Lee J. Quinton

Cutting Edge: Roles of Toll-Like Receptor 4 and IL-23 in IL-17 Expression in Response to Klebsiella pneumoniae Infection

Local production of IL-17 is a significant factor in effective host defense against Gram-negative bacteria. However, the proximal events mediating IL-17 elaboration by T cells remain unclear. In this study, we show in vivo that intact Toll-like receptor 4 signaling in the lung is required for induction of both the p19 transcript of IL-23 and IL-17 protein elaboration in response to Klebsiella pneumoniae. Although IL-17 is widely considered a CD4+ T cell product, we also demonstrate significant in vitro IL-17 production by CD8+ T cells after culture in medium from dendritic cells exposed to these bacteria. The dominant portion of this IL-17-inducing activity for both CD4+ and CD8+ T cells is IL-23. These data demonstrate the critical signaling pathway for IL-17 induction in the host response to Gram-negative pulmonary infection and suggest a direct role for IL-23 in CD8+ T cell IL-17 production.

Functions and Regulation of NF-κB RelA during Pneumococcal Pneumonia

Eradication of bacteria in the lower respiratory tract depends on the coordinated expression of proinflammatory cytokines and consequent neutrophilic inflammation. To determine the roles of the NF-κB subunit RelA in facilitating these events, we infected RelA-deficient mice (generated on a TNFR1-deficient background) with Streptococcus pneumoniae. RelA deficiency decreased cytokine expression, alveolar neutrophil emigration, and lung bacterial killing. S. pneumoniae killing was also diminished in the lungs of mice expressing a dominant-negative form of IκBα in airway epithelial cells, implicating this cell type as an important locus of NF-κB activation during pneumonia. To study mechanisms of epithelial RelA activation, we stimulated a murine alveolar epithelial cell line (MLE-15) with bronchoalveolar lavage fluid (BALF) harvested from mice infected with S. pneumoniae. Pneumonic BALF, but not S. pneumoniae, induced degradation of IκBα and IκBβ and rapid nuclear accumulation of RelA. Moreover, BALF-induced RelA activity was completely abolished following combined but not individual neutralization of TNF and IL-1 signaling, suggesting either cytokine is sufficient and necessary for alveolar epithelial RelA activation during pneumonia. Our results demonstrate that RelA is essential for the host defense response to pneumococcus in the lungs and that RelA in airway epithelial cells is primarily activated by TNF and IL-1.

Roles of Interleukin-6 in Activation of STAT Proteins and Recruitment of Neutrophils during Escherichia coli Pneumonia

Interleukin (IL)-6 concentrations are positively associated with the severity of pneumonia, and this cytokine is essential to surviving experimental pneumococcal pneumonia. The role that IL-6 plays during pneumonia and its impact during gram-negative bacterial pneumonia remain to be determined. During Escherichia coli pneumonia, IL-6-deficient mice had increased bacterial burdens in their lungs, indicating compromised host defenses. Decreased neutrophil counts in alveolar air spaces, despite normal blood neutrophil counts and survival of emigrated neutrophils, suggested that defective neutrophil recruitment was responsible for exacerbating the infection. Neutrophil recruitment requires nuclear factor (NF)- kappa B, but IL-6 was neither sufficient nor essential to induce NF- kappa B-mediated gene expression in the lungs. In contrast, IL-6 induced the phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3 in the lungs, and STAT1 and STAT3 phosphorylation during E. coli pneumonia was decreased by IL-6 deficiency. Thus, IL-6 plays essential roles in activating STAT transcription factors, enhancing neutrophil recruitment, and decreasing bacterial burdens during E. coli pneumonia.

Alcohol‐Induced Suppression of Lung Chemokine Production and the Host Defense Response to Streptococcus pneumoniae

BACKGROUNDnAcute alcohol intoxication impairs neutrophil migration in response to intrapulmonary infection with Streptococcus pneumoniae, the most common bacterial cause of pneumonia. Many of the same host defense functions that are impaired in the alcohol-intoxicated host are mechanistically associated with chemokines, a group of proinflammatory molecules that enhance neutrophil adhesion and direct neutrophil migration to sites of inflammation. The purpose of this study was to determine whether alcohol-induced chemokine suppression is responsible for impaired neutrophil recruitment into the lung during infection of the alcohol-intoxicated host.nnnMETHODSnS. pneumoniae was administered (107 colony-forming units) intratracheally 30 min after intraperitoneal injection of 20% alcohol (5.5 g/kg) or saline. Four hours after bacterial challenge, bronchoalveolar lavage fluid (BALF) was collected, and the ability of BALF to induce neutrophil chemotaxis and adhesion molecule expression was measured by using chemotactic and flow cytometric assays. In another experiment, intratracheal challenge was performed by using recombinant macrophage inflammatory protein-2 (MIP-2), and BALF neutrophils were measured.nnnRESULTSnBALF MIP-2 and cytokine-induced neutrophil chemoattractant were decreased by alcohol, and BALF from alcohol-intoxicated animals had decreased chemotactic activity for neutrophils, as well as a decreased ability to up-regulate neutrophil adhesion molecule expression, compared with controls. This decreased chemotactic activity was significantly increased in the alcohol group by repletion of chemokines to control levels. Alcohol also suppressed neutrophil recruitment after intrapulmonary challenge with MIP-2, suggesting that mechanisms other than chemokine suppression contribute to the alcohol-induced effect.nnnCONCLUSIONSnAt least two mechanisms, suppressed chemokine production and impaired neutrophil adhesion molecule expression, likely work in concert in the alcohol-intoxicated host to impair neutrophil adhesion and migration into the lung during pneumococcal infection. These alterations in neutrophil function likely increase the susceptibility of alcohol-consuming hosts to pneumonia.

