Characterization of bronchoalveolar lavage cells and macrophage-derived chemoattractant activity in pancreatic elastase-induced emphysema in hamsters

Bronchoalveolar lavage (BAL) was performed 30 days after endotracheal saline (C) or pancreatic elastase (E) administration to hamsters. E animals had twice as many cells as C animals (7.04 +/- 0.07 vs. 6.69 +/- 0.05 log10 cells, p less than 0.0001, paired t-test) and greater numbers of polymorphonuclear neutrophils (PMN) (6.61 +/- 0.05 vs. 5.27 +/- 0.04 log10 cells/animal, p less than 0.0001, t-test). On flow cytometric analysis and cell sorting, BAL cells from C animals showed a homogeneous population of cells with high light scatter and laser-excited intrinsic autofluorescence that were predominantly (greater than 99%) pulmonary alveolar macrophages (PAM). BAL cells from E animals had 80% PAM with characteristics similar to those of C animals; the remaining 20% of cells were smaller with lower forward and 90 degrees light scatter and four- to fivefold lower autofluorescence. These were found to be predominantly PMN on cytological examination of a sorted fraction. Fc receptors were expressed by greater than 95% of BAL cells from both C and E animals. To explain the altered cell population in E animals, PAM from both E and C animals were cultured in vitro, and the supernatants were assayed for neutrophil chemoattractant activity (NCA). In vitro PAM from E animals produced greater NCA than control PAM after 48 h in culture when stimulated with aggregated immunoglobulin or serum activates zymosan (p less than or equal to 0.003, ANOVA). Analyses of BAL supernatants 24 h after elastase administration indicated the presence of a heat-stable chemoattractant for PMN; such chemotactic activity, however, was not detectable 1 week or 4 weeks after the administration of elastase.     

Pulmonary recruitment, adherence, and chemotaxis of neutrophils in a rat model of cirrhosis and pneumococcal pneumonia

Cirrhotic rats have decreased pulmonary bactericidal activity and increased bacteremia after experimental pneumococcal pneumonia. To determine if this finding is due to impaired pulmonary recruitment of polymorphonuclear leukocytes (PMNL), bronchoalveolar lavage (BAL) was done on cirrhotic and normal rats after transtracheal challenge with pneumococcal types 3 and 1. Mean absolute numbers of recruited PMNL in BAL fluid (BALF) at 2, 4, 6, 8, and 24 h after 10(7) cfu of type 3 challenge were similar in cirrhotic and normal rats. In both groups, lower numbers of PMNL were recruited after challenge with 10(5) cfu of type 3. Type 1 pneumococci stimulated recruitment of similar mean absolute numbers of PMNL (x10(7] in BALF (cirrhotics vs. normals) at 24 h after challenges with 10(5) cfu (0.3 +/- 0.1 vs. 0.3 +/- 0.1) and 10(7) cfu (2.9 +/- 1.3 vs. 2.8 +/- 0.7). Peripheral blood PMNL from cirrhotic and normal rats did not differ in adherence to nylon wool columns or in chemotaxis toward lipopolysaccharide-activated normal rat serum. Thus the impaired pulmonary defense against pneumococcal pneumonia in cirrhosis is not due to deficient pulmonary PMNL recruitment.     

The effects of systemic immunization of pulmonary clearance of Pseudomonas aeruginosa

Systemic immunization with gram-negative organisms enhances the subsequent pulmonary clearance of these organisms. We studied the early time course of this phenomenon and related it to the time of appearance of polymorphonuclear leukocytes (PMN) and anti-Pseudomonas antibody in bronchoalveolar lavage (BAL). Mice were immunized intraperitoneally twice, separated by 1 wk, with 10(8) formalin-treated Pseudomonas aeruginosa. Two weeks later, they received an intrabronchial inoculum of 2.9 X 10(6) or 4.6 X 10(7) Pseudomonas organisms. Two, 4, and 6 h later, clearance and total PMN and anti-Pseudomonas antibody in the BAL were assessed. Clearance was enhanced in immunized mice at the lower inoculum. At the higher inoculum, bacteria were growing in lungs of both groups, although they were inhibited in immunized mice. Total PMN in the BAL increased progressively in both groups of mice, but net recruitment was diminished with the high inoculum. There were significant differences in the PMN in the BAL between control and immunized mice with high inoculum. Anti-Pseudomonas IgG first appeared in the BAL at 2 h, anti-Pseudomonas IgM at 6 h. These data suggest that anti-Pseudomonas IgG is an effective early pulmonary opsonin. Further, with high inoculums, immunization may aid pulmonary defenses by diminishing the magnitude of the decrement of PMN in the lung.     

