CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice

Polymorphonuclear neutrophil (PMN) extravasation/sequestration in the lung and a dysregulated inflammatory response characterize the pathogenesis of acute lung injury (ALI). Previously, we have shown that hemorrhage (Hem) serves to prime PMN such that subsequent septic challenge [cecal ligation and puncture (CLP)] produces a pathological, inflammatory response and consequent lung injury in mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) are murine CXC chemokines found elevated in the lungs and plasma following Hem/CLP and have been reported by others to share a common receptor (CXCR2). Based on these data, we hypothesize that blockade of CXCR2 immediately following Hem would suppress KC and MIP-2 priming of PMN, thereby reducing the inflammatory injury observed following CLP. To assess this, Hem mice (90 min at 35+/-5 mmHg) were randomized to receive 0, 0.4, or 1 mg antileukinate (a hexapeptide inhibitor of CXCRs) in 100 microl phosphate-bufferd saline (PBS)/mouse subcutaneously, immediately following resuscitation (Ringer's lactate-4x drawn blood volume). Twenty-four hours post-Hem, mice were subjected to CLP and killed 24 h later. The results show that blockade of CXCR2 significantly (P<0.05, Tukey's test) reduced PMN influx, lung protein leak, and lung-tissue content of interleukin (IL)-6, KC, and MIP-2 and increased tissue IL-10 levels. Plasma IL-6 was significantly decreased, and IL-10 levels increased in a dose-dependent manner compared with PBS-treated mice. A differential effect was observed in plasma levels of KC and MIP-2. KC showed a significant reduction at the 0.4 mg antileukinate dose. In contrast, plasma MIP-2 was significantly elevated at both doses compared with the PBS-treated controls. Together, these data demonstrate that blockade of CXCR2 signaling attenuates shock-induced priming and ALI observed following Hem and subsequent septic challenge in mice.     

Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency

Acute lung injury (ALI) leading to respiratory distress is a common sequela of shock/trauma, however, modeling this process in mice with a single shock or septic event is inconsistent. One explanation is that hemorrhage is often just a "priming insult," thus, secondary stimuli may be required to "trigger" ALI. To test this we carried out studies in which we assessed the capacity of hemorrhage alone or hemorrhage followed by septic challenge (CLP) to induce ALI. Lung edema, bronchoalveolar lavage interleukin (IL)-6, alveolar congestion, as well as lung IL-6, macrophage inflammatory protein (MIP)-2, and myeloperoxidase (MPO) activity were all increased in mice subjected to CLP at 24 but not 72 hours following hemorrhage. This was associated with a marked increase in the susceptibility of these mice to septic mortality. Peripheral blood neutrophils derived from 24 hours post-hemorrhage, but not Sham animals, exhibited an ex vivo decrease in apoptotic frequency and an increase in respiratory burst capacity, consistent with in vivo "priming." Subsequently, we observed that adoptive transfer of neutrophils from hemorrhaged but not sham-hemorrhage animals to neutropenic recipients reproduce ALI when subsequently septically challenged, implying that this priming was mediated by neutrophils. We also found marked general increases in lung IL-6, MIP-2, and MPO in mice deficient for toll-like receptor (TLR-4) or the combined lack of TLR-4/FasL. However, the TLR-4 defect markedly attenuated neutrophil influx into the lung while not altering the change in local cytokine/chemokine expression. Alternatively, the combined loss of FasL and TLR-4 did not inhibit the increase in MPO and exacerbated lung IL-6/MIP-2 levels even further.     

Calcitonin gene-related peptide partially reverses decreased production of chemokines KC and MIP-2 following murine sepsis

The secretion of calcitonin gene-related peptide (CGRP) and the chemokines KC and MIP-2 are increased in the animal models of endotoxemic and septic shock. We tested whether CGRP could modulate KC and MIP-2 secretion from different sources of macrophages after murine sepsis induced by cecal ligation and puncture (CLP). Macrophages were obtained from the peritoneal exudate and lung of female BALB/c mice 16 h after CLP and plated in culture with CGRP and/or LPS for 12 h. The results showed that peritoneal macrophage production of the chemokines (KC, MIP-2) and cytokines (TNF-, IL-6) was markedly decreased in CLP mice. Alveolar macrophages did not display decreased cytokine/chemokines production after CLP. CGRP (0.1 nM–10 nM) partially reversed this decreased production of LPS-induced KC and MIP-2 from peritoneal macrophages. These results suggest that CGRP might be intimately involved in recruitment of neutrophils by promoting local production of the chemokines KC and MIP-2 in murine sepsis.     

