CXCR2 ligands and G-CSF mediate PKCalpha-induced intraepidermal inflammation

Transgenic mice overexpressing PKCalpha in the epidermis (K5-PKCalpha mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCalpha is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model of cutaneous inflammation was used to define mediators of skin inflammation that may have clinical relevance. Activation of cutaneous PKCalpha increased the production of the chemotactic factors cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein 2 (MIP-2) in murine plasma. TPA treatment of cultured K5-PKCalpha keratinocytes also released KC and MIP-2 into culture supernatants through an NF-kappaB-dependent pathway. MIP-2 and KC mediated the infiltration of neutrophils into the epidermis, since this was prevented by ablating CXCR2 in K5-PKCalpha mice or administering neutralizing antibodies against KC or MIP-2. The neutrophilia resulted from PKCalpha-mediated upregulation of cutaneous G-CSF released into the plasma independent of CXCR2. These responses could be inhibited by topical treatment with a PKCalpha-selective inhibitor. Inhibiting PKCalpha also reduced the basal and TNF-alpha- or TPA-induced expression of CXCL8 in cultured psoriatic keratinocytes, suggesting that PKCalpha activity may contribute to psoriatic inflammation. Thus, skin can be the source of circulating factors that have both local and systemic consequences, and these factors, their receptors, and possibly PKCalpha could be therapeutic targets for inhibition of cutaneous inflammation.     

Role of CXCR2/CXCR2 ligands in vascular remodeling during bronchiolitis obliterans syndrome

Angiogenesis and vascular remodeling support fibroproliferative processes; however, no study has addressed the importance of angiogenesis during fibro-obliteration of the allograft airway during bronchiolitis obliterans syndrome (BOS) that occurs after lung transplantation. The ELR(+) CXC chemokines both mediate neutrophil recruitment and promote angiogenesis. Their shared endothelial cell receptor is the G-coupled protein receptor CXC chemokine receptor 2 (CXCR2). We found that elevated levels of multiple ELR(+) CXC chemokines correlated with the presence of BOS. Proof-of-concept studies using a murine model of BOS not only demonstrated an early neutrophil infiltration but also marked vascular remodeling in the tracheal allografts. In addition, tracheal allograft ELR(+) CXC chemokines were persistently expressed even in the absence of significant neutrophil infiltration and were temporally associated with vascular remodeling during fibro-obliteration of the tracheal allograft. Furthermore, in neutralizing studies, treatment with anti-CXCR2 Abs inhibited early neutrophil infiltration and later vascular remodeling, which resulted in the attenuation of murine BOS. A more profound attenuation of fibro-obliteration was seen when CXCR2(-/-) mice received cyclosporin A. This supports the notion that the CXCR2/CXCR2 ligand biological axis has a bimodal function during the course of BOS: early, it is important for neutrophil recruitment and later, during fibro-obliteration, it is important for vascular remodeling independent of neutrophil recruitment.     

Bench-to-bedside review: Acute respiratory distress syndrome – how neutrophils migrate into the lung

Acute lung injury and its more severe form, acute respiratory distress syndrome, are major challenges in critically ill patients. Activation of circulating neutrophils and transmigration into the alveolar airspace are associated with development of acute lung injury, and inhibitors of neutrophil recruitment attenuate lung damage in many experimental models. The molecular mechanisms of neutrophil recruitment in the lung differ fundamentally from those in other tissues. Distinct signals appear to regulate neutrophil passage from the intravascular into the interstitial and alveolar compartments. Entry into the alveolar compartment is under the control of CXC chemokine receptor (CXCR)2 and its ligands (CXC chemokine ligand [CXCL]1–8). The mechanisms that govern neutrophil sequestration into the vascular compartment of the lung involve changes in the actin cytoskeleton and adhesion molecules, including selectins, β₂ integrins and intercellular adhesion molecule-1. The mechanisms of neutrophil entry into the lung interstitial space are currently unknown. This review summarizes mechanisms of neutrophil trafficking in the inflamed lung and their relevance to lung injury.     

Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.     

