A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages.

Our laboratory has developed a flow cytometric assay to quantify alveolar macrophage (Mcapital EF, Cyrillic) phagocytosis of bacteria within a live animal. Mcapital EF, Cyrillics collected by bronchoalveolar lavage from rats infected transtracheally with Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry to quantify their bacterial uptake.

Repetitive organic dust exposure in vitro impairs macrophage differentiation and function.

BACKGROUND: Organic dust exposure in the agricultural industry results in significant airway disease and lung function decrease. Mononuclear phagocytes are key cells that mediate the inflammatory and innate immune response after dust exposure. OBJECTIVE: We sought to investigate the effect of organic dust extract (ODE) from modern swine operations on monocyte-derived macrophage (MDM) phenotype and function. METHODS: Peripheral blood monocytes were obtained by means of elutriation methodology (>99% CD14(+)) and differentiated into macrophages in the presence of GM-CSF (1 week) with and without ODE (0.1%). At 1 week, cells were analyzed by means of flow cytometry for cell-surface marker expression (HLA-DR, CD80, CD86, Toll-like receptor 2, Toll-like receptor 4, mCD14, and CD16), phagocytosis (IgG-opsonized zymosan particles), and intracellular killing of Streptococcus pneumoniae. At 1 week, MDMs were rechallenged with high-dose ODE (1%), LPS, and peptidoglycan (PGN), and cytokine levels (TNF-alpha, IL-6, IL-10, and CXCL8/IL-8) were measured. Comparisons were made to MDMs conditioned with heat-inactivated dust, endotoxin-depleted dust, LPS, and PGN to elucidate ODE-associated factors. RESULTS: Expression of HLA-DR, CD80, and CD86; phagocytosis; and intracellular bacterial killing were significantly decreased with ODE-challenged versus control MDMs. Responses were retained after marked depletion of endotoxin. PGN, LPS, and PGN plus LPS significantly reduced MDM surface marker expression and, except for LPS alone, also reduced phagocytosis. ODE-challenged MDMs had significantly diminished cytokine responses (TNF-alpha, IL-6, and IL-10) after repeat challenge with high-dose ODE. Cross-tolerant cytokine responses were also observed. CONCLUSION: Repetitive organic dust exposure significantly decreases markers of antigen presentation and host defense function in MDMs. Bacterial cell components appear to be driving these impaired responses.

Cirrhosis-induced defects in innate pulmonary defenses against Streptococcus pneumoniae.

BACKGROUND: The risk of mortality from pneumonia caused by Streptococcus pneumoniae is increased in patients with cirrhosis. However, the specific pneumococcal virulence factors and host immune defects responsible for this finding have not been clearly established. This study used a cirrhotic rat model of pneumococcal pneumonia to identify defect(s) in innate pulmonary defenses in the cirrhotic host and to determine the impact of the pneumococcal toxin pneumolysin on these defenses in the setting of severe cirrhosis. RESULTS: No cirrhosis-associated defects in mucociliary clearance of pneumococci were found in these studies, but early intrapulmonary killing of the organisms before the arrival of neutrophils was significantly impaired. This defect was exacerbated by pneumolysin production in cirrhotic but not in control rats. Neutrophil-mediated killing of a particularly virulent type 3 pneumococcal strain also was significantly diminished within the lungs of cirrhotic rats with ascites. Levels of lysozyme and complement component C3 were both significantly reduced in bronchoalveolar lavage fluid from cirrhotic rats. Finally, complement deposition was reduced on the surface of pneumococci recovered from the lungs of cirrhotic rats in comparison to organisms recovered from the lungs of control animals. CONCLUSION: Increased mortality from pneumococcal pneumonia in this cirrhotic host is related to defects in both early pre-neutrophil- and later neutrophil-mediated pulmonary killing of the organisms. The fact that pneumolysin production impaired pre-neutrophil-mediated pneumococcal killing in cirrhotic but not control rats suggests that pneumolysin may be particularly detrimental to this defense mechanism in the severely cirrhotic host. The decrease in neutrophil-mediated killing of pneumococci within the lungs of the cirrhotic host is related to insufficient deposition of host proteins such as complement C3 on their surfaces. Pneumolysin likely plays a role in complement consumption within the lungs. Our studies, however, were unable to determine whether pneumolysin more negatively impacted this defense mechanism in cirrhotic than in control rats. These findings contribute to our understanding of the defects in innate pulmonary defenses that lead to increased mortality from pneumococcal pneumonia in the severely cirrhotic host. They also suggest that pneumolysin may be a particularly potent pneumococcal virulence factor in the setting of cirrhosis.

Relaxin receptors in hepatic stellate cells and cirrhotic liver.