Novel chemokine responsiveness and mobilization of neutrophils during sepsis

Blood neutrophils (PMN) are usually unresponsive to CC chemokines such as monacyte chemotactic protein-1 and macrophage inflammatory protein-1 alpha. In rodents, the lung buildup of PMN as determined by myeloperoxidase (MPO) activity after airway instillation of bacterial lipopolysaccharide (LPS) was independent of MCP-1 and MIP-1 alpha. In striking contrast, during sepsis following cecal ligation and puncture (CLP), blood PMN demonstrated mRNA for CC chemokine receptors. Furthermore, PMN from CLP, but not from sham rodents, bound MCP-1 and MIP-1 alpha and responded chemotactically in vitro to both MCP-1 and MIP-1 alpha. In CCR2(-/-) mice or WT mice treated in vivo with antibodies to either MCP-1 or MIP-1 alpha, MPO activity was greatly attenuated in CLP animals. In CLP mice, increased serum IL-6 levels were found to be dependent on CCR2, MCP-1, and MIP-1 alpha. When PMN from CLP rodents were incubated in vitro with either MCP-1 or MIP-1 alpha, release of IL-6 was also shown. These findings suggest that sepsis fundamentally alters the trafficking of PMN into the lung in a manner that now engages functional responses to CC chemokines.     

Critical role of CXC chemokines in endotoxemic liver injury in mice

Tissue accumulation of leukocytes constitutes a rate-limiting step in endotoxin-induced tissue injury. Chemokines have the capacity to regulate leukocyte trafficking. However, the role of CXC chemokines, i.e., macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), in leukocyte recruitment, microvascular perfusion failure, cellular injury, and apoptosis in the liver remains elusive. Herein, mice were challenged with lipopolysaccharide (LPS) in combination with D-galactosamine, and intravital microscopy of the liver microcirculation was conducted 6 h later. It was found that immunoneutralization of MIP-2 and KC did not reduce LPS-induced leukocyte rolling and adhesion in postsinusoidal venules. In contrast, pretreatment with monoclonal antibodies against MIP-2 and KC abolished (83% reduction) extravascular recruitment of leukocytes in the livers of endotoxemic mice. Notably, endotoxin challenge increased the expression of CXC chemokines, which was mainly confined to hepatocytes. Moreover, endotoxin-induced increases of liver enzymes and hepatocellular apoptosis were decreased by more than 82% and 68%, respectively, and sinusoidal perfusion was restored in mice passively immunized against MIP-2 and KC. In conclusion, this study indicates that intravascular accumulation of leukocytes in the liver is independent of CXC chemokines in endotoxemic mice. Instead, our novel data suggest that CXC chemokines are instrumental in regulating endotoxin-induced transmigration and extravascular tissue accumulation of leukocytes. Indeed, these findings demonstrate that interference with MIP-2 and KC functions protects against septic liver damage and may constitute a potential therapeutic strategy to control pathological inflammation in endotoxemia.     

The Role of Keratinocyte-derived Chemokine in Hemorrhage-induced Acute Lung Injury in Mice

Dominant inflammatory cytokines might be different depending on the underlying causes of acute lung injury (ALI). The role of kertinocyte-derived chemokine (KC), a potent chemoattractant for neutrophils, has not been clearly established in hemorrhage-induced ALI. In this study, lung injury and cytokine expressison were evaluated in LPS- or hemorrhage-induced ALI models of BALB/c mice. The myeloperoxidase activities at 4 hr after hemorrhage and LPS-injection were 47.4±13.0 and 56.5±16.4 U/g, respectively. NF-κB activity peaked at 4 hr after hemorrhage, which was suppressed to the control level by anti-high mobility group B1 (HMGB1) antibody. Lung expressions of TNF-α, MIP-2, and IL-1β were increased by LPS injection. However, there was only a minimal increase in IL-1β and no expressions of TNF-α or MIP-2 in hemorrhage-induced ALI. In contrast, lung KC increased significantly at 4 hr after hemorrhage compared to control levels (83.1±12.3 vs. 14.2±1.6 pg/mL/mg by ELISA) (P<0.05). By immunohistochemical staining, lung neutrophils stained positive for KC. Increased KC was also observed in bronchoalveolar lavage fluid and plasma. KC plays an important role in hemorrhage-induced ALI.     

