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.     

Intraglomerular expressions of IL-1 alpha and platelet-derived growth factor (PDGF-B) mRNA in experimental immune complex-mediated glomerulonephritis.

Acute lung injury frequently develops following haemorrhage, and is characterized by increased proinflammatory cytokine levels and massive neutrophil accumulation in the lung. Blood loss produces rapid increases in tumour necrosis factor-alpha (TNF-alpha) mRNA expression among pulmonary cell populations which precede the development of lung injury. In order to examine the role of TNF-alpha in producing acute inflammatory lung injury, we treated mice following haemorrhage and resuscitation with a TNF antagonist, composed of soluble dimeric human p80 TNF receptor linked to the Fc region of human IgG1 (sTNFR:Fc). Therapy with sTNFR:Fc prevented the post-haemorrhage increases in circulating and pulmonary TNF-alpha levels normally found following blood loss. Administration of sTNFR:Fc also diminished the increase in IL-1 beta, IL-6, TNF-alpha and interferon-gamma (IFN-gamma) mRNA normally found in the lungs following haemorrhage. However, therapy with sTNFR:Fc was not associated with improvement in the histologic parameters of post-haemorrhage lung injury, such as neutrophil infiltration and interstitial oedema. In contrast to the effects of sTNFR:Fc on cytokine mRNA levels among intraparenchymal pulmonary mononuclear cells, such therapy following haemorrhage was associated with increased amounts of mRNA for TNF-alpha among peripheral blood mononuclear cells, as well as increased IFN-gamma titres in serum and bronchoalveolar lavage (BAL) specimens. These results indicate that therapy with sTNFR:Fc in the post-haemorrhage period, although capable of decreasing proinflammatory cytokine expression in the lungs, does not prevent the development of acute lung injury in this setting.     

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 (TNF_70-80) 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 TNF_70-80 would augment lung innate immunity in mice challenged with intrapulmonary Klebsiella pneumoniae. The administration of TNF_70-80 i.t. to CBA/J mice 7 days prior to, but not concomitantly with, the i.t. delivery of 3 × 10³ 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 TNF_70-80 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 TNF_70-80 intraperitoneally resulted in enhanced rather than decreased lethality of Klebsiella pneumonia compared to that for animals receiving either TNF_70-80 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.     

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.     

Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia

The adipocyte-derived hormone leptin is an important regulator of appetite and energy expenditure and is now appreciated for its ability to control innate and adaptive immune responses. We have reported previously that the leptin-deficient ob/ob mouse exhibited increased susceptibility to the Gram-negative bacterium Klebsiella pneumoniae. In this report we assessed the impact of chronic leptin deficiency, using ob/ob mice, on pneumococcal pneumonia and examined whether restoring circulating leptin to physiological levels in vivo could improve host defences against this pathogen. We observed that ob/ob mice, compared with wild-type (WT) animals, exhibited enhanced lethality and reduced pulmonary bacterial clearance following Streptococcus pneumoniae challenge. These impairments in host defence in ob/ob mice were associated with elevated levels of lung tumour necrosis factor (TNF)-alpha, macrophage inflammatory peptide (MIP)-2 [correction added after online publication 28 September 2007: definition of MIP corrected], prostaglandin E(2) (PGE(2)), lung neutrophil polymorphonuclear leukocyte (PMN) counts, defective alveolar macrophage (AM) phagocytosis and PMN killing of S. pneumoniae in vitro. Exogenous leptin administration to ob/ob mice in vivo improved survival and greatly improved pulmonary bacterial clearance, reduced bacteraemia, reconstituted AM phagocytosis and PMN H(2)O(2) production and killing of S. pneumoniae in vitro. Our results demonstrate, for the first time, that leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Further investigations are warranted to determine whether there is a potential therapeutic role for this adipokine in immunocompromised patients.     

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.     

Cyclic di-GMP stimulates protective innate immunity in bacterial pneumonia

Innate immunity is the primary mechanism by which extracellular bacterial pathogens are effectively cleared from the lung. We have previously shown that cyclic di-GMP (c-di-GMP [c-diguanylate]) is a novel small molecule immunomodulator and immunostimulatory agent that triggers protective host innate immune responses. Using a murine model of bacterial pneumonia, we show that local intranasal (i.n.) or systemic subcutaneous (s.c.) administration of c-di-GMP prior to intratracheal (i.t.) challenge with Klebsiella pneumoniae stimulates protective immunity against infection. Specifically, i.n. or s.c. administration of c-di-GMP 48 and 24 h prior to i.t. K. pneumoniae challenge resulted in significantly increased survival. Pretreatment with c-di-GMP resulted in a 5-fold reduction in bacterial CFU in the lung (P < 0.05) and an impressive >1,000-fold decrease in CFU in the blood (P < 0.01). c-di-GMP administration stimulated a robust innate response to bacterial challenge, characterized by enhanced accumulation of neutrophils and alphabeta T cells, as well as activated NK and alphabeta T lymphocytes, which was associated with earlier and more vigorous expression of chemokines and type I cytokines. Moreover, lung macrophages recovered from Klebsiella-infected mice pretreated with c-di-GMP expressed greater quantities of inducible nitric oxide synthase and nitric oxide ex vivo than did macrophages isolated from infected mice pretreated with the control, c-GMP. These findings demonstrate that c-di-GMP delivered in either a compartmentalized or systemic fashion stimulates protective innate immunity in the lung and protects mice against bacterial invasion. We propose that the cyclic dinucleotide c-di-GMP may be used clinically as an effective immunomodulator, immune enhancer, and vaccine adjuvant to protect against respiratory infection and pneumonia in humans and animals.     

