Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice.

Hepatocyte growth factor (HGF) is postulated to play an important role in the repair of pulmonary epithelium in acute lung injury. To evaluate the role of HGF in bacterial pneumonia, the kinetics of HGF production and the cellular sources of HGF have been examined in the lungs of mice that had been intratracheally challenged with Pseudomonas aeruginosa. Neutrophil accumulation in the airway occurred immediately, reached a peak at 36 h, and then progressively declined by 14 d after infection. We found a biphasic pattern of HGF messenger RNA expression and protein synthesis in the lung after bacterial infection. The first peak for HGF production was found at 6 h after infection, and the primary source of HGF was shown to be bronchial epithelial cells. Interestingly, the second peak for HGF production, which was found around 48 to 72 h after infection, was closely associated with the increase in the percentage of alveolar macrophages (AMs) that became positive for myeloperoxidase, indicating phagocytosis of apoptotic neutrophils. The cellular source of the second peak was found to be AMs. Further, murine AMs which phagocytosed apoptotic neutrophils induced higher levels of HGF production in vitro. These results strongly indicate a novel mechanism of HGF production by AMs, which are phagocytosing apoptotic neutrophils, and the pivotal role of AMs in the healing and repair of damaged pulmonary epithelium through the production of HGF.

Role of cytokine-induced neutrophil chemoattractant-2 (CINC-2) alpha in a rat model of chronic bronchopulmonary infections with Pseudomonas aeruginosa.

In order to investigate the role of the cytokine-induced neutrophil chemoattractant (CINC) in chronic bronchopulmonary infection, we developed a rat model of bronchopulmonary infection with Pseudomonas aeruginosa by using the agar bead method, and determined the kinetics of bacterial and cell number, as well as the concentrations of CINC-1, CINC-2, and CINC-3 in bronchoalveolar lavage (BAL) fluids in this model. The bacterial number in the lung rapidly increased from days 1 to 4, and declined 14 days after challenge. Neutrophil number in BAL fluid increased up to one day after challenge, and then slowly decreased during 14 days post-challenge. Among the CINCs, the local production of CINC-2 alpha sharply increased at day 1 and then decreased until day 4 post-challenge, while the local production of CINC-1 slightly increased at day 1 post-challenge. Neither CINC-2 beta nor CINC-3 were detected during the entire course of the infection. Increased CINC-2 mRNA expression in the lung tissue after challenge was associated with CINC-2 alpha production in BAL fluid. Moreover, an immunohistochemical study demonstrated the localization of CINC-1 and CINC-2 alpha primarily in alveolar macrophages and, to a much lesser extent, in bronchial epithelium of infected lung tissues, whereas CINC-2 beta and CINC-3 were not detected. When anti-CINC-1 or anti-CINC-2 alpha polyclonal antibodies were used for neutralizing neutrophil chemotactic activities in BAL fluids, the anti-CINC-2 alpha antibody inhibited 70% of the chemotactic activity in BAL fluids from infected rats at day 1 after challenge. No inhibition was observed by anti-CINC-1 antibody. These data indicate that CINC-2 alpha, which is produced by alveolar macrophages and bronchial epithelial cells, plays a pivotal role in neutrophil accumulation in the airway of a rat model of chronic bronchopulmonary infection with P. aeruginosa.

Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells.