Regulation of pulmonary fibrosis by chemokine receptor CXCR3

CXC chemokine receptor 3 (CXCR3) is the receptor for the IFN-gamma-inducible C-X-C chemokines MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. CXCR3 is expressed on activated immune cells and proliferating endothelial cells. The role of CXCR3 in fibroproliferation has not been investigated. We examined the role of CXCR3 in pulmonary injury and repair in vivo. CXCR3-deficient mice demonstrated increased mortality with progressive interstitial fibrosis relative to WT mice. Increased fibrosis occurred without increased inflammatory cell recruitment. CXCR3 deficiency resulted in both a reduced early burst of IFN-gamma production and decreased expression of CXCL10 after lung injury. We identified a relative deficiency in lung NK cells in the unchallenged CXCR3-deficient lung and demonstrated production of IFN-gamma by WT lung NK cells in vivo following lung injury. The fibrotic phenotype in the CXCR3-deficient mice was significantly reversed following administration of exogenous IFN-gamma or restoration of endogenous IFN-gamma production by adoptive transfer of WT lymph node and spleen cells. Finally, pretreatment of WT mice with IFN-gamma-neutralizing Ab's enhanced fibrosis following lung injury. These data demonstrate a nonredundant role for CXCR3 in limiting tissue fibroproliferation and suggest that this effect may be mediated, in part, by the innate production of IFN-gamma following lung injury.     

Protective ventilation of patients with acute respiratory distress syndrome

In a recent issue of the British Journal of Anaesthesia, Moloney and Griffiths reviewed clinically pertinent issues surrounding the management of the acute respiratory distress syndrome (ARDS) patient, particularly as it pertains to the treatment of ventilator induced/associated lung injury (VILI). In addition to highlighting the important observations that have contributed to further our understanding of the relationship between the mechanical ventilator and inflammatory lung injury, the authors also offer a concise reappraisal of the clinical strategies used to minimize VILI in ARDS. Special emphasis is placed on the theory of biotrauma, which attempts to explain how multi-organ failure may develop in patients who ultimately succumb to this syndrome.     

Protective ventilation of patients with acute respiratory distress syndrome

In a recent issue of the British Journal of Anaesthesia, Moloney and Griffiths reviewed clinically pertinent issues surrounding the management of the acute respiratory distress syndrome (ARDS) patient, particularly as it pertains to the treatment of ventilator induced/associated lung injury (VILI). In addition to highlighting the important observations that have contributed to further our understanding of the relationship between the mechanical ventilator and inflammatory lung injury, the authors also offer a concise reappraisal of the clinical strategies used to minimize VILI in ARDS. Special emphasis is placed on the theory of biotrauma, which attempts to explain how multi-organ failure may develop in patients who ultimately succumb to this syndrome.     

CXCR2 stimulation primes CXCR1 [Ca2+]i responses to IL-8 in human neutrophils

Neutrophil (PMN) priming and subsequent responses to the IL-8 presented on pulmonary endothelial surfaces may be crucial determinants of the development of adult respiratory distress syndrome after injury. Elevated plasma ELR+ C-X-C chemokine (CXC) levels might contribute to PMN priming after trauma, but the role of CXCs in priming circulating PMNs is unstudied. We evaluated the interactions of IL-8 and GRO-alpha in priming human PMN calcium fluxes [Ca2+]i within circulatory environments. At physiologic concentrations, GRO-alpha primes PMN for IL-8 mediated [Ca2+]i mobilization, whereas IL-8 abolishes GRO-alpha responses. Repeated GRO-alpha exposures further enhance IL-8 responses. PMN priming for IL-8 responses in normal plasma was CXCR2 dependent. CXCR2 was more responsive than CXCR1 to low levels of IL-8, together suggesting that CXCR2 is the important CXC receptor at circulating (i.e., low) agonist concentrations. CXCR1 stimulation down-regulated CXCR2 surface expression, whereas CXCR2 stimulation upregulated CXCR1 expression. GRO-alpha/ CXCR2 signaling enhanced post-receptor IL-8 initiated PMN [Ca2+]i influx as well as efflux. Sufficient stimulation of the CXCR1 terminated this cooperative relationship by downregulating surface expression of CXCR2. This study is the first to report that at physiologic concentrations, C-X-C chemokines can act on circulating human PMNs as an integrated system where CXCR2 agonists, rather than cross-desensitizing CXCR1, act to enhance signaling of IL-8 at CXCR1 both by receptor and post-receptor mechanisms. Such CXCR2 mediated priming of CXCR1/ IL-8 interaction may enhance PMN attack on the lung after injury.     

