Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.

To study the in vivo role of alveolar macrophages (AM) in gram-negative bacterial pneumonia in mice, AM were eliminated by the intratracheal (i.t.) administration of dichloromethylene diphosphonate encapsulated liposomes. Subsequently, the AM-depleted mice were infected i.t. with 100 CFU of Klebsiella pneumoniae, and the effects of AM depletion on survival, bacterial clearance, and neutrophil (polymorphonuclear leukocyte [PMN]) recruitment were assessed. It was shown that depletion of AM decreases survival dramatically, with 100% lethality at day 3 postinfection, versus 100% long-term survival in the control group. This increased mortality was accompanied by 20- to 27- and 3- to 10-fold increases in the number of K. pneumoniae CFU in lung and plasma, respectively, compared to those in nondepleted animals. This decreased bacterial clearance was not due to an impaired PMN recruitment; on the contrary, the K. pneumoniae-induced PMN recruitment in AM-depleted lungs was sevenfold greater 48 h postinfection than that in control infected lungs. Together with an increased PMN infiltration, 3- and 10-fold increases in lung homogenate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) levels, respectively, were measured. Neutralization of TNF-alpha or MIP-2, 2 h before infection, reduced the numbers of infiltrating PMN by 41.6 and 64.2%, respectively, indicating that these cytokines mediate PMN influx in infected lungs, rather then just being produced by the recruited PMN themselves. Our studies demonstrate, for the first time, the relative importance of the AM in the containment and clearance of bacteria in the setting of Klebsiella pneumonia.     

Tumor necrosis factor mediates lung antibacterial host defense in murine Klebsiella pneumonia.

Tumor necrosis factor (TNF) is a proinflammatory cytokine which has recently been shown to have beneficial effects in the setting of acquired host immunity. However, the role of TNF in innate immune responses, as in the setting of bacterial pneumonia, has been incompletely characterized. To determine the role of TNF in gram-negative bacterial pneumonia, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally, resulting in the time-dependent expression of TNF MRNA and protein within the lung. Passive immunization of animals with a soluble TNF receptor-immunoglobulin (Ig) construct (sTNFR:Fc) intraperitoneally 2 h prior to K. pneumoniae inoculation resulted in a significant reduction in bronchoalveolar lavage neutrophils, but not macrophages, at 48 h, as compared with animals receiving control IgG1. Furthermore, treatment with sTNFR:Fc resulted in 19.6- and 13.5-fold increases in K. pneumoniae CFU in lung homogenates and plasma, respectively, as compared with animals receiving control IgG1. Finally, treatment of Klebsiella-infected mice with sTNFR:Fc markedly decreased both short- and long-term survival of these animals. In conclusion, our studies indicate that endogenous TNF is a critical component of antibacterial host defense in murine Klebsiella pneumonia.     

Role of lung epithelial cells in defense against Klebsiella pneumoniae pneumonia

The airway epithelium represents a primary site for the entry of pathogenic bacteria into the lungs. It has been suggested for many respiratory pathogens, including Klebsiella pneumoniae, that adhesion and invasion of the lung epithelial cells is an early stage of the pneumonia process. We observed that poorly encapsulated K. pneumoniae clinical isolates and an isogenic unencapsulated mutant invaded lung epithelial cells more efficiently than highly encapsulated strains independent of the K type. By contrast, the unencapsulated mutant was completely avirulent in a mouse model of pneumonia, unlike the wild-type strain, which produced pneumonia and systemic infection. Furthermore, the unencapsulated mutant bound more epithelially produced complement component C3 than the wild-type strain. Our results show that lung epithelial cells play a key role as a host defense mechanism against K. pneumoniae pneumonia, using two different strategies: (i) ingestion and control of the microorganisms and (ii) opsonization of the microorganisms. Capsular polysaccharide avoids both mechanisms and enhances the virulence of K. pneumoniae.     

Lobar pneumonia in rats produced by clinical isolates of Klebsiella pneumoniae.