Erythromycin-induced suppression of pulmonary antibacterial defenses. A potential mechanism of superinfection in the lung

Erythromycin is a broad-spectrum antibiotic commonly used in patients with respiratory infections. Certain of these patients become colonized with new microorganisms and develop superinfections. Antibiotics have a number of effects other than simply killing or inhibiting the growth of bacteria and may have direct effects upon host cells, including phagocytes. In vitro and in vivo studies have demonstrated that erythromycin decreases polymorphonuclear leukocyte (PMN) directed migration. To test the hypothesis that erythromycin inhibits normal PMN migration into the alveoli in response to a bacterial challenge, mice were challenged by aerosol inhalation with Proteus mirabilis or Staphylococcus aureus and injected intravenously with erythromycin (50 or 100 mg/kg). Pulmonary bactericidal activity and total lavaged lung cell and differential counts were determined 4 h after bacterial challenge. In control mice, only 24 +/- 2% of the initial inoculum of P. mirabilis was viable at 4 h. At a dose of 100 mg/kg, lung defenses after erythromycin were ablated, allowing the proliferation of P. mirabilis to 113 +/- 5% of the initial inoculum. The number of PMN obtained by lavage after P. mirabilis challenge was also inhibited by erythromycin in a dose-dependent manner. In untreated animals, 5.0 +/- 0.2 x 10(6) PMN were recovered as compared with 3.1 +/- 0.4 x 10(6) and 1.1 +/- 0.3 x 10(6) with increasing doses of erythromycin. Intrapulmonary bactericidal activity against S. aureus was not impaired by erythromycin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between the inoculum dose of Streptococcus pneumoniae and pneumonia onset in a rabbit model.

It is generally assumed that the development of bacterial pneumonia becomes possible when the dose of inhaled or aspirated pathogens overwhelms the respiratory tract host defence system, but this hypothesis has not yet been tested either clinically or experimentally. This study evaluated inoculum dose in relation to onset of experimental pneumococcal pneumonia, and estimated the median effective dose resulting in pneumonia in healthy New Zealand White rabbits (mean+/-sd 4.75+/-0.25 kg (n = 27)). Rabbits were endobronchially inoculated with increasing doses of Streptococcus pneumoniae and pneumonia onset was observed over the following 96 h. The diagnostic approach was based on the Clinical Pulmonary Infection Score, modified for use in rabbits. Inoculation of S. pneumoniae at doses of >4.60 log(10) cfu made the development of pneumonia in rabbits more predictable (up to 90%). Lower doses of bacteria failed to cause pneumonia in 80% of inoculated animals. The median effective dose was estimated by means of logistical regression, probit analyses and the Reed-Muench method, and corresponded to 4.32, 4.38 and 4.67 log(10) cfu, respectively. It is speculated that development of pneumococcal pneumonia becomes more likely when the inoculum dose exceeds a threshold of antibacterial protection, making inoculum dose a risk factor for disease onset.     

