Role of alveolar epithelial cell intercellular adhesion molecule-1 in host defense against Klebsiella pneumoniae.

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells (AEC) in the normal alveolar space. We postulate that AEC ICAM-1 enhances the antimicrobial activity of macrophages and neutrophils in the alveolar space. Wild-type and mutant mice deficient in ICAM-1 were inoculated intratracheally with Klebsiella pneumoniae. After 10 days, 43% of the ICAM-1 mutant mice had died compared with 14% of the wild-type controls (P = 0.003). Significantly more bacteria were isolated from lungs of ICAM-1 mutant mice than controls 24 h after inoculation (log colony-forming units 5.14 +/- 0.21 vs. 3.46 +/- 0. 16, P = 0.001). However, neutrophil recruitment to the lung was not different. In similar experiments in the rat, inhibition of alveolar ICAM-1 by intratracheal administration of antibody resulted in significantly impaired clearance of K. pneumoniae. The role of phagocyte interactions with AEC ICAM-1 for antimicrobial activity was investigated in vitro using primary cultures of rat AEC that express abundant ICAM-1. Alveolar macrophage phagocytosis and killing of K. pneumoniae were increased significantly in the presence of AEC; these effects were inhibited significantly (47.5 and 52%, respectively) when AEC ICAM-1 was blocked. Similarly, neutrophil phagocytic activity for K. pneumoniae in the presence of AEC in vitro was decreased when ICAM-1 on the AEC surface was blocked. Thus in the absence of ICAM-1, there is impaired ability to clear K. pneumoniae from the lungs, resulting in increased mortality. These studies indicate that AEC ICAM-1 plays an important role in host defense against K. pneumoniae by determining the antimicrobial activity of phagocytes within the lung.

Intrapulmonary tumor necrosis factor gene therapy increases bacterial clearance and survival in murine gram-negative pneumonia.

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity in bacterial pneumonia. To augment the expression of TNF within the lung, a recombinant adenoviral vector containing the murine TNF cDNA (Ad5mTNF) has been developed, and the intratracheal administration of this vector resulted in the dose- and time-dependent expression of TNF in the lung, but not systemically. Administration of Ad5mTNF resulted in significant airspace and peribronchial inflammation, with a predominant neutrophil influx by 2 days, and mononuclear cell infiltrates by 4 to 7 days posttreatment. Importantly, the administration of Ad5mTNF at a dose of 1 x 10(8) PFU significantly improved the survival of animals challenged concomitantly with Klebsiella pneumoniae, which occurred in association with enhanced clearance of bacteria from the lung and decreased dissemination of K. pneumoniae to the bloodstream. However, the delivery of higher doses of Ad5mTNF (5 x 10(8) PFU) was not beneficial and in fact the intratracheal administration of a similar dose of control vector (Ad5LacZ) actually enhanced Klebsiella-induced lethality by impairing clearance of K. pneumoniae from the lung. Our studies suggests that the transient transgenic expression of TNF within the lung dose dependently augments antibacterial host defense in murine Klebsiella pneumonia.

Lung-specific transgenic expression of KC enhances resistance to Klebsiella pneumoniae in mice.

A vigorous host response is required to effectively clear pathogenic bacteria from the lungs and is dependent upon the recruitment and activation of neutrophils and macrophages. A family of chemotactic cytokines, referred to as chemokines, have been shown to participate in this complex protective response. In this study, we assessed the role of the C-X-C chemokine KC in lung antibacterial host defense using wild-type (wt) B6D2 mice or transgenic mice that had been bred on a B6D2 background expressing KC under the control of a Clara cell-specific promoter within the lung. The administration of Klebsiella pneumoniae to both wt and KC-transgenic mice resulted in a time-dependent expression of KC protein within the lung that peaked at 24 to 48 h postinoculation. When infected with K. pneumoniae, the KC-transgenic mice showed a striking improvement in survival compared with wt control mice. This improved survival was due to an increase in bacterial clearance, which occurred in association with a vigorous recruitment of neutrophils in the KC-transgenic mice compared with their wt control counterparts. No differences in the lung levels of the specific cytokines TNF-alpha, IFN-gamma, IL-12, and IL-10 were noted. However, inducible macrophage inflammatory protein-2 levels were significantly decreased in the KC-transgenic mice compared with the wt mice. This study indicates that the compartmentalized overexpression of KC in vivo results in increased lung bacterial clearance and improved survival, which occurs in association with enhanced polymorphonuclear leukocyte influx to the lung.

Ethanol feeding impairs innate immunity and alters the expression of Th1- and Th2-phenotype cytokines in murine Klebsiella pneumonia.

The prolonged and excessive consumption of alcohol has been shown to predispose the host to a variety of infectious complications, which may be due, in part, to the inability to produce important activating cytokines. In this study, we assessed the effect of chronic alcohol ingestion on bacterial clearance, survival, and cytokine mRNA and protein expression in mice with Klebsiella pneumonia. Two-week ethanol feeding resulted in substantial impairment in the clearance of K. pneumoniae and decreased survival, compared with CD-1 mice receiving an isocaloric diet without ethanol. No differences were noted between control and ethanol groups in the total numbers or percent of bronchoalveolar lavage fluid neutrophils or macrophages at 24 and 48 hr post-intratracheal K. pneumoniae. Importantly, the lungs of alcohol-fed mice with Klebsiella pneumonia displayed a decrease or delay in the expression of interleukin (IL)-12 p35 and p40 mRNA and interferon-gamma mRNA, respectively, as well as reduced IL-12 and interferon-gamma protein levels, compared with controls. Conversely, a time-dependent increase in lung IL-10 mRNAand protein was noted in ethanol-fed animals, compared with control animals challenged with K. pneumoniae. In summary, our studies indicate that ethanol ingestion results in a profound suppression of lung bacterial clearance and decreased survival in Klebsiella pneumonia, which occurs in association with a shift in the balance of lung cytokine mRNA and protein expression favoring Th2- rather than Th1-phenotype cytokines.