Transtracheal instillation of clinical isolate Klebsiella pneumoniae serotype 1 (KP1) into the lungs of rats resulted in the production of a characteristic, chronic lobar pneumonia. To further examine this phenomenon, two variants of this organism were employed in this experimental model. These variants differed markedly in capsule size, colony morphology, and in virulence, as determined by mouse lethality tests. The ability of these strains to establish a lobar pneumonia in rats correlated with the virulence of the respective organisms as monitored by intraperitoneal injection in mice. The 50% lethal doses in mice were 4.9 x 10(1) colony-forming units (CFU) for the more virulent KP1 strain (KP1-O) and 1.42 x 10(5) CFU for the less virulent variant (KP1-T). In the rat lung model, marked lung pathology was evident by day 6 with a KP1-O inoculum of 5 x 10(2) CFU, whereas KP1-T caused little or no lung pathology when delivered transtracheally at a concentration of 7 x 10(6) CFU. Two relatively nonvirulent variants of K. pneumoniae serotype 2 were also used in this rat lung model and were found not to produce a lobar pneumonia even when delivered in large doses. These results indicate that a chronic lobar pneumonia can be established in a rat model if the appropriate organism is employed and the virulence of K. pneumoniae injected intraperitoneally into mice is an excellent indicator of an organism's potential to cause lobar pneumonia in rats.     

Macrophage Inflammatory Protein 1α/CCL3 Is Required for Clearance of an Acute Klebsiella pneumoniae Pulmonary Infection

The objective of these studies was to determine the role of macrophage inflammatory protein 1alpha/CCL3 in pulmonary host defense during Klebsiella pneumoniae infection. Following intratracheal inoculation, 7-day survival of CCL3(-/-) mice was less than 10%, compared to 60% for CCL3(+/+) mice. Survival of CCR5(-/-) mice was equivalent to that of controls, indicating that the enhanced susceptibility of CCL3(-/-) mice to K. pneumoniae is mediated via another CCL3 receptor, presumably CCR1. At day 3, CFU burden in the lungs of CCL3(-/-) mice was 800-fold higher than in CCL3(+/+) mice, demonstrating that CCL3 is critical for control of bacterial growth in the lung. Surprisingly, CCL3(-/-) mice had no differences in the recruitment of monocytes/macrophages and even showed enhanced neutrophil recruitment at days 1, 2, and 3 postinfection, compared to CCL3(+/+) mice. Therefore, the defect in clearance was not due to insufficient recruitment of leukocytes. No significant differences in cytokine levels of monocyte chemoattractant protein 1 (MCP-1), interleukin 12, gamma interferon, or tumor necrosis factor alpha in lung lavages were found between CCL3(+/+) and CCL3(-/-) mice. CCL3(-/-) alveolar macrophages were found to have significantly lower phagocytic activity toward K. pneumoniae than CCL3(+/+) alveolar macrophages. These findings demonstrate that CCL3 production is critical for activation of alveolar macrophages to control the pulmonary growth of the gram-negative bacterium K. pneumoniae.     

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.     

Surfactant protein D enhances phagocytosis and killing of unencapsulated phase variants of Klebsiella pneumoniae

Pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin (collectin) that is secreted into the alveoli and distal airways of the lung. We have studied the interactions of SP-D and alveolar macrophages with Klebsiella pneumoniae, a common cause of nosocomial pneumonia. SP-D does not agglutinate encapsulated K. pneumoniae but selectively agglutinates spontaneous, unencapsulated phase variants, such as Klebsiella strain K50-3OF, through interactions with their lipopolysaccharides (LPS). These effects are calcium dependent and inhibited with maltose but not lactose, consistent with involvement of the SP-D carbohydrate recognition domain. Precoating of K50-3OF with SP-D enhances the phagocytosis and killing of these organisms by rat alveolar macrophages in cell culture and stimulates the production of nitric oxide by the NR-8383 rat alveolar macrophage cell line. SP-D similarly enhances the NO response to K50-3OF LPS adsorbed to Latex beads under conditions where soluble LPS or SP-D, or soluble complexes of SP-D and LPS, do not stimulate NO production. Our studies demonstrate that interactions of SP-D with exposed arrays of Klebsiella LPS on a particulate surface can enhance the host defense activities of alveolar macrophages and suggest that activation of macrophages by SP-D requires binding to microorganisms or other particulate ligands. Because unencapsulated phase variants are likely to be responsible for the initial stages of tissue invasion and infection, we speculate that SP-D-mediated agglutination and/or opsonization of K. pneumoniae is an important defense mechanism for this respiratory pathogen in otherwise healthy individuals.     