Effects of systemic and local CXC chemokine administration on the ethanol-induced suppression of pulmonary neutrophil recruitment.

BACKGROUNDnAcute alcohol intoxication impairs the neutrophil response to intrapulmonary infection, resulting in impaired host defense and increased patient morbidity and mortality. We recently showed that intratracheal (IT) chemokine administration promotes pulmonary neutrophil migration in rats and that this process is enhanced by systemic administration of the Glu-Leu-Arg (ELR+) and CXC chemokine cytokine-induced neutrophil chemoattractant (CINC). Here we hypothesized that exogenous chemokine administration would mitigate the suppressive effect of alcohol on neutrophil recruitment into the lung.nnnMETHODSnMacrophage inflammatory protein-2 (MIP-2), a rat ELR+ CXC chemokine, or live Klebsiella pneumoniae (K. pneumoniae) was administered it to induce alveolar neutrophil migration in the absence or presence of acute ethanol intoxication. Depending on the experimental protocol, rats received either intravenous (IV) CINC or IT chemokines (CINC and MIP-2) 20 min after it MIP-2 or K. pneumoniae. Rats were euthanized 90 min or four hr after the first IT injection for sample collection.nnnRESULTSnNeutrophil counts were significantly elevated in bronchoalveolar lavage fluid (BALF) of rats receiving IT MIP-2 compared with vehicle-treated rats, and this response was significantly decreased in animals pretreated with ethanol. CINC IV enhanced the neutrophil response to IT MIP-2 in both the absence and presence of acute ethanol intoxication. In rats challenged with K. pneumoniae, ethanol pretreatment significantly reduced BALF levels of CINC and MIP-2, suppressed alveolar neutrophil recruitment, and decreased whole-lung myeloperoxidase activity. CINC IV did not alter BALF neutrophil counts in the absence or presence of ethanol administration 4 hr after IT K. pneumoniae. Alternatively, IT chemokine instillation partially restored BALF neutrophil recruitment but not whole-lung myeloperoxidase activity in ethanol-treated rats.nnnCONCLUSIONSnEthanol significantly inhibits the pulmonary inflammatory responses to both MIP-2 and K. pneumoniae. Exogenous chemokine administration may be a useful means to enhance host defenses in the ethanol-intoxicated host, although the results of this study also indicate that ethanol intoxication can impair neutrophil recruitment, independent of its effects on local chemotactic gradients.

Interferon-γ Enhances the Pulmonary CXC Chemokine Response to Intratracheal Lipopolysaccharide Challenge

CXC chemokines are major chemoattractants for pulmonary polymorphonuclear leukocyte (PMNL) recruitment. To study the effects of interferon (IFN)-gamma on the pulmonary chemokine response to lipopolysaccharide (LPS) challenge, rats were treated with intratracheal IFN-gamma (1x10(5) U/rat) 24 h before an intratracheal LPS (100 microg/rat) challenge. Intratracheal LPS caused significant increases in both cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 in bronchoalveolar lavage (BAL) fluid and pulmonary PMNL recruitment. IFN-gamma enhanced these responses. IFN-gamma also increased LPS-induced tumor necrosis factor (TNF)-alpha in BAL fluid. LPS-induced TNF-alpha and CINC mRNA expression in alveolar macrophages was increased by IFN-gamma. CD11b/c and CD18 expression on circulating PMNLs was not affected by IFN-gamma, nor was the chemotaxis of these cells. IFN-gamma increases the pulmonary CXC chemokine response, which may serve as one mechanism underlying enhanced PMNL delivery into the lung.

249 THE LIPOPOLYSACCHARIDE BINDING PROTEIN RESPONSE TO INTRATRACHEAL LIPOPOLYSACCHARIDE

Lipopolysaccharide binding protein (LBP) is an acute-phase glycoprotein that facilitates lipopolysaccharide (LPS) activation of immune cells through interactions with CD14 and toll-like receptor 4. In response to conditions such as sepsis, trauma, and ARDS, LBP concentrations increase in both plasma and bronchoalveolar lavage fluid (BALF). Less is known, however, about the role or distribution of this protein in response to localized intrapulmonary infection. The purpose of our current study was to characterize the local and systemic LBP responses to intratracheal (i.t.) LPS. C3HEN mice received i.t. LPS (10 μg) and were sacrificed at 0 h, 4 h, 8 h, 24 h, and 48 h. BALF and plasma were collected at each time point and analyzed for the content of LBP and several proinflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6). Livers and spleens were harvested for analysis of LBP mRNA. LBP concentrations were increased in plasma by 8 h after i.t. LPS and remained elevated throughout the entire observation period. Changes in plasma LBP paralleled similar increases in liver LBP mRNA expression. TNF, G-CSF, and IL-6 were all increased in BALF following the i.t. LPS challenge. However, while the TNF response remained compartmentalized within the alveolar space, G-CSF and IL-6 levels were elevated both locally in BALF and systemically in plasma. From these data, we conclude that LBP is systemically expressed in response to i.t. LPS. Furthermore, we speculate that the systemic LBP response may be initiated in the liver by pulmonary-derived IL-6, a cytokine known to potently induce hepatic LBP synthesis.