CXCL1/CXCR2在羊瘙痒因子感染小鼠脑组织中的分布特征研究

目的 探究朊病毒感染小鼠脑组织CXC趋化因子配体1 (CXCL1)与CXC趋化因子受体2 (CXCR2)的分布特征。方法 通过免疫组织化学、免疫组织荧光双染实验明确羊瘙痒因子139A及ME7感染终末期小鼠脑组织中CXCL1/CXCR2的分布特征,确定CXCL1/CXCR2的靶细胞及与羊瘙痒因子样朊蛋白(PrPSc)沉积的关系。结果 通过全脑区免疫组化染色发现,CXCL1/CXCR2在羊瘙痒因子139A及ME7感染终末期小鼠脑组织中的含量明显升高,主要分布在海马、皮层、丘脑、小脑及延髓5个脑区。CXCL1与小胶质细胞和神经元细胞存在共定位,而CXCR2与神经元细胞存在共定位。在羊瘙痒因子139A及ME7感染终末期小鼠脑组织中CXCL1、CXCR2和PrP^Sc三者存在明显共定位。结论 CXCL1/CXCR2分布于朊病毒感染小鼠脑组织中朊病毒病理特征集中的脑区。     

CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow

Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR⁺ chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2^–/– and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2^–/– neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR⁺ chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF–induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.     

Differential effects of macrophage inflammatory chemokine-2 and keratinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 +/- 5 mmHg and were then administered anti-MIP- 2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringer's lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; microU/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (increased MPO, increased % esterase+ cells in tissue) into the lung and local tissue inflammation (increased IL-6/MIP-2, decreased IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.     

Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling

OBJECTIVE: To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK). DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the mice. INTERVENTIONS: In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. MEASUREMENTS AND MAIN RESULTS: The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues. CONCLUSIONS: Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.     

Nonpeptide CXCR2 antagonist prevents neutrophil accumulation in hyperoxia-exposed newborn rats

Neutrophil influx in lung injury is controlled in part by chemokines acting through the receptor, CXCR2. To avoid adverse effects of steroids typically used to modify inflammation, we evaluated the effects of competitive blockade of CXCR2 in rats on neutrophil function in vitro and on neutrophil influx in vivo in hyperoxia-induced newborn lung injury, a model of bronchopulmonary dysplasia. In vitro, SB-265610 antagonizes rat cytokine-induced neutrophil chemoattractant-1 (CINC-1)-induced calcium mobilization, IC50 = 3.7 nM, and rat neutrophil chemotaxis in a concentration-dependent manner, IC50 = 70 nM. In vivo, newborn rats exposed to 95% O2 for 8 days had increased lung neutrophil content. Injection with 1 to 3 mg/kg SB-265610 on days 3 to 5 reduced hyperoxia-induced neutrophil accumulation in bronchoalveolar lavage and whole lung myeloperoxidase accumulation at the highest doses. To determine whether these effects might be due in part to increased neutrophil apoptosis, peripheral neutrophils were cultured with and without SB-265610. Apoptosis was assessed by morphology, viability, and terminal transferase deoxyuridine triphosphatidyl nucleotide nick-end labeling. Treatment of neutrophils with CINC-1 reduced apoptosis compared with untreated neutrophils. SB-265610 reduced the antiapoptotic effect of CINC-1 to the levels of those untreated with CINC-1. A selective CXCR2 antagonist may be useful in diseases where neutrophil-mediated exacerbation is present.     

CXCL1 and its receptor, CXCR2, mediate murine sickle cell vaso-occlusion during hemolytic transfusion reactions

Hemolytic transfusion reactions (HTRs) can produce serious and potentially life-threatening complications in sickle cell disease (SCD) patients; however, the mechanisms underlying these complications remain undetermined. We established a model of alloimmune, IgG-mediated HTRs in a well-characterized humanized murine model of SCD. HTRs induced acute vaso-occlusive crisis (VOC), resulting in shortened survival of SCD mice. Acute VOC was associated with elevated circulating inflammatory chemokine levels, including striking elevation of the levels of the neutrophil chemoattractant CXCL1. Recombinant CXCL1 administration was sufficient to induce acute VOC in SCD mice, characterized by leukocyte recruitment in venules, capture of circulating red blood cells, reduction of venular flow, and shortened survival. In contrast, blockade of the CXCL1 receptor, CXCR2, prevented HTR-elicited acute VOC and prolonged survival in SCD mice. These results indicate that CXCL1 is a key inflammatory mediator of acute VOC in SCD mice. Targeted inhibition of CXCL1 and/or CXCR2 may therefore represent a new therapeutic approach for acute VOC in SCD patients.     