The relationship between bronchoalveolar neutrophil recruitment and bronchoconstriction induced by a soluble extract of cotton bracts

Byssinosis is characterized by an obstructive bronchitis in some workers exposed to cotton dust. Mechanisms of airway alterations induced by cotton dust are unknown. To study mechanisms of these alterations, we administered an aqueous extract of cotton bract (CBE) to normal human subjects. After CBE inhalation, we demonstrated reproducible reductions in flow rates of variable magnitude. The percentage of polymorphonuclear leukocytes (PMN) recovered by bronchoalveolar lavage (BAL) and total PMN per milliliter BAL fluid were increased after challenge with CBE, and these parameters were correlated (r = 0.556 and r = 0.553, respectively) with the degree of bronchoconstriction induced by CBE. In addition, chemotactic factors for PMN were noted in BAL fluid and in supernatant from BAL cell cultures. The degree of BAL fluid chemotactic activity correlated with the degree of bronchoconstriction induced by CBE. Assessment of molecular sieve column effluent demonstrated BAL fluid to have 3 distinct peaks of chemotactic activity (approximate molecular weight, greater than 25,000, 10,000, and less than 1,000 daltons) and cell culture supernatant to contain 1 peak (approximate MW, 10,000 daltons). Complement activation as one source of BAL chemotactic activity was suggested by almost complete inhibition of BAL chemotactic activity after incubation with antibody to the fifth component of complement (C5) and detection of C5a des Arg in BAL fluid from some subjects challenged with CBE. These findings suggest that exposure of volunteer subjects to CBE results in a bronchoalveolar inflammatory response related to both complement activation and chemotactic factor synthesis by luminal cells.     

Alcohol-induced suppression of lung chemokine production and the host defense response to Streptococcus pneumoniae

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

The pulmonary clearance of smooth and rough strains of Pseudomonas aeruginosa

Upper airway colonization with rough strains of Pseudomonas is associated with clinical deterioration in patients with cystic fibrosis. These rough strains are less toxic than smooth strains in the burned mouse model and in vitro assays. We measured the 4-h pulmonary clearance of 10(4) and 10(6) rough and smooth Pseudomonas after intrabronchial inoculation. After 10(4) Pseudomonas, rough strains were cleared more efficiently than smooth strains (89 +/- 13% versus 140 +/- 19% of the original inoculum, respectively, p less than 0.05). This was associated with more total bronchoalveolar lavage PMNs after inoculation with rough as compared with smooth Pseudomonas (1.60 +/- 0.38 x 10(5) versus 0.55 +/- 0.09 x 10(5), respectively, p less than 0.05). After inoculation with 10(6) Pseudomonas, rough were cleared less efficiently than smooth Pseudomonas (380 +/- 45% versus 134 +/- 12% of the original inoculum, respectively, p less than 0.05). There was no difference in the BAL PMNs of the animals inoculated with either bacterial strain at this inoculum (8.1 +/- 0.6 x 10(5) and 9.4 +/- 1.0 x 10(5) for the rough and smooth Pseudomonas, respectively). When PMN differences were abolished in C5-deficient mice, 10(4) rough were cleared less efficiently than smooth Pseudomonas. The relatively poor clearance of rough Pseudomonas could not be explained by in vitro differences in PMN killing or BAL toxicity or by intrinsic growth rates.     

Alcohol intoxication inhibits pulmonary S100A8 and S100A9 expression in rats challenged with intratracheal lipopolysaccharide

BACKGROUND: Alcohol is known to inhibit the recruitment of polymorphonuclear leukocytes (PMNs) into tissue sites including the lung. During infection and inflammation, recruited neutrophils (PMNs) release S100 proteins that function to promote the recruitment of additional phagocytes. METHODS AND RESULTS: This study investigated the effects of alcohol intoxication on S100 protein production in the lung in response to lipopolysaccharide (LPS). Animals were administered alcohol (5.5 g/kg) or saline 30 minutes before intratracheal challenge with LPS (100 microg/rat). Alcohol suppressed PMN recruitment into the lung following intratracheal LPS, which was associated with an inhibition of increase in S100A8 levels in both the bronchoalveolar lavage (BAL) fluid and lysates of cells recovered by BAL at 90 minutes and 4 hours post-LPS challenge. S100A8 and S100A9 mRNA expression in cells recovered by BAL was significantly up-regulated at both 90 minutes and 4 hours after the LPS challenge, and alcohol also suppressed this response. In addition, intratracheal LPS caused a transient increase in S100A8 mRNA expression in circulating leukocytes at 90 minutes after the challenge. Similarly, this LPS-induced up-regulation of S100A8 mRNA expression was inhibited in rats intoxicated with alcohol. CONCLUSION: These data show that alcohol inhibits the S100 protein response in the lung, which may serve as a mechanism underlying alcohol-induced suppression of PMN recruitment into the terminal airways during pulmonary infection.     

