Regulation of lipopolysaccharide-induced lung inflammation by plasminogen activator Inhibitor-1 through a JNK-mediated pathway

The neutrophil is of undoubted importance in lung inflammation after exposure to LPS. We have shown recently that systemic inhibition of JNK decreased neutrophil recruitment to the lung after exposure to LPS, although the mechanisms underlying this inhibition are incompletely understood. As plasminogen activator inhibitor-1 (PAI-1) accentuates cell migration, with JNK activation recently shown to up-regulate PAI-1 expression, this suggested that systemic JNK inhibition may down-regulate LPS-induced pulmonary neutrophil recruitment through a decrease in PAI-1 expression. We show in this study that exposure of mice to aerosolized LPS increased PAI-1 expression in the lung and alveolar compartment, which was decreased by pretreatment with the JNK inhibitor SP600125. Exogenous, intratracheally administered PAI-1 prevented the inhibition of pulmonary neutrophil recruitment in the setting of systemic JNK inhibition, thereby suggesting a role for PAI-1 in the JNK-mediated pathway regulating LPS-induced neutrophil recruitment. In addition, PAI-1(-/-) mice had a decrease in neutrophil recruitment to the alveolar compartment after exposure to LPS, compared with wild-type controls, further suggesting a role for PAI-1 in LPS-induced lung inflammation. An increase in the intravascular level of KC is a likely mechanism for the inhibition of pulmonary neutrophil recruitment after LPS exposure in the setting of decreased PAI-1 expression, as systemic KC levels after exposure to LPS were increased in PAI-1-deficient mice and in mice pretreated with SP600125, with augmentation of intravascular KC levels inhibiting neutrophil recruitment to the lung after exposure to LPS.     

IL-17 and TNF-α sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin-dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.     

Species specificity of ADAM10 and ADAM17 proteins in interleukin-6 (IL-6) trans-signaling and novel role of ADAM10 in inducible IL-6 receptor shedding

Hypomorphic ADAM17(ex/ex) mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17(ex/ex) mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.     

Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice

Intratracheal instillation of the monocyte chemoattractant JE/monocyte chemoattractant protein (MCP)-1 in mice was recently shown to cause increased alveolar monocyte accumulation in the absence of lung inflammation, whereas combined JE/MCP-1/lipopolysaccharide (LPS) challenge provoked acute lung inflammation with early alveolar neutrophil and delayed alveolar monocyte influx. We evaluated the role of resident alveolar macrophages (rAM) in these leukocyte recruitment events and related phenomena of lung inflammation. Depletion of rAM by pretreatment of mice with liposomal clodronate did not affect the JE/MCP-1-driven alveolar monocyte accumulation, despite the observation that rAM constitutively expressed the JE/MCP-1 receptor CCR2, as analyzed by flow cytometry and immunohistochemistry. In contrast, depletion of rAM largely suppressed alveolar cytokine release as well as neutrophil and monocyte recruitment profiles upon combined JE/MCP-1/LPS treatment. Despite this strongly attenuated alveolar inflammatory response, increased lung permeability was still observed in rAM-depleted mice undergoing JE/MCP-1/LPS challenge. Lung leakage was abrogated by codepletion of circulating neutrophils or administration of anti-CD18. Collectively, rAM are not involved in JE/MCP-1-driven alveolar monocyte recruitment in noninflamed lungs but largely contribute to the alveolar cytokine response and enhanced early neutrophil and delayed monocyte influx under inflammatory conditions (JE/MCP-1/LPS deposition). Loss of lung barrier function observed under these conditions is rAM independent but involves circulating neutrophils via beta(2)-integrin engagement.     