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661-1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of alpha2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV(-/-) neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV(-/-) neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV(-/-) leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV(-/-) leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of alpha2,3-linked sialic acid-specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.     

Ventilation-induced neutrophil infiltration and apoptosis depend on apoptosis signal-regulated kinase 1 pathway

OBJECTIVE: Positive pressure ventilation with large tidal volumes has been shown to cause release of cytokines, including macrophage inflammatory protein (MIP)-2, a functional equivalent of human interleukin-8, neutrophil infiltration, and apoptosis. The mechanisms regulating ventilation-induced cytokine production and lung cell death are unclear. Based on our previous in vitro and in vivo models of lung cell stretch, we hypothesized that high tidal volume ventilation-induced MIP-2 production, neutrophil infiltration, and apoptosis are dependent on the activation of apoptosis signal-regulated kinase 1 (ASK1), the upstream activator of c-Jun N-terminal kinase (JNK). DESIGN:: Prospective, controlled animal experiment. SETTING: University research laboratory. SUBJECTS: Male C57BL/6 mice, weighting 20-25 g. INTERVENTIONS: C57BL/6 mice were exposed to high tidal volume (30 mL/kg) or low tidal volume (6 mL/kg) mechanical ventilation for 15 mins to 5 hrs. MEASUREMENTS AND MAIN RESULTS: High tidal volume ventilation induced MIP-2 messenger RNA expression, MIP-2 protein production, neutrophil migration into the lung, airway epithelial cell apoptosis, and activation of ASK1, JNK, and activator protein (AP)-1 DNA binding in a dose-dependent and time-dependent manner. ASK1 inhibition with thioredoxin attenuated high tidal volume ventilation-induced MIP-2 messenger RNA expression, MIP-2 protein production, neutrophil migration into the lung, airway epithelial cell apoptosis, activation of JNK, and AP-1 DNA binding. CONCLUSIONS: Our data showed that high tidal volume ventilation-induced MIP-2 production, neutrophil sequestration, and apoptotic cell death were dependent, in part, on activation of the ASK1/JNK/AP-1 pathway.     

Lung and liver injury following hepatic ischemia/reperfusion in the rat is increased by exogenous lipopolysaccharide which also increases hepatic TNF production in vivo and in vitro

Hepatic ischemia/reperfusion (I/R) results in tumor necrosis factor (TNF) release. Kupffer cells (KC) are one source of this TNF. This study investigates the effects of hepatic I/R combined with lipopolysaccharide (LPS) on the lung and liver injury that follow hepatic I/R and on hepatic release of TNF, epithelial neutrophil activating protein (ENA-78), and macrophage inflammatory protein-2 (MIP-2). The effects of these experimental conditions on TNF production by primary rat KC in vitro were also investigated. Rats were subjected to hepatic I/R alone, hepatic I/R + LPS, sham laparotomy alone, or sham laparotomy + LPS and pulmonary MPO, pulmonary microvascular permeability, hepatic neutrophil influx, hepatic injury, and hepatic TNF, ENA-78, and MIP-2 production were measured. These experiments demonstrated that hepatic I/R in conjunction with LPS results in a more severe lung and liver injury and increased hepatic TNF, ENA-78, and MIP-2 release. The effects of these experimental conditions on rat KC TNF production demonstrated that hepatic I/R + LPS results in a more significant release of TNF as compared to LPS alone or I/R alone. Hepatic I/R plus LPS results in a more severe lung and liver injury and is likely secondary to a more significant and prolonged release of TNF by KC. This may provide a mechanism for development of multiple organ system failure in some patients undergoing hepatic resection, hepatic transplantation, complex vascular operations, or in the setting of hypovolemic shock. Portal endotoxemia related to mesenteric venous congestion or other systemic insults may have a significant impact on post-operative complications and recovery in the setting of a local or global hepatic I/R injury.     