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airway of patients with chronic airway diseases. To investigate the role of P. aeruginosa infection in IL-8 production in the airway of these patients, we examined whether cell lysates of P. aeruginosa could cause IL-8 production from human bronchial epithelial cells. Diluted sonicated supernatants of P. aeruginosa (SSPA) with a mucoid or nonmucoid phenotype stimulated human bronchial epithelial (BET-1A) cells to produce IL-8. In this study, we have purified a 59-kDa heat-stable protein with IL-8-inducing activity from the SSPA by sequential ion-exchange chromatography. The N-terminal sequence of this purified protein completely matched a sequence at the N-terminal part of the mature protein of nitrite reductase from P. aeruginosa. In addition, immunoblotting with a polyclonal immunoglobulin G (IgG) against recombinant Pseudomonas nitrite reductase demonstrated a specific binding to the purified protein. Furthermore, the immunoprecipitates of the SSPA with a polyclonal IgG against recombinant nitrite reductase induced a twofold-higher IL-8 production in the BET-1A cell culture than did the immunoprecipitates of the SSPA with a control IgG. These lines of evidence confirmed that Pseudomonas nitrite reductase was responsible for IL-8 production in the BET-1A cells. The purified nitrite reductase induced maximal expression of IL-8 mRNA in the BET-1A cells at 1 to 3 h after stimulation, and the IL-8 mRNA expression declined by 8 h after stimulation. New protein translation was not required for nitrite reductase-mediated IL-8 mRNA expression in the BET-1A cells. Nitrite reductase stimulated the BET-1A cells, as well as human alveolar macrophages, pulmonary fibroblasts, and neutrophils, to produce IL-8. In contrast, nitrite reductase induced significant levels of tumor necrosis factor alpha and IL-1beta protein only in human alveolar macrophages. These data support the notion that nitrite reductase from P. aeruginosa induces the production of inflammatory cytokines by respiratory cells and may contribute to the pathogenesis of chronic airway diseases and persistent P. aeruginosa infection.

Endogenous tumor necrosis factor (TNF) alpha mediates neutrophil accumulation at the mid-phase of a murine model of Pseudomonas aeruginosa pneumonia.

To determine the role of endogenous tumor necrosis factor (TNF) alpha on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF alpha activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa-infected mice. In the case of fatal pneumonia, the TNF alpha activity in the BALFs appeared within 3 hr, peaked at 6-12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF alpha activity was detected in the plasma. The elevation of TNF alpha activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF alpha activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6-12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF alpha activity in the BALFs with anti-murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0-49.7% at 6 hr and 49.3-54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti-murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre-immune serum). These data suggest that intra-alveolar TNF alpha plays an important role in causing lung neutrophil accumulation at the mid-phase of murine P. aeruginosa pneumonia.

A monoclonal antibody reactive with a common epitope of Moraxella (Branhamella) catarrhalis lipopolysaccharides.

A hybrid cell line producing a monoclonal antibody (MAb) against Moraxella (Branhamella) catarrhalis lipopolysaccharide (LPS) was established. The specificity of the MAb 1B12 to purified rough LPSs from six strains of M. catarrhalis was ascertained by enzyme-linked immunosorbent assay (ELISA), competitive-inhibition ELISA, and immunoblotting. MAb 1B12 bound to live bacterial cells and culture supernatants from a total of 34 strains of M. catarrhalis, including 12 strains with different LPS serotypes. No cross-reactions with smooth and rough LPSs from selected enterobacterial and nonenterobacterial strains, with other respiratory pathogens, or with Neisseria species were observed. These data suggest that MAb 1B12 recognizes a common epitope of M. catarrhalis LPS which differs from serotype determinants.

Airway interleukin-8 in elderly patients with bacterial lower respiratory tract infections.

We investigated the interleukin-8 (IL-8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents. The IL-8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens. We also demonstrated that human recombinant IL-8 at a range of 6.25-25 ng/ml significantly enhanced opsonophagocytic killing of P. aeruginosa immunotype-1 strain by human neutrophils in the presence of a serotype-specific anti-lipopolysaccharide monoclonal antibody and fresh normal human serum. These data suggest that the level of IL-8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL-8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.

Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases.

To evaluate of the role of interleukin-8 (IL-8), a chemotactic cytokine, in the continuous neutrophil accumulation in the airways of patients with chronic airway disease (CAD) and persistent Pseudomonas aeruginosa infection, we investigated the cell population, IL-8 levels, IL-1 beta levels, tumor necrosis factor (TNF) activities, and neutrophil elastase (NE) activities of bronchoalveolar lavage (BAL) fluids in 17 CAD patients (with P. aeruginosa infections [CAD+PA], n = 9; without any bacterial infections [CAD-PA], n = 8) and 8 normal volunteers. We found significant elevations of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from CAD patients, in the following rank order: CAD+PA > CAD-PA > normal volunteers. IL-1 beta/albumin ratios were elevated only in CAD+PA, while no TNF bioactivity was detected in BAL fluids. The neutrophil numbers correlated significantly with the IL-8/albumin ratios and NE/albumin ratios in the BAL fluids of CAD patients. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in BAL fluids, the mean reduction rate of NCF activities in CAD+PA patients was significantly higher than that in CAD-PA patients. We also evaluated the effects of low-dose, long-term erythromycin therapy in BAL fluids from three CAD+PA and two CAD-PA patients. Treatment with erythromycin caused significant reductions of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from these patients. To elucidate the mechanism of erythromycin therapy, we also examined whether erythromycin suppressed IL-8 production by human alveolar macrophages and neutrophils in vitro. We demonstrated a moderate inhibitory effect of erythromycin on IL-8 production in Pseudomonas-stimulated neutrophils but not in alveolar macrophages. Our data support the view that persistent P. aeruginosa infection enhances IL-8 production and IL-8-derived NCF activity, causing neutrophil accumulation in the airways and the progressive lung injuries observed in patients with CAD. The clinical efficacy of erythromycin therapy for CAD patients might be partly mediated through a reduced IL-8 production, diminishing neutrophil accumulation and NE release in the airways.