Divergent role of gamma interferon in a murine model of pulmonary versus systemic Klebsiella pneumoniae infection

Klebsiella pneumoniae is a leading cause of both community-acquired and nosocomial gram-negative-bacterial pneumonia. A further clinical complication of pulmonary K. pneumoniae infection is dissemination of bacteria from the lung into the peripheral blood, resulting in bacteremia concurrent with the localized pulmonary infection. Here, we report studies detailing the divergent role of gamma interferon (IFN-gamma) in pulmonary versus systemic K. pneumoniae infection. Intratracheal inoculation of IFN-gamma knockout mice resulted in significantly increased mortality compared to that observed for wild-type infected animals. Increased mortality correlated with a 100-fold increase in pulmonary bacteria within 2 days postinfection and upregulation of lung-associated interleukin-10 (IL-10) mRNA. Interestingly, IFN-gamma knockout mice had a twofold reduction in plasma aminospartate transferase activity, indicating diminished liver injury following peripheral blood bacterial dissemination. To study the host response towards blood-borne bacteria in the absence of the ongoing pulmonary infection, intravenous inoculation studies were initiated. IFN-gamma knockout mice were no more susceptible to intravenous infection than their wild-type counterparts. The consistent observation in IFN-gamma knockout mice was for improved survival correlating with increased clearance of blood- and liver-associated bacteria. Intravenous inoculation resulted in a two- to threefold increase in hepatic IL-10 production 24 and 48 h postinfection. Liver injury was also significantly reduced in IFN-gamma knockout mice. These data indicate that IFN-gamma secretion is a critical mediator in the resolution of localized gram-negative pulmonary pneumonia. Surprisingly, host responses towards systemic infection with the same bacteria appear to be IFN-gamma independent.     

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.     

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.     

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.     

Molecular analysis of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia

Klebsiella pneumoniae is a common cause of gram-negative bacterial nosocomial pneumonia. Two surface polysaccharides, lipopolysaccharide (LPS) O side chain and capsular polysaccharide (CPS), are critical for the microorganism in causing sepsis, but little is known about their role in pneumonia. To investigate their contribution in the pathogenesis of K. pneumoniae pneumonia, we characterized the host response to bacterial challenge with a highly virulent clinical isolate or with isogenic insertion-duplication mutants deficient in CPS or LPS O side chain in a murine model of pneumonia. Animals challenged intratracheally with the wild-type or LPS O side chain-deficient strain developed pneumonia and became bacteremic before death. Extensive lung lesions as well as pleuritis, vasculitis, and edema were observed by histopathological examination, and polymorphonuclear infiltration was also demonstrated. In contrast, none of the animals challenged with the unencapsulated strain developed pneumonia or bacteremia. Examination of tissue from this group did not identify lung lesions, and none of the infected animals died. Analysis of the early host defense mechanisms that contributed to the clearance of the unencapsulated mutant showed that the levels of C3 deposited on the unencapsulated mutant surface were threefold higher than those for the wild-type and LPS O side chain-deficient strains. Furthermore, phagocytosis of the unencapsulated mutant by human alveolar macrophages (AM) was more efficient than that of the wild-type and LPS O side chain-deficient strains. We conclude that CPS, but not LPS O side chain, is essential for Klebsiella pneumonia because it modulates the deposition of C3 and protects the microorganisms against human AM phagocytosis.     

Role of inflammatory mediators in resistance and susceptibility to pneumococcal infection

Variations in the host response during pneumonia caused by Streptococcus pneumoniae in susceptible (CBA/Ca) and resistant (BALB/c) inbred mouse strains were investigated. Significant differences were detected in survival time, core body temperature, lung-associated and systemic bacterial loads, mast cell numbers, magnitude and location of cytokine production, lung disruption, and ability of isolated lung cells to release the cytokine tumor necrosis factor (TNF) alpha in vitro. Overall, the results indicate that the reduced capacity of CBA/Ca mice to induce rapid TNF activity within the airways following infection with S. pneumoniae may be a factor in their elevated susceptibility to pneumococcal pneumonia.     

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 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.     

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.     

