CX3CR1 gene has been deleted and replaced with a cDNA encoding enhanced green fluorescent protein (eGFP) were treated with METH and examined for striatal neurotoxicity. METH depleted DA, caused microglial activation, and increased body temperature in CX3CR1 knockout mice to the same extent and over the same time course seen in wild-type controls. The effects of METH in CX3CR1 knockout mice were not gender-dependent and did not extend beyond the striatum. Striatal microglia expressing eGFP constitutively show morphological changes after METH that are characteristic of activation. This response was restricted to the striatum and contrasted sharply with unresponsive eGFP-microglia in surrounding brain areas that are not damaged by METH. We conclude from these studies that CX3CR1 signaling does not modulate METH neurotoxicity or microglial activation. Furthermore, it appears that striatal-resident microglia respond to METH with an activation cascade and then return to a surveying state without undergoing apoptosis or migration.

CXCR2 may be critical in recruiting neutrophils (PMN) into the inflamed colon in DSS‐induced colitis

Dextran sodium sulphate (DSS)‐induced colitis in mice is characterized by PMN infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To address this, we determined the role of CXCR2 in DSS‐induced colitis by neutralizing CXCR2 or using CXCR2 KO (knockout) mice. DSS (5% w/v) was administered through drinking water to CXCR2 KO and Balb/c mice (WT) for 5 days followed by tap water for 1 day. In the neutralization study, DSS‐treated or control WT mice were injected i.p with goat serum or goat anti‐CXCR2 serum and disease severity was assessed. DSS‐treated CXCR2 KO mice failed to recruit PMN into the mucosa and had less severe crypt damage with no ulceration compared with WT mice. In the neutralization experiments, goat serum‐injected mice receiving DSS lost weight and showed severe clinical illness. Histological observation revealed edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, the anti‐CXCR2 serum‐treated mice showed less clinical illness with no weight loss. This was associated with less histological damage and a lack of PMN infiltration. Our experiments suggest a role for CXCR2 in DSS‐induced colitis and that it may be critical in recruiting PMN into the inflamed mucosa. Funded by the CCFC and the Swedish Medical Research Council, and an IWK Health Centre Fellowship to SMF.

Chemokine Receptor CX3CR1 Mediates Skin Wound Healing by Promoting Macrophage and Fibroblast Accumulation and Function

Wounds heal through a highly regulated, self-limited inflammatory response, however, precise inflammatory mediators have not been fully delineated. In this study, we report that in a mouse model of excisional skin wound healing the chemokine CX3CL1 and its receptor CX3CR1 were both highly induced at wound sites; CX3CL1 colocalized with macrophages and endothelial cells, whereas CX3CR1 colocalized mainly with macrophages and fibroblasts. Loss of CX3CR1 function delayed wound closure in both CX3CR1 knockout (KO) mice and in wild-type mice infused with anti-CX3CR1-neutralizing Ab. Conversely, transfer of bone marrow from donor wild-type mice, but not from donor CX3CR1 KO mice, restored wound healing to normal in CX3CR1 KO-recipient mice. Direct effects of CX3CR1 disruption at the wound site included marked reduction of macrophages and macrophage products, such as TGF-beta1 and vascular endothelial growth factor. Consistent with this, we observed reduced alpha-smooth muscle actin (a marker for myofibroblasts) and collagen deposition in skin from wounded CX3CR1 KO mice, as well as reduced neovascularization. Together, the data support a molecular model of skin wound repair in which CX3CR1 mediates direct recruitment of bone marrow-derived monocytes/macrophages which release profibrotic and angiogenic mediators.

Delayed and Deficient Dermal Maturation in Mice Lacking the CXCR3 ELR-Negative CXC Chemokine Receptor

Replacement of wounded skin requires the initially florid cellular response to abate and even regress as the dermal layer returns to a relatively paucicellular state. The signals that direct this "stop and return" process have yet to be deciphered. CXCR3 chemokine receptor and its ligand CXCL11/IP-9/I-TAC are expressed by basal keratinocytes and CXCL10/IP-10 by keratinocytes and endothelial cells during wound healing in mice and humans. In vitro, these ligands limit motility in dermal fibroblasts and endothelial cells. To examine whether this signaling pathway contributes to wound healing in vivo, full-thickness excisional wounds were created on CXCR3 wild-type (+/+) or knockout (-/-) mice. Even at 90 days, long after wound closure, wounds in the CXCR3(-/-) mice remained hypercellular and presented immature matrix components. The CXCR3(-/-) mice also presented poor remodeling and reorganization of collagen, which resulted in a weakened healed dermis. This in vivo model substantiates our in vitro findings that CXCR3 signaling is necessary for inhibition of fibroblast and endothelial cell migration and subsequent redifferentiation of the fibroblasts to a contractile state. These studies establish a pathophysiologic role for CXCR3 and its ligand during wound repair.