Therapeutic effects of a human antiflagella monoclonal antibody in a neutropenic murine model of Pseudomonas aeruginosa pneumonia.

Human immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) reactive with type-specific Pseudomonas aeruginosa lipopolysaccharide (LPS) and flagella were compared for their protective activities against Fisher immunotype 2 P. aeruginosa pneumonia in neutropenic mice. The activity of the antiflagella MAb at a dose of 500 micrograms per mouse was comparable to that of the anti-LPS MAb at the same dose. In vivo protection was correlated with bacterial density in the lung tissue and blood of infected mice. In vitro data suggested that the protective activity of the antiflagella MAb was due more to inhibition of bacterial motility than to opsonophagocytosis of bacteria by alveolar macrophages. In contrast, the protective activity of the anti-LPS MAb was primarily related to alveolar macrophage-mediated opsonophagocytosis. Antiflagella MAb at a dose of 500 micrograms combined with oral sparfloxacin at a subtherapeutic dose of 62.5 micrograms produced a significant increase in survival (P < 0.05) compared with that produced by either agent alone or no treatment. The additive effects between the antiflagella MAb and sparfloxacin at sub-MICs on the inhibitory effects of bacterial motility supported the in vivo effect of the combination. These data suggest that human isotype-matched antiflagella and anti-LPS MAbs have similar protective activities against Pseudomonas pneumonia in neutropenic mice, despite discrete mechanisms of antibody-matched protection. In addition, in vivo synergy was demonstrated between antiflagella MAb and sparfloxacin in this model.

Emergence and persistence of Pseudomonas aeruginosa in the cystic fibrosis airway.

Colonization in the respiratory tracts of cystic fibrosis (CF) patients by mucoid Pseudomonas aeruginosa correlates with the progression of bronchial airway pathology. There is a direct correlation between the incidence of Pseudomonas colonization and age, clinical score, extent of pulmonary disease, severity of radiographic changes, and level of serum immunoglobulins. The central propensity to Pseudomonas colonization in patients with CF is not freely understood, but we discuss the acquisition and persistence of P aeruginosa in the CF airway. Elucidation of pathogenetic mechanisms of CF inflammatory airways disease is the first essential step to initiating novel therapies. It has been difficult to prove that the ability of P aeruginosa to adhere to the respiratory epithelium and provide selective advantage for this gram-negative bacillus over other potential pathogens for infection in the CF airway. However, flexible filaments (pili) extending from the Pseudomonas cell wall are thought to medicate epithelial cell adherence for nonmucoid P aeruginosa, and similarly, the gelatinous exopolysaccharide alginate produced by mucoid variants of P aeruginosa seems to be the adhesive to tracheal cells. Following the signal event of adherence, this bacterial pathogen competes successfully for iron cofactor and multiplies, releasing proteases with broad substrate specificities that dramatically alter the airway antiprotease screen, and the pathogen creates defects in local antibacterial defenses. Lung inflammation in CF is characterized by massive neutrophil infiltration. Although critical to host defense, neutrophils also cause progressive airway damage by release of bioactive lipids, oxygen metabolites, and granule enzymes such as hydrolases, myeloperoxidase (MPO), lysozyme, and neutral serine proteases. The necessarily circumscribed discussion that follows will focus narrowly on the host cell-derived factors (macrophages and neutrophils) proposed as important components in this pathogenetic scheme.