Neutrophil chemotactic factors in bacterial pneumonia

The influx of neutrophils into the lung is a prominent feature in patients with bacterial pneumonia. Since neutrophils migrate in response to chemotactic factors, chemotactic activity was evaluated in bronchoalveolar lavage (BAL) fluid obtained from 12 patients with bacterial pneumonia and ten normal control subjects. Chemotactic activity was greatly elevated in the BAL fluid of the pneumonia patients compared with control subjects (p less than 0.01). To partially characterize the chemotactic factors present in the lavage fluid of the patient group, molecular sieve chromatography was performed on the lavage fluid, and at least three peaks of chemotactic activity were identified. Since the molecular weight of the smaller peaks approximated the molecular weight of two known chemotactic factors, C5a and leukotriene B4, these factors were measured in lavage fluid by radioimmunoassay. C5a was detectable in none of the normal subjects but was detectable in four of 14 BAL samples obtained from the patients. Leukotriene B4 was detectable in all subjects and was significantly elevated in the pneumonia patients (552 +/- 95 vs 81 +/- 16 pg/ml, p less than 0.01). These findings demonstrate that elevated neutrophil chemotactic activity is present in the lungs of patients with bacterial pneumonia and suggest that C5a and leukotriene B4 may account, at least in part, for this increase.     

Intrapulmonary chemotaxins in the normal and in the cyclophosphamide-treated host

The kinetics of intrapulmonary chemotactic activity and the generation of a pulmonary polymorphonuclear leukocytic (PMN) response during experimental Pseudomonas aeruginosa pneumonia were studied in normal and in cyclophosphamide-treated guinea pigs. In normal animals, chemotactic activity for PMN appeared in airways promptly (2 h) after infection and preceded the influx of PMN to infected airways. Week-long regimens of intraperitoneally administered cyclophosphamide, in dosages of 7.5 mg/kg/day (low-dose) or 15 mg/kg/day (high-dose), resulted in systemic myelosuppression accompanied by a dose-related decrease in recruitment of PMN to infected airways. The chemotactic activity assayed in bronchoalveolar fluids obtained from low-dose-treated animals was not affected by cyclophosphamide. However, chemotactic activity in bronchoalveolar fluids was significantly reduced (p less than 0.01) in animals receiving the high-dose cyclophosphamide regimen. Gel chromatography of bronchoalveolar fluids from infected animals revealed that a high molecular weight (20,000 daltons or greater) and a low molecular weight (5,000 daltons) chemotactic factor were present in normal specimens, and that both were absent from specimens obtained from animals receiving the high-dose treatment. Hemolytically active C5 was detected in infected bronchial fluids, but cyclophosphamide treatment did not reduce amounts of C5 in infected airways. These data suggest that in addition to myelosuppression, cyclophosphamide treatment impairs the capacity for pulmonary inflammation by reducing the normal intrapulmonary chemotactic gradient during infection.     

Impairment of antibacterial defense mechanisms of the lung by extrapulmonary infection

To determine whether extrapulmonary infection alters antibacterial defenses of the lung, we challenged mice with peritonitis due to Escherichia coli by aerosol inhalation with either Staphylococus aureus or Pseudomonas aeruginosa. In animals without peritonitis, 14% +/- 5% and 11% +/- 1% of the initially deposited viable S. aureus and P. aeruginosa, respectively, remained in the lungs at 4 hr. In contrast, in mice with peritonitis, at 4 hr 45% +/- 9% of the staphylococci were recoved, and the P. aeruginosa had increased to 948% +/- 354% of the initial inoculum. Proliferation of P. aeruginosa in mice with peritonitis was associated with impaired recruitment of polymorphonuclear neutrophils (PMNs) into the lungs. In contrast, a noninfectious stimulus induced more PMNs into the peritoneal cavity than did intraabdominal sepsis but only minimally impaired PMN recruitment into the lungs after aerosol challenge with P. aeruginosa. Sterile intraperitoneal stimulation did not significantly impair intrapulmonary killing of P. aeruginosa. Levels of antigenic C3 and functionally active C5 were significantly depleted in mice with peritonitis due to E. coli. We conclude that the systemic effects of sepsis, including complement depletion, contribute to the decreased pulmonary PMN recruitment and to impaired intrapulmonary bacterial killing of animals with peritonitis due to E. coli.     