The role of CXC chemokine receptor 2 inPseudomonas aeruginosacorneal infection

Pseudomonas is one of the leading causes of contact lens-related microbial keratitis. Despite the use of antibiotics, the host inflammatory response continues to cause damage to the cornea, which may lead to blindness. CXCR2-binding chemokines have been implicated in the pathogenesis of Pseudomonas keratitis, and the exact role of this receptor remains to be elucidated. Corneas of CXCR2 knockout and wild-type mice (Cmkar 2-/- and Cmkar 2+/+) were scratched, and 2x10(6) CFU/mL Pseudomonas 6294 or 6206 was added to corneas. Twenty-four hours postinfection, mice were killed, and eyes were harvested for enumeration of bacteria, myeloperoxidase (MPO) levels, and inflammatory mediators. Cmkar 2-/- had 20- to 100-fold more bacteria than Cmkar 2+/+ mice. There were no differences in MPO levels between gene knockout and Cmkar 2+/+ mice. Histology revealed PMN were restricted to the limbal area. Levels of CXCR2 chemokines (keratinocyte-derived chemokine and MIP-2) were elevated significantly in gene knockout mice. A lack of CXCR2 leads to an inability to control bacterial numbers as a result of the inability of PMN to reach the site of infection in the avascular cornea. These results imply that CXCR2 is critical to the extravasation of neutrophils into the avascular cornea.

Human CXCR2 (hCXCR2) takes over functionalities of its murine homolog in hCXCR2 knockin mice

Human CXCR2 (hCXCR2) has been implicated in diverse inflammatory diseases. When roles of this receptor studied in animal models are extrapolated into men, large species differences in expression of the receptor and its ligands must be considered. These differences seriously weaken conclusions toward the role of hCXCR2 in the development of human diseases. It furthermore hampers straightforward testing of CXCR2 antagonists, especially when compounds discriminate between human and other species' receptors. Using gene targeting in embryonic stem cells, a hCXCR2 knockin mouse strain was generated in which endogenous murine CXCR2 (mCXCR2) sequences are replaced by the hCXCR2 gene. Correct targeting and expression on neutrophils were confirmed by Southern blot and immunohistochemical analyses. A phenotypic analysis of the hCXCR2 knockin mice, in comparison to wild-type and CXCR2 knockout mice, confirmed proper function of the hCXCR2 gene. In vivo migratory responses of neutrophils were intact in hCXCR2 knockin mice. Finally, an experiment with a CXCR2 antagonist demonstrated that the knockin model is indeed useful for in vivo evaluation of low-molecular weight compounds. In conclusion, our data unequivocally show that hCXCR2 can functionally replace mCXCR2, making this an attractive model to test novel pharmaceuticals designed to antagonize human CXCR2 in vivo.

Intranasal inoculation of Chlamydia trachomatis mouse pneumonitis agent induces significant neutrophil infiltration which is not efficient in controlling the infection in mice.

Previous studies have shown that chlamydial infection is accompanied by significant infiltration of neutrophils at the site of infection. However, the role of neutrophils in host defence against chlamydial infection is not clearly understood. Using genetically different inbred mouse strains and CXCR-2 gene knockout (KO) mice, we examined the mechanism for neutrophil recruitment and the role of neutrophils during chlamydial lung infection. Our data showed that C3H mice exhibited significantly higher and more persistent neutrophil infiltration in the lung than did C57BL/6 mice following Chlamydia trachomatis mouse pneumonitis infection. The massive neutrophil infiltration in C3H mice was paralleled by high-level expression of CXCR-2 and its ligands, CXC chemokines (macrophage inflammatory protein 2, cytokine-induced neutrophil attractant (KC) and lipopolysaccharide-induced CXC chemokine), and proinflammatory cytokines (tumour necrosis factor-alpha, interleukin-1 and interleukin-6) in the lung. Although much greater infiltration of neutrophils was observed in C3H mice than in C57BL/6 mice, the former mice had more severe disease and higher in vivo chlamydial growth than the latter. Moreover, CXCR-2 KO mice, which revealed a dramatic reduction in neutrophil activity, showed comparable chlamydial infection to wild-type mice. These results suggest that neutrophils are not efficient for controlling chlamydial lung infection.