Activity of cefepime and carbapenems in experimental pneumonia caused by porin-deficient Klebsiella pneumoniae producing FOX-5 β-lactamase

The in-vivo activities of cefepime, imipenem and meropenem against the porin-deficient strain Klebsiella pneumoniae C2 and its derivative K. pneumoniae C2(pMG252) coding for the AmpC-type beta-lactamase FOX-5 were determined. Bactericidal activities were determined with the kill-curve method. A pneumonia model in guinea-pigs was developed, and Cmax, t(1/2) and DeltaT/MIC were calculated for the three agents tested. Animals were treated for 72 h with sterile saline (control group) or with cefepime, imipenem or meropenem (240 mg/kg/day, intramuscularly, three times daily). Bacterial counts in lungs (log10 CFU/g tissue) were determined by serial dilution. MICs (mg/L) of cefepime, imipenem and meropenem against K. pneumoniae C2/K. pneumoniae C2(pMG252), determined by macrodilution, were: 0.5/4, 0.5/0.5 and 0.25/0.5, respectively. Bacterial counts in the lungs of animals infected with K. pneumoniae C2 and treated with antimicrobial agents were always lower than in the control group (cefepime, 4.4 +/- 0.5; imipenem, 4.6 +/- 0.4; meropenem, 4.7 +/- 0.5; control group, 5.6 +/- 0.8; p <0.01). No significant differences were observed among the groups receiving therapy (p >0.05). Bacterial lung clearance was higher in treated animals than in control animals following infection with K. pneumoniae C2(pMG252) (cefepime, 4.5 +/- 0.4; imipenem, 4.0 +/- 0.3; meropenem, 4.6 +/- 0.4; control group, 6.1 +/- 0.6; p <0.01), with imipenem producing better clearance than either cefepime or meropenem (p <0.05). Thus, in the guinea-pig pneumonia model, cefepime, imipenem and meropenem were each effective against the porin-deficient K. pneumoniae strain C2 and its derivative expressing the plasmid-mediated AmpC type beta-lactamase FOX-5.     

Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice

To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.     

Role of tumor necrosis factor alpha in pathogenesis of pneumococcal pneumonia in mice.

The production and role of tumor necrosis factor alpha (TNF-alpha) in pneumococcal pneumonia were investigated in a mouse pneumonia model. When approximately 10(6) CFU of Streptococcus pneumoniae TUM19 were used to inoculate CBA/J mice intranasally, TNF-alpha levels in the lungs and serum began to increase from 1 and 3 days after infection, respectively, concomitantly with the increase in bacterial counts in the lungs. Anti-TNF-alpha antibody accelerated bacterial proliferation in the blood and the death of the mice. Although serum levels of immunoglobulin G antibody against the infecting bacteria were not affected by the anti-TNF-alpha antibody treatment, neutrophil counts in the blood were decreased by the treatment. These results suggest that TNF-alpha produced in the course of pneumococcal pneumonia prevents bacteremia by increasing the number of neutrophils in the blood.     

LPS-binding protein-deficient mice have an impaired defense against Gram-negative but not Gram-positive pneumonia

LPS-binding protein (LBP) can facilitate the transfer of cell wall components of both Gram-negative bacteria (LPS) and Gram-positive bacteria (lipoteichoic acid) to inflammatory cells. Although LBP is predominantly produced in the liver, recent studies have indicated that this protein is also synthesized locally in the lung by epithelial cells. To determine the role of LBP in the immune response to pneumonia, LBP gene-deficient (-/-) and normal wild-type (WT) mice were intra-nasally infected with either Streptococcus pneumoniae or Klebsiella pneumoniae, common Gram-positive and Gram-negative pathogens, respectively. Pneumococcal pneumonia was associated with a 7-fold rise in LBP concentrations in bronchoalveolar lavage fluid of WT mice; LBP-/- mice infected with S. pneumoniae showed a similar survival and a similar bacterial burden in their lungs 48 h post-infection. In Klebsiella pneumonia, however, LBP-/- mice demonstrated a diminished survival together with an enhanced bacterial outgrowth in their lungs. These data suggest that LBP is important for a protective immune response in Klebsiella pneumonia, but does not contribute to an effective host response in pneumococcal pneumonia.     