The role of CD44 in the acute and resolution phase of the host response during pneumococcal pneumonia

Streptococcus pneumoniae is the most prevalent pathogen causing community-acquired pneumonia. CD44 is a transmembrane adhesion molecule, expressed by a wide variety of cell types, that has several functions in innate and adaptive immune responses. In this study, we tested the hypothesis that CD44 is involved in the host response during pneumococcal pneumonia. On intranasal infection with a lethal dose of S. pneumoniae CD44-knockout (KO) mice showed a prolonged survival when compared with wild-type mice, which was accompanied by a diminished pulmonary bacterial growth and reduced dissemination to distant body sites. Whereas, proinflammatory cytokine responses and lung pathology were not affected, CD44 deficiency resulted in increased early neutrophil influx into the lung. In separate experiments, we confirmed a detrimental role of CD44 in host defense against pneumococci during sublethal pneumonia, as demonstrated by an improved capacity of CD44 KO mice to clear a low infectious dose. In addition, CD44 appeared important for the resolution of lung inflammation during sublethal pneumonia, as shown by histopathology of lung tissue slides. In conclusion, we show here that CD44 facilitates bacterial outgrowth and dissemination during pneumococcal pneumonia, which in lethal infection results in a prolonged survival of CD44 KO mice. Moreover, during sublethal pneumonia CD44 contributes to the resolution of the inflammatory response.     

白细胞介素22及其受体在大鼠肺炎克雷伯菌肺炎中的表达

目的 探讨白细胞介素22(IL-22)及IL-22受体IL-10R2在大鼠肺炎克雷伯菌肺炎发病过程中表达的意义。方法 50只SPF级SD大鼠按随机数字法分为肺炎组(n=25)和对照组(n=25)。肺炎组通过气管内注射肺炎克雷伯菌建立大鼠肺炎克雷伯菌肺炎模型,对照组则滴入等体积无菌生理盐水。造模后4h、1、2、3、4d通过观察大鼠的一般情况、肺组织大体和病理切片、肺组织细菌菌落计数对肺炎模型进行评价。采用实时荧光定量PCR和ELISA分别检测肺炎组和对照组大鼠造模后4h、1、2、3、4d肺组织IL-10R2 mRNA、外周血IL-22的表达水平。应用直线相关方法分析菌落计数与IL-22及其受体的相关性。结果肺炎组大鼠造模后4h出现活动度下降、聚集成团、竖毛现象;大体肺组织明显充血水肿,显微镜下肺泡结构破坏、肺泡腔及间质显著的充血及水肿、显著的中性粒细胞浸润。肺炎组大鼠肺组织均可培养出细菌,造模后4h细菌菌落计数最高[(0.94±0.02)×105 CFU/g],之后逐渐降低,至4d时降至最低[(0.07±0.00)×105 CFU/g],证实肺炎模型制备成功。与对照组比较,肺炎组IL- 10R2mRNA的表达量于造模后4h开始升高,于第3天达峰值（12.95±0.65比0.31±0.31,P＜0.05）,其后开始下降。肺炎组外周血中IL-22均低于同时间点对照组。肺炎组外周血中IL-22自造模后第3天较4h开始明显升高且随时间的延长逐渐升高。肺炎组中IL-10R2 mRNA、外周血IL-22的表达水平均与同时间点肺组织菌落计数呈负相关（r=-0.64,P=0.01;r=-0.59,P=0.00）。结论 IL-10R2在肺炎克雷伯菌肺炎中高表达,而外周血中IL-22可能由于消耗导致降低,但可随肺炎的好转回升。IL-22是肺炎克雷伯茵肺炎早期的炎性反应因子,在体内具有清除细菌的作用。     

ALVEOLAR DESTRUCTION IN EXPERIMENTAL KLEBSIELLA PNEUMONIA

Sequential histological changes of the lungs were studied in experimental Klebsiella pneumonia, using untreated control mice, cyclophosphamide-treated mice, and carrageenan-treated mice. Cyclophosphamide was used to deplete polymorphonuclear leukocytes and monocytes, and carrageenan was used to deplete mononuclear phagocytes selectively. At 72 hours, varying degree of alveolar necrosis could be seen in untreated control mice. However, the lung lesions of cyclophosphamide- or carrageenan-treated mice were significantly different from those of the control mice. The lung lesions of cyclophosphamide-treated mice indicated that destruction of the alveolar septa was not induced by K. pneumoniae itself but by inflammatory cells, because the alveolar walls were preserved very well in spite of considerable bacterial multiplication in alveolar lumina until infiltration of inflammatory cells occurred. The lung lesions of carrageenan-treated mice showed that alveolar spaces were packed with polymorphonuclear leukocytes, but the alveolar walls were preserved very well as far as the authors could tell after examining the lung lesions by silver impregnation staining. These results suggest that macrophages rather than polymorphonuclear leukocytes and organisms play an important role in alveolar injury in experimental Klebsiella pneumonia.