Differential regulation of neutrophil-activating chemokines by IL-6 and its soluble receptor isoforms

Interleukin-6 signaling via its soluble receptor (sIL-6R) differentially regulates inflammatory chemokine expression and leukocyte apoptosis to coordinate transition from neutrophil to mononuclear cell infiltration. sIL-6R activities may, however, be influenced in vivo by the occurrence of two sIL-6R isoforms that are released as a consequence of differential mRNA splicing (DS) or proteolytic cleavage (PC) of the cognate IL-6R (termed DS- and PC-sIL-6R). Using human peritoneal mesothelial cells and a murine model of peritoneal inflammation, studies described in this work have compared the ability of both isoforms to regulate neutrophil recruitment. In this respect, DS- and PC-sIL-6R were comparable in their activities; however, these studies emphasized that IL-6 trans signaling differentially controls neutrophil-activating CXC chemokine expression. In vitro, stimulation of mesothelial cells with IL-6 in combination with either DS-sIL-6R or PC-sIL-6R showed no induction of CXC chemokine ligand (CXCL)1 (GRO alpha) and CXCL8 (IL-8), whereas both isoforms enhanced CXCL5 (ENA-78) and CXCL6 (granulocyte chemotactic protein-2) expression. Moreover, when complexed with IL-6, both isoforms specifically inhibited the IL-1 beta-induced secretion of CXCL8. These findings were paralleled in vivo, in which induction of peritoneal inflammation in IL-6-deficient (IL-6(-/-)) mice resulted in enhanced keratinocyte-derived chemokine and macrophage-inflammatory protein-2 (the murine equivalent of CXCL1 and CXCL8) levels, but reduced LPS-induced CXC chemokine (the murine equivalent of CXCL5) expression. Reconstitution of IL-6 signaling in IL-6(-/-) mice with IL-6 and its soluble receptor isoforms corrected this chemokine imbalance and suppressed overall neutrophil infiltration. These data confirm that sIL-6R-mediated signaling primarily limits neutrophil influx; however, induction of CXCL5 and CXCL6 may regulate other neutrophil responses.     

ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.     

The guidance receptor neogenin promotes pulmonary inflammation during lung injury

Lung injury is marked by a persistent self-propagating inflammation within the pulmonary tissue that is initiated by the migration of leukocytes into the alveolar space. Recent work has demonstrated that neuronal guidance proteins are involved into the orchestration of leukocyte migration. Neogenin is a crucial guidance receptor for axonal migration, yet its role during leukocyte migration and acute inflammation is to date unknown. Here, we report that neogenin influences neutrophil migration across endothelial HMEC-1 and alveolar A549 monolayers in vitro. In vivo, Neo1(-/-) mice demonstrated 59% reduced cell count, 41% reduced TNF-α, and 76% reduced IL-6 levels within the alveolar space during lung injury. In studies employing chimeric animals, the presence of Neo1(-/-) bone marrow was associated with a 42% reduction of cell count and reduced inflammatory changes within pulmonary tissue during lung injury. The functional inhibition of neogenin through antibody injection confirmed these results and the role of neogenin for the inflammatory changes within the alveolar space. Previously unappreciated, the guidance receptor neogenin has a significant effect on the orchestration of leukocyte migration and the control of acute inflammation.     

Systemic inhibition of the angiotensin-converting enzyme limits lipopolysaccharide-induced lung neutrophil recruitment through both bradykinin and angiotensin II-regulated pathways