Acute Lung Inflammation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055-Induced Pneumonia and Sepsis in BALB/c Mice: A Comparative Study

Lungs play an important role in the body's defense against a variety of pathogens, but this network of immune system-mediated defense can be deregulated during acute pulmonary infections. The present study compares acute lung inflammation occurring during Klebsiella pneumoniae B5055-induced pneumonia and sepsis in BALB/c mice. Pneumonia was induced by intranasal instillation of bacteria (10⁴ cfu), while sepsis was developed by placing the fibrin-thrombin clot containing known amount of bacteria (10² cfu) into the peritoneal cavity of animals. Mice with sepsis showed 100% mortality within five post-infection days, whereas all the animals with pneumonia survived. In animals suffering from K. pneumoniae B5055-induced pneumonia, all the inflammatory parameters (TNF-α, IL-1α, MPO, MDA, and NO) were found to be maximum till third post-infection day, after that, a decline was observed, whereas in septic animals, all the above-mentioned markers of inflammation kept on increasing. Histopathological study showed presence of alternatively activated alveolar macrophages (or foam cells) in lungs of mice with pneumonia after third post-infection day, which might have contributed to the induction of resolution of inflammation, but no such observation was made in lungs of septic mice. Hence, during pneumonia, controlled activation of macrophages may lead to resolution of inflammation.     

Interleukin-17 and lung host defense against Klebsiella pneumoniae infection

Bacterial pneumonia remains an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Cytokines and chemokines are critical molecules expressed in response to invading pathogens and are necessary for normal lung bacterial host defenses. Here we show that interleukin (IL)-17, a novel cytokine produced largely by CD4+ T cells, is produced in a compartmentalized fashion in the lung after challenge with Klebsiella pneumoniae. Moreover, overexpression of IL-17 in the pulmonary compartment using a recombinant adenovirus encoding murine IL-17 (AdIL-17) resulted in the local induction of tumor necrosis factor-alpha, IL-1beta, macrophage inflammatory protein-2, and granulocyte colony-stimulating factor (G-CSF); augmented polymorphonuclear leukocyte recruitment; and enhanced bacterial clearance and survival after challenge with K. pneumoniae. However, simultaneous treatment with AdIL-17 provided no survival benefit after intranasal K. pneumoniae challenge. These data show that IL-17 may have a role in priming for enhanced chemokine and G-CSF production in the context of lung infection and that optimally timed gene therapy with IL-17 may augment host defense against bacterial pneumonia.     

Pulmonary and systemic host response to Streptococcus pneumoniae and Klebsiella pneumoniae bacteremia in normal and immunosuppressed mice

Mortality related to bacteremic pneumonia remains high, and the role of sepsis in inflammation, pulmonary injury, and death remains unclear, mostly in leukopenic states. In the present study, the microbiology, histopathology, and host response to Streptococcus pneumoniae and Klebsiella pneumoniae infection were determined in an experimental model of bacteremia in immunocompetent and leukopenic mice. Leukocyte depletion by cyclophosphamide did not impair the early clearance of pneumococci from blood but facilitated growth in lungs. By contrast, klebsiellae rapidly grew in blood of leukopenic mice. These observations suggest that tissue-based phagocytes and circulating leukocytes, respectively, play prominent roles in S. pneumoniae and K. pneumoniae eradication. The kinetics of leukocyte recruitment in lungs during S. pneumoniae bacteremia suggested early strong inflammation in immunocompetent mice that is associated with tumor necrosis factor alpha release and histological disorders, including cell debris and surfactant in alveolar spaces. Leukocyte depletion further stimulated pulmonary capillary leakage both in S. pneumoniae and K. pneumoniae bacteremia, which seemed attributable to bacterial virulence factors. Nitric oxide production did not differ significantly among groups. Leukopenia and low platelet counts characterized the late stage of bacteremia for both strains, but only K. pneumoniae altered renal function. Understanding the pathogenesis of bacteremia will help establish beneficial therapies for both sepsis and pneumonia.     