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.     

Fingerprinting of the TLR4-induced acute inflammatory response

Intensive scientific efforts in the past decades have helped shed light into the pathogenesis of endotoxin-induced inflammation. We have used multiplexing bead-based assays to characterize the responses in two models of in vivo LPS challenge. C57BL/6 mice were either injected intraperitoneally (endotoxemia) or intratracheally (acute lung injury; ALI) with lipopolysaccharide (LPS). The time courses (1 h–24 h) of the following 20 inflammatory mediators in plasma or broncho-alveolar lavages were simultaneously analyzed: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-12(p40), IL-13, Eotaxin (CCL11), G-CSF, GM-CSF, IFN-γ, KC (CXCL1), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5) and TNF-α. While significant inductions of all mediators were found, substantial differences in their absolute concentrations, time points of maximal concentrations and clearances were observed. There were also notable variations in the patterns of several cytokines/chemokines when samples from endotoxemia and LPS-ALI were compared. These data may be helpful in defining analytic strategies including selection of optimal time points for studying the host immune response to endotoxin.     

Differential contribution of bacterial N-formyl-methionyl-leucyl- phenylalanine and host-derived CXC chemokines to neutrophil infiltration into pulmonary alveoli during murine pneumococcal pneumonia

Despite the development of new potent antibiotics, Streptococcus pneumoniae remains the leading cause of death from bacterial pneumonia. Polymorphonuclear neutrophil (PMN) recruitment into the lungs is a primordial step towards host survival. Bacterium-derived N-formyl peptides (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) and host-derived chemokines (KC and macrophage inflammatory protein 2 [MIP-2]) are likely candidates among chemoattractants to coordinate PMN infiltration into alveolar spaces. To investigate the contribution of each in the context of pneumococcal pneumonia, CD1, BALB/c, CBA/ca, C57BL/6, and formyl peptide receptor (FPR)-knockout C57BL/6 mice were infected with 10(6) or 10(7) CFU of penicillin/erythromycin-susceptible or -resistant serotype 3 or 14 S. pneumoniae strains. Antagonists to the FPR, such as cyclosporine H (CsH) and chenodeoxycholic acid, or neutralizing antibodies to KC and MIP-2 were injected either 1 h before or 30 min after infection, and then bronchoalveolar lavage fluids were obtained for quantification of bacteria, leukocytes, and chemokines. CsH was effective over a short period after infection with a high inoculum, while anti-CXC chemokine antibodies were effective after challenge with a low inoculum. CsH prevented PMN infiltration in CD1 mice infected with either serotype 3 or 14, whereas antichemokine antibodies showed better efficacy against the serotype 3 strain. When different mouse strains were challenged with serotype 3 bacteria, CsH prevented PMN migration in the CD1 mice only, whereas the antibodies were effective against CD1 and C57BL/6 mice. Our results suggest that fMLP and chemokines play important roles in pneumococcal pneumonia and that these roles vary according to bacterial and host genetic backgrounds, implying redundancy among chemoattractant molecules.     

CXC Chemokine Redundancy Ensures Local Neutrophil Recruitment during Acute Inflammation

Previous publications demonstrated that elevated systemic levels of interleukin (IL)-8 decrease local neutrophil recruitment. We tested whether sustained, high plasma levels of IL-8 would prevent local inflammation after inflammatory insults. Mice carrying the transgene for human IL-8 were separated on the basis of their plasma levels of IL-8 into IL-8-positive (plasma levels >90 ng/ml) and IL-8-negative (IL-8 below detection). Presence of the IL-8 transgene did not improve survival or morbidity nor did it alter peritoneal neutrophil recruitment induced by the cecal ligation and puncture model of sepsis. In an acute lung injury model created by intratracheal injection of acid, IL-8-positive mice showed no reduction in alveolar neutrophil recruitment. There was no difference in the local recruitment of neutrophils when either thioglycollate or glycogen was injected intraperitoneally. We examined the chemotactic response to murine chemokines to test how neutrophil recruitment occurs in the setting of elevated plasma IL-8 and found that neutrophils from both IL-8-positive and -negative mice respond equally well to recombinant KC or macrophage inflammatory protein (MIP)-2. We measured KC and MIP-2 in the peritoneum after thioglycollate injection and demonstrated that IL-8-positive mice have significantly higher levels of the chemokines compared to the IL-8-negative mice. Antibody inhibition of KC and MIP-2 in the IL-8-positive mice significantly decreased peritoneal neutrophil recruitment in response to thioglycollate, clarifying their important role in the local neutrophil recruitment. Our data demonstrate that despite the presence of high plasma levels of IL-8, neutrophils may still be recruited to sites of local inflammation because of chemokine redundancy.     