Recruitment of neutrophils to the lung is a sentinel event in acute lung inflammation. Identifying mechanisms that regulate neutrophil recruitment to the lung may result in strategies to limit lung damage and improve clinical outcomes. Recently, the renin angiotensin system (RAS) has been shown to regulate neutrophil influx in acute inflammatory models of cardiac, neurologic, and gastrointestinal disease. As a role for the RAS in LPS-induced acute lung inflammation has not been described, we undertook this study to examine the possibility that the RAS regulates neutrophil recruitment to the lung after LPS exposure. Pretreatment of mice with the angiotensin-converting enzyme (ACE) inhibitor enalapril, but not the anti-hypertensive hydralazine, decreased pulmonary neutrophil recruitment after exposure to LPS. We hypothesize that inhibition of LPS-induced neutrophil accumulation to the lung with enalapril occurred through both an increase in bradykinin, and a decrease in angiotensin II (ATII), mediated signaling. Bradykinin receptor blockade reversed the inhibitory effect of enalapril on neutrophil recruitment. Similarly, pretreatment with bradykinin receptor agonists inhibited IL-8-induced neutrophil chemotaxis and LPS-induced neutrophil recruitment to the lung. Inhibition of ATII-mediated signaling, with the ATII receptor 1a inhibitor losartan, decreased LPS-induced pulmonary neutrophil recruitment, and this was suggested to occur through decreased PAI-1 levels. LPS-induced PAI-1 levels were diminished in animals pretreated with losartan and in those deficient for the ATII receptor 1a. Taken together, these results suggest that ACE regulates LPS-induced pulmonary neutrophil recruitment via modulation of both bradykinin- and ATII-mediated pathways, each regulating neutrophil recruitment by separate, but distinct, mechanisms.     

A macrophage subpopulation recruited by CC chemokine ligand-2 clears apoptotic cells in noninfectious lung injury

CC chemokine ligand-2 (CCL2)/monocyte chemoattractant protein (MCP)-1 expression is upregulated during pulmonary inflammation, and the CCL2-CCR2 axis plays a critical role in leukocyte recruitment and promotion of host defense against infection. The role of CCL2 in mediating macrophage subpopulations in the pathobiology of noninfectious lung injury is unknown. The goal of this study was to examine the role of CCL2 in noninfectious acute lung injury. Our results show that lung-specific overexpression of CCL2 protected mice from bleomycin-induced lung injury, characterized by significantly reduced mortality, reduced neutrophil accumulation, and decreased accumulation of the inflammatory mediators IL-6, CXCL2 (macrophage inflammatory protein-2), and CXCL1 (keratinocyte-derived chemokine). There were dramatic increases in the recruitment of myosin heavy chain (MHC) II IA/IE(int)CD11c(int) cells, exudative macrophages, and dendritic cells in Ccl2 transgenic mouse lungs both at baseline and after bleomycin treatment compared with levels in wild-type mice. We further demonstrated that MHCII IA/IE(int)CD11c(int) cells engulfed apoptotic cells during acute lung injury. Our data suggested a previously undiscovered role for MHCII IA/IE(int)CD11c(int) cells in apoptotic cell clearance and inflammation resolution.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses

Leukocyte migration into the epithelial compartment is an important feature in the active phase of mycobacterial infections. In this study, we used the Transwell model to investigate the mechanisms behind mycobacteria-induced leukocyte recruitment and investigated the role of TLR2 and TLR4 in this process. Infection of epithelial cells resulted in significantly increased secretion of the neutrophil chemotactic CXCL8 and IL-6, but no secretion of monocyte chemotactic CCL2 or TNF-α was observed. In contrast to epithelial response, mycobacteria-infected neutrophils and monocytes secreted all these cytokines. Corresponding with epithelial cytokine response, mycobacterial infection of the epithelial cells increased neutrophil diapedesis, but decreased monocyte recruitment. However, monocyte recruitment towards mycobacteria infected epithelial cells significantly increased following addition of neutrophil pre-conditioned medium. Mycobacterial infection also increases alveolar epithelial expression of TLR2, but not TLR4, as analyzed by flow cytometry, Western blotting and visualized by confocal microscopy. Blocking of TLR2 inhibited neutrophil recruitment and cytokine secretion, while blocking of TLR4 had a lesser effect. To summarize, we found that primary alveolar epithelial cells produced a selective TLR2-dependent cytokine secretion upon mycobacterial infection. Furthermore, we found that cooperation between cells of the innate immunity is required in mounting proper antimicrobial defence.     

Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis

Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses. We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic. Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2(+) alveolar macrophage population, and CCR2-deficient mice transfused with CCR2(+) mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin. Our data show that alveolar monocyte recruitment is strictly dependent on CCR2. LPS-induced neutrophil migration to the lungs is CCR2 independent. However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified. We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation.     

Both hemopoietic and resident cells are required for MyD88-dependent pulmonary inflammatory response to inhaled endotoxin

Inhaled endotoxin induces an inflammatory response that contributes to the development and severity of asthma and other forms of airway disease. Here, we show that inhaled endotoxin-induced acute bronchoconstriction, TNF, IL-12p40, and KC production, protein leak, and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecule MyD88. Bronchoconstriction, inflammation, and protein leak are normal in Toll/IL-1R domain-containing adaptor inducing IFN-beta-deficient mice. MyD88 is involved in TLR, but also in IL-1R-associated kinase 1-mediated IL-1R and -18R signaling. We exclude a role for IL-1 and IL-18 pathways in this response, as IL-1R1 and caspase-1 (ICE)-deficient mice develop lung inflammation while TLR4-deficient mice are unresponsive to inhaled LPS. Significantly, using bone marrow chimera, we demonstrate that both hemopoietic and resident cells are necessary for a full MyD88-dependent response to inhaled endotoxin; bronchoconstriction depends on resident cells while cytokine secretion is mediated by hemopoietic cells.     

Death receptors mediate the adverse effects of febrile-range hyperthermia on the outcome of lipopolysaccharide-induced lung injury

We have shown that febrile-range hyperthermia enhances lung injury and mortality in mice exposed to inhaled LPS and is associated with increased TNF-α receptor activity, suppression of NF-κB activity in vitro, and increased apoptosis of alveolar epithelial cells in vivo. We hypothesized that hyperthermia enhances lung injury and mortality in vivo by a mechanism dependent on TNF receptor signaling. To test this, we exposed mice lacking the TNF-receptor family members TNFR1/R2 or Fas (TNFR1/R2(-/-) and lpr) to inhaled LPS with or without febrile-range hyperthermia. For comparison, we studied mice lacking IL-1 receptor activity (IL-1R(-/-)) to determine the role of inflammation on the effect of hyperthermia in vivo. TNFR1/R2(-/-) and lpr mice were protected from augmented alveolar permeability and mortality associated with hyperthermia, whereas IL-1R(-/-) mice were susceptible to augmented alveolar permeability but protected from mortality associated with hyperthermia. Hyperthermia decreased pulmonary concentrations of TNF-α and keratinocyte-derived chemokine after LPS in C57BL/6 mice and did not affect pulmonary inflammation but enhanced circulating markers of oxidative injury and nitric oxide metabolites. The data suggest that hyperthermia enhances lung injury by a mechanism that requires death receptor activity and is not directly associated with changes in inflammation mediated by hyperthermia. In addition, hyperthermia appears to enhance mortality by generating a systemic inflammatory response and not by a mechanism directly associated with respiratory failure. Finally, we observed that exposure to febrile-range hyperthermia converts a modest, survivable model of lung injury into a fatal syndrome associated with oxidative and nitrosative stress, similar to the systemic inflammatory response syndrome.     

Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute lung injury (ALI), these data provide further evidence that AKI may cause ALI.     

IL-23-dependent IL-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory Mycoplasma pneumoniae infection