Effects of the combination of lipopolysaccharide-specific monoclonal antibodies and sparfloxacin against Pseudomonas aeruginosa pneumonia in neutropenic mice.

The effects of the combination of a murine monoclonal antibody (MAb) specific for the O side chain of Pseudomonas aeruginosa Fisher immunotype 1 lipopolysaccharide and sparfloxacin in a neutropenic mouse model of P. aeruginosa pneumonia were examined. Under the condition that neither MAb at a dose of 500 micrograms per mouse administered intravenously nor a suboptimal dose of oral sparfloxacin (5 mg/kg of body weight) protected mice from challenge with a fatal dose, the combination therapy with MAb and sparfloxacin caused a significant increase in the survival rate (P less than 0.001 compared with either treatment alone). The effect of the combination was closely correlated to bacterial killing in plasma and lung tissue of infected mice. In vitro, a significant MAb-dependent, complement-mediated killing of P. aeruginosa was documented in the presence of sparfloxacin at one-half the MIC, while the killing was not observed in the absence of sparfloxacin. These in vivo and in vitro data suggest the usefulness of combination therapy with a lipopolysaccharide-reactive immunoglobulin G MAb and sparfloxacin in neutropenic patients with P. aeruginosa pneumonia.

[Biochemical analysis of lipopolysaccharides from respiratory pathogenic Branhamella catarrhalis strains and the role of anti-LPS antibodies in Branhamella respiratory infections].

We characterized lipopolysaccharides (LPSs) from respiratory pathogenic Branhamella catarrhalis (BC) strains, and evaluated the protective property of anti-BC LPS antibody in BC respiratory infections. LPSs from four strains of BC were lipooligosaccharide having no O-side chain and a M(r) of 3 KDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All of them produced different patterns, showing two to four bands on SDS-PAGE. We found high level of anti-BC IgG antibody in convalescent sera from a patient with BC respiratory tract infection by ELISA. This IgG antibody recognized BC LPSs on Western blots. Two respiratory pathogens of BC (strains; 87-122, 88-23) were tested in a bactericidal assay employing a convalescent sera. 87-122 strain was susceptible to antibody-dependent, complement-mediated killing, while 88-23 strain was resistant. The killing of 87-122 strain was inhibited by addition of the homologous BC LPS to the convalescent sera in a dose-dependent manner. These data support that anti-BC LPS antibody may mediate complement-lysis of some strains of BC, and play a protective role in BC respiratory infections.

[Serum sensitivity of Pseudomonas aeruginosa isolated from sputum as a virulence factor in the lower respiratory tract].

To elucidate the clinical significance of serum-sensitivity of respiratory pathogenic Pseudomonas aeruginosa (P. aeruginosa) strains, we examined serum-sensitivity of P. aeruginosa isolated from 16 patients with lower respiratory tract infections and clinical backgrounds of these patients. We also evaluated the virulence of four serum-resistant and four serum-sensitive P. aeruginosa strains in murine pneumonia model induced by intratracheal challenge, and the silver-stained profiles of purified lipopolysaccharide (LPS) from these strains. Serum-sensitive strains were isolated only from patients with chronic bronchitis, bronchiectasis, and diffuse panbronchiolitis colonized with P. aeruginosa, and rarely caused pneumonias, while serum-resistant strains caused pneumonias in some cases. Intratracheal challenge of mice with 5 x 10(7) cfu per mouse of a serum resistant strain caused fatal hemorrhagic pneumonia with bacteremia. In contrast, the same dose of a serum-sensitive strain provided non-fatal pneumonia without bacteremia. LD50 of serum-sensitive strains in a murine model of P. aeruginosa pneumonia were at least 2-10 times higher than those of serum-resistant strain. The LPS profiles of two serum-resistant strains and one serum-sensitive strain showed ladder-like patterns. The similar analysis demonstrated that one serum-sensitive strain was lack of ladder-like patterns. These data support that serum-sensitive P. aeruginosa strains are less virulent than serum-resistant P. aeruginosa strains in the lower respiratory tract, and serum sensitivity of P. aeruginosa strains is determined by the structure of the O-side chain of LPS; either lack of the O-side chain or the presence of sparse O-side chain.