Aminophylline-induced suppression of pulmonary antibacterial defenses

Respiratory infections are frequently observed in patients with chronic obstructive pulmonary disease, indicating that host defenses are compromised. Antibacterial defenses of the lung against such infections include the alveolar macrophage and polymorphonuclear leukocytes (PMN) that migrate into the lung to provide auxiliary phagocytic defenses. To test the hypothesis that aminophylline acutely impairs pulmonary antibacterial defenses, mice were challenged by aerosol inhalation with Staphylococcus aureus or Proteus mirabilis and injected intraperitoneally with aminophylline (20, 40, or 80 mg/kg). Pulmonary bactericidal activity and total lavaged lung cell and differential counts were determined 4 h after bacterial challenge. The highest dose of aminophylline suppressed the killing of S. aureus so that 55 +/- 5% of the initial viable bacteria remained as compared with 22 +/- 4% in the control animals. In contrast, there was a dose-related suppression of pulmonary antibacterial defenses against gram-negative bacteria. With doses of 40 and 80 mg/kg, lung defenses were ablated, allowing the proliferation of P. mirabilis to 115 +/- 9% and 253 +/- 9%, respectively, the control value being 26 +/- 3%. The number of PMN obtained by lavage after aerosol challenge with P. mirabilis was also inhibited by aminophylline in a dose-dependent manner. From the lungs of untreated animals 5.0 +/- 0.3 X 10(6) PMN were recovered as compared with 3.3 +/- 0.1 X 10(6), 2.5 +/- 0.2 X 10(6), and 1.8 +/- 0.1 X 10(6), respectively, with increasing doses of aminophylline. The bactericidal activity of lavaged PMN from the lungs of aminophylline-treated rats challenged with the gram-negative bacterium in vivo was significantly depressed when compared with that in control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Granulocyte depletion prevents tumor necrosis factor-mediated acute lung injury in guinea pigs

To examine the role of polymorphonuclear neutrophils (PMN) and other granulocytes in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we compared the permeability edema and pulmonary histopathology in normal (granulocyte sufficient) guinea pigs and in granulocytopenic guinea pigs treated with TNF. Circulating granulocytes were depleted with cyclophosphamide. Two groups of normal animals were treated with either saline (PMN+/Control) or 1.4 x 10(6) U/kg recombinant human TNF (PMN+/TNF). Three granulocytopenic groups were treated with either saline (PMN-/Control), TNF (PMN-/TNF), or intravenous infusion of 2 x 10(9) E. coli strain J96 (PMN-/Sepsis). We measured the amount of 125I-labeled albumin in bronchoalveolar lavage (BAL) fluid and whole lung tissue and the wet/dry lung weight ratio to assess pulmonary transvascular protein flux and edema. We also quantified PMN in BAL fluid and fixed lung tissue. There were no statistically significant differences in any of these parameters between the PMN+/Control, PMN-/Control, or PMN-/TNF groups, except that the PMN+/Control predictably had more PMN/alveolus than the PMN- groups. However, both the PMN+/TNF and the PMN-/Sepsis groups had increased amounts of 125I-labeled albumin in BAL fluid and lung tissue (p less than 0.01) and increased wet/dry lung weight ratios (p less than 0.05), compared to all other groups. Histopathologically, capillary congestion and moderate inflammation were seen in the PMN+/TNF group, and acute inflammation and gross alveolar hemorrhage were seen in the PMN-/Sepsis group.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of hyperoxia on pulmonary clearance of Pseudomonas aeruginosa