CXCL1/KC and CXCL5/LIX are selectively produced by corneal fibroblasts and mediate neutrophil infiltration to the corneal stroma in LPS keratitis

The severity of corneal inflammation depends on the activity of infiltrating neutrophils responding to chemotactic factors such as CXC chemokines. This study examines the relative contribution of CXCL1/keratinocyte-derived chemokine (KC), CXCL2/monocyte-inhibitory protein-2 (MIP-2), and CXCL5/LPS-induced chemokine (LIX) in neutrophil recruitment to the corneal stroma during LPS keratitis, where neutrophils infiltrate the corneal stroma at 6 h after LPS injection and peak at 24 h. Consistent with this timeframe, KC was detected after 3 h, reached peak levels at 24 h, and decreased thereafter. In contrast, LIX production was not detected until 8 h after injection and peaked at 24 h. MIP-2 was detected at 3 h but did not reach the levels of KC and LIX. Cell types associated with corneal inflammation produced markedly different chemokines in vitro: Murine corneal fibroblasts (MK/T-1) produced LIX and KC in response to LPS but did not produce MIP-2, whereas peritoneal macrophages and neutrophils produced MIP-2 and KC but did not produce LIX. To determine the role of these chemokines in neutrophil recruitment to the cornea, anti-LIX, anti-KC, or anti-MIP-2 was injected into the corneal stroma of enhanced GFP chimeric mice prior to LPS, and total cell and neutrophil infiltration was examined. Antibody to LIX and KC, injected individually or in combination, significantly inhibited neutrophil recruitment to the cornea, whereas anti-MIP-2 had no inhibitory effect. Together, these findings demonstrate cell-specific production of CXC chemokines and show that LIX and KC mediate neutrophil recruitment into the cornea during LPS keratitis.     

Neutrophil Sequestration in Liver and Lung is Differentially Regulated by C-X-C Chemokines During Experimental Peritonitis

C-X-C chemokines play an important role in the migration and activation of neutrophils (PMNs) during an inflammatory event. We measured mRNA and protein expression of the murine C-X-C chemokines macrophage inflammatory protein-2 (MIP-2) and KC in the lungs, liver, blood, and peritoneal cavity of Swiss Webster mice after cecal ligation and puncture (CLP). Neutralizing antibodies to MIP-2 and KC were also used to determine the biological effects of these chemokines on neutrophil sequestration and organ injury in the CLP model. The data showed that early after CLP, MIP-2 mRNA and protein were expressed predominantly by the lung, whereas KC mRNA and protein were expressed by the liver. Inhibition of MIP-2 reduced both lung neutrophil sequestration and peritoneal neutrophil migration. Inhibition of KC had no effect on overall neutrophil sequestration in liver but reduced injury as measured by serum transaminases. An early survival benefit was found with anti-KC treatment, although overall survival was not different. Our study showed a differential expression by organs of C-X-C chemokines during sepsis and suggested that such chemokine effects are tissue-specific.     

Expression profiles of cytokines and chemokines in murine MDR1a-/- colitis

Objective: MDR1a-/- mice spontaneously develop colitis as the result of imperfect epithelial barrier derived from MDR1a deficiency in the large intestine; however, the pathogenesis is not well understood. This study investigated the expression profiles of cytokines and chemokines in murine MDR1a-/- colitis. Methods: MDR1a-/- and wild-type FVB mice were monitored from the 6^th to the 16^th week of age. Production of various cytokines and chemokines in the large intestine and mesenteric lymph node (MLN) cells was examined. Results: Inflammatory cell infiltration, IL-1β production, and MPO activity were aberrantly enhanced in the tissue of MDR1a-/- mice. Under various stimuli, MLN cells produced higher levels of Th1-type (IFN-γ, IL-2, and IL-12) and proinflammatory (IL-1β and TNF-α) cytokines. Inflammatory chemokines MIP-2/CXCL2, KC/CXCL1, MIP-1α/CCL3, MCP-1/CCL2, and RANTES/CCL5 were also markedly upregulated in the tissue. Conclusion: Since the expression profiles of cytokines and chemokines correspond well with those in human IBD, MDR1a-/- mouse is a useful model for the analysis of IBD pathophysiology. Received 12 May 2006; returned for revision vision 15 July 2006; accepted by I. Ahnfelt-R?nne 18 January 2007     