T-Cell Properties Determine Disease Site, Clinical Presentation, and Cellular Pathology of Experimental Autoimmune Encephalomyelitis

Two distinct clinical phenotypes of experimental autoimmune encephalomyelitis are observed in BALB interferon-gamma knockout mice immunized with encephalitogenic peptides of myelin basic protein. Conventional disease, characterized by ascending weakness and paralysis, occurs with greater frequency after immunizing with a peptide comprising residues 59 to 76. Axial-rotatory disease, characterized by uncontrolled axial rotation, occurs with greater frequency in mice immunized with a peptide corresponding to exon 2 of the full length 21.5-kd protein. The two clinical phenotypes are histologically distinguishable. Conventional disease is characterized by inflammation and demyelination primarily in spinal cord, whereas axial-rotatory disease involves inflammation and demyelination of lateral medullary areas of brain. Both types have infiltrates in which neutrophils are a predominating component. By isolating T cells and transferring disease to naive recipients, we show here that the type of disease is determined entirely by the inducing T cell. Furthermore, studies using CXCR2 knockout recipients, unable to recruit neutrophils to inflammatory sites, show that although neutrophils are critical for some of these T cells to effect disease, there are also interferon-gamma-deficient T cells that induce disease in the absence of both interferon-gamma and neutrophils. These results highlight the multiplicity of T-cell-initiated effector pathways available for inflammation and demyelination.

Acute exposure to CXC chemokine ligand 10, but not its chronic astroglial production, alters synaptic plasticity in mouse hippocampal slices

Brain levels of CXC chemokine ligand 10 (CXCL10) are elevated in a number of neuropathological conditions. To determine its impact on neuronal function, we measured synaptic transmission and plasticity in hippocampal slices prepared from transgenic (TG) mice with chronic astroglial production of CXCL10. We also tested the acute effect of recombinant CXCL10 applied to slices from normal C57Bl/6J mice, CXCL10 TG mice and CXCR3 knock out (KO) mice. Chronic production of CXCL10 did not alter synaptic plasticity. By contrast, exogenous CXCL10 (10 ng/ml) significantly inhibited long-term potentiation (LTP) in slices from normal C57Bl/6J mice and CXCL10 TG. The effect was probably receptor-mediated because CXCL10-induced inhibition of LTP was not observed in CXCR3 KO mice. Our findings suggest that acute exposure to CXCL10 alters synaptic plasticity via CXCR3 in mouse hippocampus.

Prevention of angiogenesis by naked DNA IL-12 gene transfer: angioprevention by immunogene therapy.

IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-gamma and angiostatic CXCR3 chemokine ligands. Naked DNA intramuscular injection of an expression vector plasmid producing IL-12 resulted in significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant, inhibition. Control plasmid injection did not affect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell-depleted C57/bl and nude mice as well as in IFN-gamma(-/-) and CXCR3(-/-) knockout mice, indicating that NK- and/or T-cell-initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. Finally, IL-12 plasmid DNA gene transfer significantly prevented the growth and vascularization of highly angiogenic KS-Imm Kaposi's sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. These data suggest that a preventive gene therapy approach using antiangiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angioimmunoprevention.

Impaired healing of nitrogen mustard wounds in CXCR2 null mice.

To examine the significance of chemokine activation of CXCR2 in wound healing after chemical burn, cutaneous injury was created by topical application of nitrogen mustard on CXCR2 wild type (+/+), heterozygous (+/-), and knockout (-/-) mice. Wounds were analyzed histologically for neutrophil and monocyte infiltration and for reepithelialization at postwound days 4, 7, and 10. Neutrophil recruitment to the wound site was reduced through postwound day 7 in CXCR2 -/- mice as indicated by myeloperoxidase assay and by visual quantitation. Because there is always concern that mice with targeted deletion of a specific receptor may undergo developmental adaptations to offset the loss of the receptor, we also accessed chemical wound repair in the presence of a small molecule antagonist of CXCR2. Dietary supplementation with a CXCR2 antagonist (SB-265610) during the wound repair process also markedly delayed healing parameters in CXCR2 +/+ mice, even greater than treatment with glucocorticoids. These parallel studies further establish that mice deficient in CXCR2 function exhibit delayed cutaneous wound healing that may be primarily linked to impaired neutrophil recruitment after chemical burn with nitrogen mustard. Thus, there may be a potential therapeutic benefit of treating nitrogen mustard-induced skin lesions with agonists of CXCR2 to facilitate the wound repair process.

Secondary lymphoid tissue chemokine (CCL21) activates CXCR3 to trigger a Cl- current and chemotaxis in murine microglia.

Microglial cells represent the major immunocompetent element of the CNS and are activated by any type of brain injury or disease. A candidate for signaling neuronal injury to microglial cells is the CC chemokine ligand CCL21, given that damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that a local application of CCL21 for 30 s triggered a Cl(-) conductance with lasted for tens of minutes. This response was sensitive to the Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamide-4'-isothiocyanatostilbene, 2,2'-disulfonic acid. Moreover, CCL21 triggered a chemotaxis response, which was sensitive to Cl(-) channel blockers. In microglial cells cultured from CCR7 knockout mice, CCL21 produced the same type of Cl(-) current as well as a chemotaxis response. In contrast, in microglial cells from CXCR3 knockout mice, CCL21 triggered neither a Cl(-) conductance nor a chemotaxis response after CCL21 application. We conclude that the CCL21-induced Cl(-) current is a prerequisite for the chemotaxis response mediated by the activation of CXCR3 but not CCR7 receptors, indicating that in brain CCL21 acts via a different receptor system than in lymphoid organs.

CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses.

We have developed a mouse brain abscess model by using Staphylococcus aureus, one of the main etiologic agents of brain abscesses in humans. Direct damage to the blood-brain barrier was observed from 24 h to 7 days after S. aureus exposure as demonstrated by the accumulation of serum IgG in the brain parenchyma. Evaluation of brain abscesses by immunohistochemistry and flow cytometry revealed a prominent neutrophil infiltrate. To address the importance of neutrophils in the early containment of S. aureus infection in the brain, mice were transiently depleted of neutrophils before implantation of bacteria-laden beads. Neutrophil-depleted animals consistently demonstrated more severe brain abscesses and higher CNS bacterial burdens compared with control animals. S. aureus led to the induction of numerous chemokines in the brain, including macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL1, monocyte chemoattractant protein-1/CCL2, and TCA-3/CCL1, within 6 h after bacterial exposure. These chemokines also were expressed by both primary cultures of neonatal mouse microglia and astrocytes exposed to heat-inactivated S. aureus in vitro. Because neutrophils constitute the majority of the cellular infiltrate in early brain abscess development, subsequent analysis focused on MIP-2 and KC/CXCL1, two neutrophil-attracting CXC chemokines. Both MIP-2 and KC protein levels were significantly elevated in the brain after S. aureus exposure. Neutrophil extravasation into the brain parenchyma was impaired in CXCR2 knockout mice and was associated with increased bacterial burdens. These studies demonstrate the importance of the CXCR2 ligands MIP-2 and KC and neutrophils in the acute host response to S. aureus in the brain.

Delayed Wound Healing in CXCR2 Knockout Mice

Previous studies demonstrated that the CXC chemokine, MGSA/GRO-alpha and its receptor, CXCR2, are expressed during wound healing by keratinocytes and endothelial cells at areas where epithelialization and neovascularization occur. The process of wound healing is dependent on leukocyte recruitment, keratinocyte proliferation and migration, and angiogenesis. These processes may be mediated in part by CXC chemokines, such as interleukin-8 and MGSA/GRO-alpha. To examine further the significance of CXC chemokines in wound healing, full excisional wounds were created on CXCR2 wild-type (+/+), heterozygous (+/-), or knockout (-/-) mice. Wounds were histologically analyzed for neutrophil and monocyte infiltration, neovascularization and epithelialization at days 3, 5, 7, and 10 postwounding. The CXCR2 -/- mice exhibited defective neutrophil recruitment, an altered temporal pattern of monocyte recruitment, and altered secretion of interleukin-1beta. Significant delays in wound healing parameters, including epithelialization and decreased neovascularization, were also observed in CXCR2 -/- mice. In vitro wounding experiments with cultures of keratinocytes established from -/- and +/+ mice revealed a retardation in wound closure in CXCR2 -/- keratinocytes, suggesting a role for this receptor on keratinocytes in epithelial resurfacing that is independent of neutrophil recruitment. These in vitro and in vivo studies further establish a pathophysiologic role for CXCR2 during cutaneous wound repair.

ELR-CXC CHEMOKINES CAUSE TRANSPLANTATION INJURY THROUGH CXCR2-MEDIATED NEUTROPHIL RECRUITMENT.

173 Transplantation injury is an important cause of graft nonfuction and has been attributed to early neutrophil infiltration. Since chemokines are mediators of leukocyte infiltration, and may play a role in transplantation injury, we examined chemokine mRNA levels in cardiac isografts immediately after transplantation. C57BL/6 hearts were heterotopically transplanted to C57BL/6 recipients and then removed at various times following transplantation and the total RNA extracted. The mRNA expression of the neutrophil attracting ELR-CXC chemokines LIX, KC and MIP-2 as well as the monocyte attractant JE was then analyzed by Northern analysis. All chemokines were upregulated in isografted hearts within 3 hours after transplantation and reached maximum expression at either 3 hours (KC and MIP-2) or 6-12 hours (LIX and JE) then declined through 24 hours. Neutrophils and monocytes infiltrated the organ in a pattern consistent with chemokine expression. To test the importance of ELR-CXC chemokines in transplantation injury, transplants were performed in CXCR2 knockout mice. CXCR2 is the only receptor for LIX, KC, and MIP-2. In these mice, neutrophil infiltration was abolished though chemokine expression remained unchanged. These results demonstrate that early chemokine expression mediates the initial neutrophil infiltration that causes transplantation injury.