Among the toxic effects of hyperoxia may be impaired pulmonary clearance of gram-negative bacteria. To better define this effect, we exposed BALB/c mice to 100% O2 for 24, 48, or 72 hr and intrabronchially inoculated them with 10(6) Pseudomonas aeruginosa. Clearance was assessed 4 hr later by quantitative lung cultures of air- and O2-exposed mice. Clearance was first reduced at 48 and 72 hr in mice exposed to O2. To determine the mechanism responsible, we measured bronchoalveolar lavage neutrophil (PMN) counts and neutrophil chemotactic activity at 0, 2, and 4 hr after instillation of P. aeruginosa into mice first exposed to air or O2 for 48 hr. Air-exposed mice had more PMNs than did O2-exposed mice after challenge (13.3 +/- 2.1 X 10(5) vs. 4.4 +/- 0.6 X 10(5)). There was no difference in neutrophil chemotactic activity between air- and O2-exposed mice at any time, although chemotactic activity increased in both groups after challenge. Our data suggest that hyperoxia impairs pulmonary clearance of P. aeruginosa by decreasing the influx of PMNs and that this effect is not due to diminished chemotactic activity in bronchoalveolar lavage fluid.     

Acute cigarette smoke exposure in dogs: the inflammatory response

Acute cigarette smoke causes polymorphonuclear leukocyte (neutrophil, PMN) recruitment to the lung followed by loss of elastase from the recruited cells. Dogs were exposed to cigarette smoke with different oxidant content, bronchoalveolar lavage (BAL) was performed, and the cell distribution in the recovered alveolar lining fluid was analyzed. Exposures were 1, 3, or 6 cigarettes on one or multiple days with a maximum dose of 42 cigarettes. The mean percent PMN present in control lavage was 2.01%, while the mean percent PMN recovered in BAL after a dose of 42 1R1 cigarettes was 13.05%. Recoverable PMN, after a single exposure to three 1R1 cigarettes, also increased from 1.7 to 10.4% by 15 h after cessation of smoke exposure. The cell response for multiple (2 and 7) day exposures was similar. The elastase content per BAL neutrophil decreased relative to peripheral blood PMN from the same animals. No free elastolytic activity was found in BAL, but PMN elastase antigen was present. Increased frequency of cigarette smoke exposure delayed the return to homeostatic cell conditions. The increased PMN accumulation observed may result in an increased proteolytic load in the pulmonary interstitium and contribute to the pathogenesis of emphysema.     

Modulation by pentobarbital of neutrophil responses to inhaled E. coli endotoxin in sheep: role of lung epithelium.

Neutrophils (PMNs) are implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). The role of the epithelium in the modulation of PMN migration within the lungs was examined. Epithelial integrity and PMN concentrations in the lung air spaces and lymph were measured in sheep anaesthetized with either halothane (1-2.5%) or intravenous pentobarbital (12+/-4 mg x kg(-1) x h(-1)). Ventilation with an aerosol containing 25 mg Escherichia Coli endotoxin (lipopolysaccharide; LPS) effected neutrophil recruitment to the air spaces. Lymphatic clearance of aerosolized 99mTc-DTPA provided an index of epithelial integrity. Three hours after the deposition of LPS, the lung lining fluid of sheep anaesthetized with halothane (n=7) had 4.9+/-3.2x10(6) PMN.mL(-1), but the lung lymph had almost no PMNs (3+/-8%). Sheep anaesthetized with pentobarbital (n=6) had fewer PMNs in the air spaces (2.4+/-1.2X10(6) mL(-1)) and more PMNs in the lung lymph (30+/-20%). Control sheep (n=5) that received no LPS had almost no PMNs in the airspaces or lung lymph, regardless of the anaesthesia. Three additional sheep that remained awake after receiving LPS also had no PMNs in the lung lymph. The PMN fraction in the lung lymph correlated well with the extra-alveolar epithelial permeability measured by lymphatic clearance of aerosolized diethylenetriamine penta-acetic acid (r=0.81, p<0.001). Aerosolized lipopolysaccharide recruits neutrophils into the lungs of sheep, but they appear to remain in the airspaces unless extra-alveolar permeability is increased by agents such as pentobarbital.     