肠系膜淋巴管结扎对大鼠急性肺损伤的影响

目的：探讨结扎肠系膜淋巴管对失血-脂多糖（LPS）致大鼠急性肺损伤（ALI）的拮抗作用。方法：雄性Wistar大鼠45只，均分为结扎组、未结扎组、假手术组，以失血、LPS复制二次打击模型，结扎组行肠系膜淋巴管结扎术致肠淋巴液断流。在手术创伤后24 h，所有大鼠颈总动脉放血，进行血气分析；从左肺收集支气管肺泡灌洗液（BALF），观察WBC、NO及其合酶、SOD、MDA以及肺泡通透性指数等指标的水平；右肺制备10%组织匀浆，检测MPO、ATPase活性等指标；观察右肺后叶超微结构。结果：二次打击后，未结扎组动脉血PaCO2、BALF中细胞总数及PMN、NO2-/NO3-、NOS、MDA含量以及肺匀浆MPO活性、肺通透性指数均显著高于假手术组，动脉血pH、PaO2、BALF中SOD、肺匀浆ATPase活性显著低于假手术组（P<0.01，P<0.05）；结扎组大鼠BALF中细胞总数及PMN、MDA、NO2-/NO3-含量、肺通透性指数均显著高于假手术组，BALF中SOD活性显著低于假手术组（P<0.01，P<0.05）。但结扎组大鼠动脉血pH、PaO2、肺匀浆ATPase活性显著高于未结扎组，动脉血PaCO2、BALF中细胞总数及PMN、NO2-/NO3-、NOS、MDA含量、肺通透性指数及肺匀浆MPO显著低于未结扎组（P<0.01，P<0.05）；且肺血管内皮细胞损伤程度较未结扎组轻微。结论：肠系膜淋巴管结扎可减轻失血-LPS致大鼠的急性肺损伤。提示二次打击的肠系膜淋巴液在大鼠急性肺损伤的发病过程中发挥着重要作用。     

The role of CXC chemokine receptor 2 in Pseudomonas aeruginosa corneal 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.     

Role of inducible nitric oxide synthase in the regulation of neutrophil migration in zymosan-induced inflammation.

In the present study, by comparing the responses in wild-type mice and mice lacking the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the role played by iNOS in the regulation of polymorphonuclear granulocyte (PMN) accumulation and chemokine production in the mouse peritoneal cavity in response to administration of zymosan (0.2 mg). Zymosan injection induced the production of nitric oxide, and triggered a time-dependent PMN immigration into the peritoneal cavity. This response was associated with increases in the level of the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-2, monocyte chemo-attractant protein (MCP)-1 and cytokine-induced neutrophil chemo-attractant (KC), as measured in the peritoneal cavities. Injection of zymosan also induced a time-dependent increase in the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in the peritoneal cavity. When comparing the response between wild-type and iNOS knockout (KO) mice, we observed that the low-level PMN accumulation measured at 1 hr was slightly but significantly increased in the absence of functional iNOS. On the other hand, the delayed response (2-4 hr after zymosan) of PMN accumulation was suppressed in the iNOS KO mice. The early enhancement of PMN infiltration in the iNOS-deficient mice was associated with increased peritoneal levels of MIP-2, KC and IL-10 proteins. The delayed suppression of PMN infiltration was associated with reduced MIP-2 and IL-10 levels in the peritoneal cavity. The lack of iNOS did not affect the release of MIP-1alpha and MCP-1 at any of the time-points studied. The current data demonstrate that iNOS regulates the production of certain CXC (but not CC) proinflammatory chemokines, the production of IL-10 and exerts a biphasic regulatory effect on PMN accumulation in zymosan-induced acute inflammation.     