Pharmacodynamic and protective properties of a murine lipopolysaccharide-specific monoclonal antibody in experimental Pseudomonas aeruginosa pneumonia in mice.

We employed a Pseudomonas aeruginosa mouse pneumonia model to evaluate the ability of a murine monoclonal antibody (MAb) specific for the O-side chain of P. aeruginosa Fisher Immunotype-1 lipopolysaccharide (LPS) to achieve and sustain therapeutic levels in plasma and lung tissue, reduce bacterial populations in the lung, and prevent pneumonia-associated mortality. An IgG3 MAb (Y1-5A4) administered to mice i.v. over a dose range of 125-1,000 micrograms/mouse produced plasma and lung tissue levels at 2 hr of 61-507 micrograms/ml and 4.3-150 micrograms/g, respectively. The 1,000 micrograms MAb dose reduced bacterial counts in lung tissue (log10 cfu/g +/- S.D.) and blood (log10 cfu/ml +/- S.D.) 20 hr post-treatment (18 hr post-challenge) from 10.00 +/- 0.66 to 7.66 +/- 0.91 (P less than 0.01) and from 4.39 +/- 0.81 to less than 3.0, respectively. Administration of MAb to mice in doses of 125-500 micrograms 2 hr prior to a 3 x 50% lethal bacterial challenge produced significant protection against death, with a calculated 50% protective dose of 167 micrograms. Protection was noted following administration of 1,000 micrograms of MAb up to 6 hr after bacterial challenge (P less than 0.05, compared with untreated control). Histological examination of lung tissue from infected mice revealed less acute inflammation, necrosis, and hemorrhage in MAb-treated compared with untreated control animals and greater localization of Pseudomonas antigen within the phagocytic cells in alveolar space. These findings document the in vivo therapeutic efficacy of an LPS-specific IgG MAb in a murine model of acute P. aeruginosa pneumonia, based in part upon the achievability of effective MAb concentrations in plasma and lung tissue.

[The reliability of fine needle aspiration cytology in the diagnosis of breast cancer].

Between September 1981 and December 1987, 1023 palpable breast tumors were submitted for cytological evaluation by fine needle aspiration before surgery (carcinoma: 793 cases, benign breast diseases: 230 cases). Clinical factor relating to the success of the aspirations were evaluated. Cases were regarded as positive when patients had a grade IV or more rating by Papanicolaou classification. The sensitivity was shown to be 73% (578/793); the specificity being 98% (226/230). A significantly lower rate of positive cancer identification occurred in smaller tumors (less than 2 cm, P less than 0.01) or scirrhous carcinomas (P less than 0.05). The comparison of positive rates between the aspiration cytology (66%) and the stamp cytology (95%) in 258 breast cancers (P less than 0.01) indicates the limited value of cytological diagnosis of breast cancers by fine needle aspiration.

Elevation of sialyl stage-specific mouse embryonic antigen levels in pleural effusion in patients with adenocarcinoma of the lung.

Sialyl stage-specific mouse embryonic antigen (SSEA-1) levels were measured in pleural effusions obtained from patients with lung cancer and benign pulmonary disease, using a solid-phase immunoradiometric sandwich assay. The mean (+/- SEM) levels (unit/ml) of pleural fluid sialyl SSEA-1 were 3620 +/- 1419 in adenocarcinoma (n = 25), 123 +/- 30 in nonadenocarcinoma (n = 13) and 95 +/- 19 in benign pulmonary disease (n = 13), respectively. The positive rate was 64% in adenocarcinoma, 7.7% in nonadenocarcinoma, and 0% in benign pulmonary disease, respectively, when a cutoff level was defined as the mean + 3 SD value (300 unit/ml) based on pleural fluid sialyl SSEA-1 levels in benign pulmonary disease. There was a significant positive correlation between pleural fluid levels of sialyl SSEA-1 and those of carcinoembryonic antigen in adenocarcinoma patients (r = 0.8246, P less than 0.01). Pleural fluid sialyl SSEA-1 levels correlated with cytologic findings in adenocarcinoma patients. These observations suggest that sialyl SSEA-1 in pleural effusion is a useful marker to discriminate malignant from nonmalignant and adenocarcinoma from nonadenocarcinoma of the lung.