Efficacy of recombinant human granulocyte colony-stimulating factor in a murine model of pneumococcal pneumonia: effects of lung inflammation and timing of treatment

The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a murine model of pneumococcal pneumonia was examined. Intranasal inoculations were 10(7) cfu/mouse (high inoculum) and 5 x 10(4) cfu/mouse (low inoculum) of Streptococcus pneumoniae, which induced severe or mild lung inflammation, respectively. With the low inoculum, rhG-CSF significantly improved survival when initiated 24 h or 10 min before, but not when initiated 24 h after, infection. Pretreatment with rhG-CSF significantly increased myeloperoxidase (MPO) activity in lungs 8 h after the infection and increased circulating neutrophil count 24, 48, and 72 h after infection. In contrast, rhG-CSF did not improve survival of animals infected with the high inoculum and did not increase MPO activity or neutrophil count in blood over those of sham-treated controls. These data strongly suggest that the severe inflammatory response typically observed in pneumococcal pneumonia recruits a maximum number of neutrophils in the lungs and thus masks the beneficial effect of rhG-CSF.     

The cardiac glycoside digoxin disrupts host defense in experimental pneumococcal pneumonia by impairing neutrophil mobilization

Normal CD-1 mice were administered digoxin (4 micrograms/kg/24 h) and infected with type 3 Streptococcus pneumoniae in order to assess the effects of the cardiac glycoside on the chemotactic responsiveness of peripheral blood neutrophils and the mobilization of granulocytes from storage pools. The chemotactic responses to autologous zymosan-activated serum (C5a) by neutrophils obtained from uninfected digoxin-treated and control animals were similar; comparable observations were made with circulating granulocytes isolated from animals at 24 or 48 h after intratracheal challenge with 5 x 10(5) colony-forming units (cfu) of bacteria. However, at 4 and 6 h after intratracheal pneumococcal challenge, the number of immature neutrophils in the peripheral blood was significantly lower in the glycoside-treated animals versus controls; at 24 and 48 h, these differences were not apparent. Following the intravenous inoculation of pneumococci, the number of circulating immature neutrophils was also found to be significantly lower at 4 and 24 h in animals given the cardiac glycoside versus controls. We conclude that digoxin disrupts host defense in experimental pneumococcal pneumonia and bacteremia by impairing the mobilization of neutrophils.     

Enhanced PMN response in chronic bronchitis and community-acquired pneumonia.

Chronic bronchitis is a frequent underlying disease in community-acquired pneumonia (CAP). It is unclear to what extent an impaired or exaggerated innate immune response contributes to disease manifestations and severity. To assess the role of neutrophil activation and recruitment during acute pneumonic episodes, peripheral polymorphonulcear neutrophil (PMN) activation, chemotactic activity, interleukin-8 (CXCL-8) and CXCL-8 receptor (CXCR) expression and apoptosis rate were evaluated in CAP patients with and without chronic bronchitis. In addition, the expression of CXCRs and CXCL-8 was assessed on pulmonary neutrophils in chronic bronchitis patients to compare the activation of the chemokine system in different compartments. CAP severity was assessed by the simplified acute physiology score II and the prognosis of disease was assessed by the pneumonia severity index (PSI). An increased chemotactic activity of PMN from chronic bronchitis patients with CAP was found, which was not related to the expression of CXCRs. In addition, a decreased apoptosis rate of PMN was observed. Chemotactic activity was related to the PSI. Comparison of peripheral and pulmonary PMN revealed enhanced CXCL-8 levels and a decreased CXCR expression in the lung. In conclusion, neutrophil function in patients with chronic bronchitis and community-acquired pneumonia is characterised by an increased chemotactic activity combined with a decreased apoptosis rate. The downregulation of interleukin-8 receptors in the pulmonary compartment deserves further investigation.