Pathogenesis of pneumococcal pneumonia in cyclophosphamide-induced leukopenia in mice

Streptococcus pneumoniae pneumonia frequently occurs in leukopenic hosts, and most patients subsequently develop lung injury and septicemia. However, few correlations have been made so far between microbial growth, inflammation, and histopathology of pneumonia in specific leukopenic states. In the present study, the pathogenesis of pneumococcal pneumonia was investigated in mice rendered leukopenic by the immunosuppressor antineoplastic drug cyclophosphamide. Compared to the immunocompetent state, cyclophosphamide-induced leukopenia did not hamper interleukin-1 (IL-1), IL-6, macrophage inflammatory protein-1 (MIP-1), MIP-2, and monocyte chemotactic protein-1 secretion in infected lungs. Leukopenia did not facilitate bacterial dissemination into the bloodstream despite enhanced bacterial proliferation into lung tissues. Pulmonary capillary permeability and edema as well as lung injury were enhanced in leukopenic mice despite the absence of neutrophilic and monocytic infiltration into their lungs, suggesting an important role for bacterial virulence factors and making obvious the fact that neutrophils are ultimately not required for lung injury in this model. Scanning and transmission electron microscopy revealed extensive disruption of alveolar epithelium and a defect in surfactant production, which were associated with alveolar collapse, hemorrhage, and fibrin deposits in alveoli. These results contrast with those observed in immunocompetent animals and indicate that leukopenic hosts suffering from pneumococcal pneumonia are at a higher risk of developing diffuse alveolar damage.     

Nitric oxide exerts distinct effects in local and systemic infections with Streptococcus pneumoniae

Nitric oxide (NO) is known to be involved in the immune response against a range of organisms. Little is known about the effects of nitric oxide in pneumococcal infections. We have now investigated the role of nitric oxide in local and systemic infections caused by Streptococcus pneumoniae in NOS2 deficient mice. Although a deficiency in NO does not affect survival of mice during pneumococcal pneumonia, NO does control pneumococcal viability within the lung airways and tissue. Bronchoalveolar lavage fluid (BALF) from NOS2-deficient mice contained significantly elevated TNF activity, IFNgamma and total protein during mid/late infection. Incubation of S. pneumoniae with the NO donor SNAP revealed a direct anti-pneumococcal effect for NO in vitro. Deficiency in NOS2 did not affect bacteraemia following intranasal infection. In contrast NOS2-deficient mice were significantly less susceptible to intravenous infection with S. pneumoniae than were wild type mice and were able to control pneumococcal viability within the bloodstream. Our results indicate that NO is required within the lungs for anti-bacterial activity during the pneumococcal pneumonia but during Gram-positive bacteraemia NO is associated with increased bacterial loads and reduced survival.     

Intrapulmonary expression of macrophage inflammatory protein 1alpha (CCL3) induces neutrophil and NK cell accumulation and stimulates innate immunity in murine bacterial pneumonia

Macrophage inflammatory protein 1alpha (MIP-1alpha) (CCL3) is an important mediator of leukocyte recruitment and activation in a variety of inflammatory states, including infection. A recombinant human type 5 adenovirus containing the murine MIP-1alpha cDNA (AdMIP-1alpha) was constructed to determine the effect of transient intrapulmonary expression of MIP-1alpha on leukocyte recruitment, activation, and bacterial clearance in a murine model of Klebsiella pneumoniae pneumonia. The intratracheal administration of AdMIP-1alpha resulted in both time- and dose-dependent expression of MIP-1alpha mRNA and protein within the lung. Importantly, the intrapulmonary overexpression of MIP-1alpha resulted in a maximal 35- and 100-fold reduction in lung and blood bacterial burden, respectively, in animals cochallenged with K. pneumoniae, which was associated with a significant increase in neutrophil and activated NK cell accumulation. Furthermore, the transgenic expression of MIP-1alpha during bacterial pneumonia resulted in enhanced expression of gamma interferon mRNA, compared to that observed in Klebsiella-challenged animals pretreated with control vector. These findings indicate an important role for MIP-1alpha in the recruitment and activation of selected leukocyte populations in vivo and identify this cytokine as a potential immunoadjuvant to be employed in the setting of localized bacterial infection.