Pharmacological characterization of Sch527123, a potent allosteric CXCR1/CXCR2 antagonist

In neutrophils, growth-related protein-alpha (CXCL1) and interleukin-8 (CXCL8), are potent chemoattractants (Cytokine 14:27-36, 2001; Biochemistry 42:2874-2886, 2003) and can stimulate myeloperoxidase release via activation of the G protein-coupled receptors CXCR1 and CXCR2. The role of CXCR1 and CXCR2 in the pathogenesis of inflammatory responses has encouraged the development of small molecule antagonists for these receptors. The data presented herein describe the pharmacology of 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide (Sch527123), a novel antagonist of both CXCR1 and CXCR2. Sch527123 inhibited chemokine binding to (and activation of) these receptors in an insurmountable manner and, as such, is categorized as an allosteric antagonist. Sch527123 inhibited neutrophil chemotaxis and myeloperoxidase release in response to CXCL1 and CXCL8 but had no effect on the response of these cells to C5a or formyl-methionyl-leucyl-phenylalanine. The pharmacological specificity of Sch527123 was confirmed by testing in a diversity profile against a panel of enzymes, channels, and receptors. To measure compound affinity, we characterized [(3)H]Sch527123 in both equilibrium and nonequilibrium binding analyses. Sch527123 binding to CXCR1 and CXCR2 was both saturable and reversible. Although Sch527123 bound to CXCR1 with good affinity (K(d) = 3.9 +/- 0.3 nM), the compound is CXCR2-selective (K(d) = 0.049 +/- 0.004 nM). Taken together, our data show that Sch527123 represents a novel, potent, and specific CXCR2 antagonist with potential therapeutic utility in a variety of inflammatory conditions.     

Keratinocyte growth factor pretreatment is associated with decreased macrophage inflammatory protein-2alpha concentrations and reduced neutrophil recruitment in acid aspiration lung injury

A two-hit model of acid aspiration was used to examine the effect of keratinocyte growth factor (KGF) on chemokine levels and neutrophil recruitment into the lung. Mice were subjected to cecal ligation and puncture and then either KGF or saline, intratracheally (i.t.). Forty-eight hours later, the mice were given i.t. acid. After 8 h, neutrophil counts in bronchoalveolar lavage (BAL) fluid were significantly decreased in animals pretreated with KGF (23 +/- 4 x 10(3)/mouse) compared with saline (74 +/- 2 x 10(3)/mouse). In addition, the BAL fluid IL-6 levels were decreased in the KGF-treated group (88+/- 44 pg/mL) compared with the saline group (166 +/- 34 pg/mL). To examine the mechanism behind the KGF-induced reduction in neutrophil influx, the murine chemokines KC and macrophage inflammatory protein (MIP)-2alpha were measured. KC levels in plasma and BAL fluid were not significantly different between the treatment groups. Likewise, levels of MIP-2alpha in plasma were not affected by KGF treatment. However, 8 h after acid aspiration, MIP-2alpha concentrations were significantly lower in the KGF-treated group. The ratio of MIP-2alpha in BAL fluid versus plasma was lower in the KGF group (0.72 +/- 0.28) than in the saline group at 3 h (2.23 +/- 0.93) and also significantly lower in the KGF group (3.02 +/- 0.78) compared with the saline group (6.23 +/- 1.19) at 8 h. In this study, KGF pretreatment after acid aspiration was associated with reduced neutrophil recruitment into the lung and a decrease in MIP-2alpha gradients between BAL fluid and plasma.     

The CXCR3 targeting chemokine CXCL11 has potent antitumor activity in vivo involving attraction of CD8+ T lymphocytes but not inhibition of angiogenesis

The IFN-gamma-inducible and CXCR3-targeting human CXC chemokines CXCL9 (Mig) and CXCL10 (IP10) have potent antitumor activity through attraction of cytotoxic T lymphocytes and inhibition of angiogenesis. The more recently identified CXCR3-targeting chemokine CXCL11 (I-TAC/IP9) proved to be a more potent chemokine than CXCL9 and CXCL10 in vitro, both in chemotaxis assays with CXCR3+ T lymphocytes and in calcium mobilization experiments. However, its antitumor activity in vivo has not been shown so far. To investigate this, mice were challenged with EL4 T-cell lymphoma cells, genetically modified to produce murine CXCL11. Tumor growth curves showed complete rejection of CXCL11-producing tumors but not of control tumors. Tumor infiltrate analysis by flow cytometry showed a clear correlation between rejection of CXCL11-producing tumors and an increase of tumor-infiltrating CD8+CXCR3+ as well as CD8+CXCR3- T lymphocytes. In vivo CD8 T-cell depletion completely abrogated the antitumor effect. No difference in angiogenesis between control and CXCL11-producing tumors was observed. In survivors, rechallenge experiments with wild-type tumor cells suggested development of protective antitumor immunity involving tumor-specific IFN-gamma production by CD8+ T lymphocytes. These experiments show, for the first time, antitumor activity of CXCL11 in vivo, which warrants exploration for its potential role in anticancer immunotherapy.