Regulatory effects of iNOS on acute lung inflammatory responses in mice

The role of endogenous NO in the regulation of acute lung injury is not well defined. We investigated the effects of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the acute inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) into wild-type (WT) mice and mice deficient in iNOS (iNOS(-/-)) or eNOS (eNOS(-/-)). Endpoints of inflammatory injury were myeloperoxidase (MPO) content and leak of albumin into lung. Inflammatory injury was similar in WT and eNOS(-/-) mice but was substantially increased in iNOS(-/-) mice. Bronchoalveolar lavage (BAL) fluids of iNOS(-/-) and WT mice showed similar levels of CXC chemokines (MIP-2, KC) but enhanced levels of CC chemokines (MCP-1, MCP-3). Increased lung content of MPO in iNOS(-/-) mice was reduced by anti-MCP-1 to values found in WT mice. In vitro stimulation of microvascular endothelial cells with LPS and IFN gamma revealed elevated production of CXC and CC chemokines in cells from iNOS(-/-) mice when compared to endothelial cells from iNOS(+/+) mice. Peritoneal macrophages from iNOS(-/-) donors also revealed increased production of CC chemokines after stimulation with LPS and interferon (IFN gamma). These data indicate that absence of iNOS causes enhanced lung inflammatory responses in mice which may be related to enhanced production of MCP-1 by endothelial cells and macrophages. It appears that iNOS affects the lung inflammatory response by regulating chemokine production.     

CXC chemokine receptor CXCR2 is essential for protective innate host response in murine Pseudomonas aeruginosa pneumonia

Pulmonary infection due to Pseudomonas aeruginosa has emerged as a leading cause of mortality. A vigorous host response is required to effectively clear the organisms from the lungs. This host defense is dependent on the recruitment and activation of neutrophils and macrophages. A family of chemotactic cytokines (chemokines) has been shown to participate in this protective response. In this study, we assessed the role of the ELR(+) (glutamic acid-leucine-arginine motif positive) CXC chemokines and their CXC chemokine receptor (CXCR2) in lung antibacterial host defense. The intratracheal administration of Pseudomonas to mice resulted in the time-dependent influx of neutrophils to the lung, peaking at 12 to 24 h after inoculation. The influx of neutrophils was associated with a similar time-dependent expression of the ELR(+) CXC chemokines, KC, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Selective neutralization of MIP-2 or KC resulted in modest changes in neutrophil influx but no change in bacterial clearance or survival. However, neutralization of CXCR2 resulted in a striking increase in mortality, which was associated with a marked decrease in neutrophil recruitment and bacterial clearance. Conversely, the site-specific transgenic expression of KC resulted in enhanced clearance of bacteria after Pseudomonas challenge. This study indicates that ELR(+) CXC chemokines are critical mediators of neutrophil-mediated host defense in Pseudomonas pneumonia.     

Staphylococcal enterotoxin B-induced acute inflammation is inhibited by dexamethasone: important role of CXC chemokines KC and macrophage inflammatory protein 2

This study was conducted to examine the anti-inflammatory mechanisms of dexamethasone during leukocyte recruitment and expression of the CXC chemokines macrophage inflammatory protein 2 (MIP-2) (CXCL2) and cytokine-induced neutrophil chemoattractant (KC) (CXCL1) in staphylococcal enterotoxin B (SEB)-induced acute inflammation. To do this, SEB was injected into murine air pouches with or without dexamethasone pretreatment for 2 h. SEB induced infiltration of leukocytes in a dose- and time-dependent manner, with the maximal response observed after 4 h of treatment with 10 microg of SEB. The recruited leukocytes comprised more than 77% neutrophils. Moreover, SEB challenge (10 microg) provoked time-dependent secretion of CXC chemokines, which peaked after 1 h. Local administration of antibodies against MIP-2 and KC significantly reduced SEB-triggered neutrophil accumulation by 38 and 59%, respectively. Dexamethasone (10 mg kg(-1)) significantly decreased neutrophil recruitment by 82% and reduced secretion of MIP-2 and KC by 89 and 85%, respectively, in response to SEB challenge. Our data demonstrate that dexamethasone potently inhibits neutrophil recruitment in SEB-induced inflammation. Moreover, we provide evidence that MIP-2 and KC are key mediators in the neutrophil response to SEB. Furthermore, our findings demonstrate that dexamethasone attenuates SEB-induced expression of MIP-2 and KC. Thus, this study elucidates important signaling pathways of SEB-induced neutrophil recruitment and anti-inflammatory mechanisms of action of dexamethasone.