Intrapulmonary delivery of tumor necrosis factor agonist peptide augments host defense in murine gram-negative bacterial pneumonia.

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity in Klebsiella pneumonia. Recently, a TNF agonist peptide consisting of the 11-amino-acid TNF binding site (TNF70-80) has been shown to possess many of the leukocyte-activating properties of TNF without the associated toxicity when administered locally or systemically. Given the beneficial effects of TNF in gram-negative pneumonia, we hypothesize that the intratracheal (i.t.) administration of TNF70-80 would augment lung innate immunity in mice challenged with intrapulmonary Klebsiella pneumoniae. The administration of TNF70-80 i.t. to CBA/J mice 7 days prior to, but not concomitantly with, the i.t. delivery of 3 x 10(3) CFU of K. pneumoniae resulted in a marked increase in survival compared to that of animals receiving a control peptide i.t. In addition, pretreatment with TNF70-80 resulted in improved bacterial clearance, which occurred in association with enhanced lung myeloperoxidase activity (as a measure of lung polymorphonuclear leukocyte influx), and increased expression of the important activating cytokines TNF, macrophage inflammatory protein-2, interleukin-12, and gamma interferon compared that for animals receiving control peptide. Finally, the administration of TNF70-80 intraperitoneally resulted in enhanced rather than decreased lethality of Klebsiella pneumonia compared to that for animals receiving either TNF70-80 or control peptide i.t. Our studies suggest that the intrapulmonary, but not systemic, administration of the TNF agonist peptide may serve as an important immunoadjuvant in the treatment of murine Klebsiella pneumonia.

Anti-interleukin-12 therapy protects mice in lethal endotoxemia but impairs bacterial clearance in murine Escherichia coli peritoneal sepsis.

The overzealous production of proinflammatory cytokines in sepsis can result in shock, multiorgan dysfunction, and even death. In this study we assessed the role of endogenously produced interleukin (IL)-12 in murine models of endotoxemia and Gram-negative peritoneal sepsis. Initial studies indicated that intraperitoneal lipopolysaccharide (LPS) administration to mice induced a significant time-dependent increase in plasma, lung, and liver IL-12 levels. Passive immunization with anti-IL-12 serum intraperitoneally before LPS resulted in a marked reduction in plasma levels of tumor necrosis factor and interferon-gamma. Furthermore, we observed an increase in endotoxin-induced mortality in mice transiently overexpressing murine IL-12 using a recombinant adenoviral vector (Ad5 mIL-12) administered intraperitoneally. Neutralization of tumor necrosis factor or interferon-gamma in animals overexpressing IL-12 resulted in significant reductions in LPS-induced mortality, suggesting that the mechanism whereby IL-12 increases LPS-induced mortality is primarily mediated by the enhancement of these cytokines. In contrast, we observed no survival benefit in animals passively immunized with anti-IL-12 serum before the intraperitoneal administration of 2 x 10(8) live Escherichia coli. Interestingly, there was an approximately 70-fold increase in peritoneal fluid E. coli colony-forming units and the early onset of bacteremia in animals treated with anti-IL-12 serum, as compared with control animals. These results indicate that IL-12 is produced in response to LPS exposure, and the neutralization of this cytokine improves survival in endotoxin-challenged animals. However, IL-12 represents an essential component of antibacterial host defense, as anti-IL-12 therapy results in significant impairment in the host's ability to clear Gram-negative bacterial infection.

Nitric oxide is required for effective innate immunity against Klebsiella pneumoniae.

Nitric oxide (NO) has been associated with protection against various parasitic and viral infections and may play a similar role in bacterial infections. We studied the role of NO in host defense against Klebsiella pneumoniae infection in the lung. Initial studies demonstrated a time-dependent increase in NO production of the lungs of CBA/J mice following the intratracheal administration of K. pneumoniae (7 x 10(2) CFU). To assess the role of NO in Klebsiella pneumonia, mice were treated intraperitoneally with either L-NAME (N-omega-nitro-L-arginine methylester), a competitive inhibitor of NO synthesis, or D-NAME, an inert enantiomer. The treatment of Klebsiella-infected mice with L-NAME resulted in a 10- and 46-fold increase in K. pneumoniae CFU in lungs and blood, respectively, at 48 h post-K. pneumoniae inoculation compared to treatment of mice with D-NAME. In addition, a greater-than-twofold increase in mortality was evident in L-NAME-treated mice compared to the mortality in control animals. No significant difference in bronchoalveolar lavage inflammatory cell profiles was noted between L-NAME- and D-NAME-treated mice with Klebsiella pneumonia. Interestingly, increased levels of tumor necrosis factor, gamma interferon, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 mRNA and protein were noted in infected mice treated with L-NAME compared to the levels in mice treated with D-NAME. Importantly, the in vitro incubation of murine alveolar macrophages with L-NAME, but not with D-NAME, resulted in a significant impairment in both the phagocytosis and killing of K. pneumoniae. In total, these results suggest that NO plays a critical role in antibacterial host defense against K. pneumoniae, in part by regulating macrophage phagocytic and microbicidal activity.