IL-23 induces IL-17 production in activated CD4+ T cells and participates in host defense against many encapsulated bacteria. However, whether the IL-23/IL-17 axis contributes to a Mycoplasma pneumoniae (Mp)-induced lung inflammation (e.g., neutrophils) has not been addressed. Using an acute respiratory Mp infection murine model, we found significantly up-regulated lung IL-23p19 mRNA in the early phase of infection (4h), and alveolar macrophages were an important cell source of Mp-induced IL-23. We further showed that Mp significantly increased IL-17 protein levels in bronchoalveolar lavage (BAL). Lung gene expression of IL-17, IL-17C and IL-17F was also markedly up-regulated by Mp in vivo. IL-17 and IL-17F were found to be derived mainly from lung CD4+ T cells, and were increased upon IL-23 stimulation in vitro. In vivo blocking of IL-23p19 alone or in combination with IL-23/IL-12p40 resulted in a significant reduction of Mp-induced IL-17 protein and IL-17/IL-17F mRNA expression, which was accompanied by a trend toward reduced lung neutrophil recruitment, BAL neutrophil activity, and Mp clearance. However, IL-23 neutralization had no effect on Mp-induced lung IL-17C mRNA expression. These results demonstrate that IL-17/IL-17F production is IL-23-dependent in an acute Mp infection, and contributes to neutrophil recruitment and activity in the lung defense against the infection.     

Lung injury during LPS-induced inflammation occurs independently of the receptor P2Y<Subscript>1</Subscript>

Disruption of the lung endothelial and epithelial barriers during acute inflammation leads to excessive neutrophil migration. It is likely that activated platelets promote pulmonary recruitment of neutrophils during inflammation, and previous studies have found that anti-platelet therapy and depletion of circulating platelets have lung-protective effects in different models of inflammation. Because ADP signaling is important for platelet activation, I investigated the role of the ADP-receptor P2Y₁, a G protein-coupled receptor expressed on the surface of circulating platelets, during lipopolysaccharide (LPS)-induced inflammation and lung injury in P2Y₁-null and wild-type mice. Systemic inflammation was induced by a single intraperitoneal dose of LPS (3 mg/kg), and the mice were analyzed 24 h posttreatment. The data show that the LPS-induced inflammation levels were comparable in the P2Y₁-null and wild-type mice. Specifically, splenomegaly, counts of circulating platelets and white blood cells (lymphocytes and neutrophils), and assessments of lung injury (tissue architecture and cell infiltration) were similar in the P2Y₁-null and wild-type mice. Based on my results, I conclude that lung injury during LPS-induced inflammation in mice is independent of P2Y₁ signaling. I propose that if a blockade of purinergic signaling in platelets is a potential lung-protective strategy in the treatment of acute inflammation, then it is more likely to be a result of the disruption of the signaling pathway mediated by P2Y₁₂, another G protein-coupled receptor that mediates platelet responses to ADP.     

IL-17, produced by lymphocytes and neutrophils,is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4(+) and CD8(+) cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4(+) cells, but not spleen CD8(+) or airway neutrophils. We have shown that IL-17, produced mainly by CD4(+) cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.     

Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8

Interleukin (IL)-8 from pulmonary epithelial cells has been suggested to play an important role in the airway inflammation, although the mechanism remains unclear. We envisioned a possibility that pulmonary epithelial CCR3 could be involved in secretion and regulation of IL-8 and promote lipopolysaccharide (LPS)-induced lung inflammation. Human bronchial epithelial cell line NCI-H292 and alveolar type II epithelial cell line A549 were used to test role of CCR3 in production of IL-8 at cellular level. In vivo studies were performed on C57/BL6 mice instilled intratracheally with LPS in a model of acute lung injury (ALI). The activity of a CCR3-specific inhibitor (SB-328437) was measured in both in vitro and in vivo systems. We found that expression of CCR3 in NCI-H292 and A549 cells were increased by 23% and 16%, respectively, 24 h after the challenge with LPS. LPS increased the expression of CCR3 in NCI-H292 and A549 cells in a time-dependent manner, which was inhibited significantly by SB-328437. SB-328437 also diminished neutrophil recruitment in alveolar airspaces and improved LPS-induced ALI and production of IL-8 in bronchoalveolar lavage fluid. These results suggest that pulmonary epithelial CCR3 be involved in progression of LPS-induced lung inflammation by mediating release of IL-8. CCR3 in pulmonary epithelia may be an attractive target for development of therapies for ALI.