The polypeptide hormone relaxin has antifibrotic effects on a number of tissues, including the liver. Central to the progression of hepatic fibrosis is the transdifferentiation of hepatic stellate cells (HSC) from a quiescent state to an activated, myofibroblastic phenotype that secretes fibrillar collagen. Relaxin inhibits markers of HSC activation, but relaxin receptor expression in the liver is unclear. The purpose of this study was to determine the expression of the relaxin receptors LGR7 and LGR8 in activated HSC. Production of cAMP was induced by treatment of HSC with relaxin, or the relaxin-related peptides InsL3 or relaxin-3, selective activators of LGR8 and LGR7, respectively. Quiescent HSC expressed low levels of LGR7 but not LGR8. During progression to the activated phenotype, expression of both receptors increased markedly. Immunocytochemistry confirmed the presence of both receptors in activated HSC. In normal rat liver, LGR7, but not LGR8, was expressed at low levels. In cirrhotic liver, expression of both receptors significantly increased. Neither receptor was detectable in normal liver by immunohistochemistry, but both LGR7 and LGR8 were readily detectable in cirrhosis. These results were confirmed in human cirrhotic tissue, with the additional finding of occasional perisinusoidal LGR7 immunoreactivity in non-cirrhotic tissue. In conclusion, the expression of LGR7 and LGR8 is increased with activation of HSC in culture. Cirrhosis also caused increased expression of both receptors. Therefore, agents that stimulate LGR8 and LGR7 may be therapeutically useful to limit the activation of hepatic stellate cells in liver injury.

Smoke exposure and ethanol ingestion modulate intrapulmonary polymorphonuclear leukocyte killing, but not recruitment or phagocytosis.

BACKGROUND: People who smoke and abuse alcohol are uniquely susceptible to pulmonary infections caused by Streptococcus pneumoniae, the pneumococcus. The primary cellular defense against pneumococci within the lungs is the polymorphonuclear leukocyte (PMN). Cigarette smoke and ethanol (EtOH) are known to alter certain PMN functions, but little is known about their concurrent effects. METHODS: Male Sprague-Dawley rats were exposed twice daily for 8 weeks to cigarette smoke (smoke-exposed) or room air (sham-exposed). During the final week of exposure, the rats were pair-fed a liquid diet containing either 36 or 0% EtOH calories. Polymorphonuclear leukocytes were prerecruited into the rats' lungs by transtracheal injection of lipopolysaccharide. Five hours later, the rats were infected transtracheally with S. pneumoniae, and PMN recruitment, phagocytosis, and bactericidal activity were quantified within their lungs. Chemokine levels were also measured in bronchoalveolar lavage fluids, lung homogenates, and sera. RESULTS: Neither PMN recruitment nor phagocytic uptake of pneumococci was altered by EtOH ingestion or smoke exposure. Killing of the organisms, however, was significantly decreased in sham-exposed, but not smoke-exposed, rats ingesting EtOH. Parallel results were determined for serum cytokine-induced neutrophil chemoattractant-1 (CINC-1), with EtOH ingestion significantly decreasing the levels in sham-exposed, but not smoke-exposed, rats. Pulmonary levels of macrophage inflammatory protein-2 (MIP-2) and CINC-1 were highly elevated by the combination of EtOH and smoke. CONCLUSIONS: One week of EtOH ingestion by rats impaired the ability of their PMNs to kill S. pneumoniae within their lungs. This was not due to decreased recruitment of the PMNs to the lungs or to diminished phagocytosis of intrapulmonary pneumococci. The addition of twice-daily cigarette smoke exposure to this short-term EtOH ingestion model restored PMN bactericidal ability to levels observed in the absence of either treatment. These EtOH-induced and smoke-induced alterations in PMN killing may be related to alterations in both pulmonary and systemic inflammatory chemokine levels.

A novel flow cytometric assay for measurement of in vivo pulmonary neutrophil phagocytosis.

BACKGROUND: Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs) isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS) to recruit PMNs to their lungs. They are then infected with live 5(-and 6) carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. RESULTS: The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. CONCLUSION: This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

Relaxin receptor expression in hepatic stellate cells and in cirrhotic rat liver tissue.

Relaxin has antifibrotic effects on the hepatic stellate cells (HSCs) responsible for collagen deposition in cirrhosis. The expression of relaxin receptors LGR7 and LGR8 in HSCs and liver disease was examined. Activated and quiescent HSCs expressed LGR7, whereas only activated HSCs expressed LGR8. Relaxin, relaxin-3, or InsL3 treatment increased cAMP, suggesting activation of both receptors. LGR8 and LGR7 were present in cirrhotic rat liver, but were undetectable in normal liver. In conclusion, both LGR7 and LGR8 are expressed in activated HSCs and cirrhotic liver, suggesting that relaxin, InsL3, or relaxin-3 may be useful in the treatment of hepatic fibrosis.

Smoke exposure exacerbates an ethanol-induced defect in mucociliary clearance of Streptococcus pneumoniae.

BACKGROUND: Alcoholics and smokers are particularly susceptible to pulmonary infections caused by Streptococcus pneumoniae, the pneumococcus. Infection begins when pneumococci colonizing the nasopharynx are aspirated into the lower respiratory tract. The major host defense against this movement is the mucociliary clearance apparatus. Both cigarette smoke and ethanol (EtOH) exposure alter ciliary beating and protein kinase activity in the respiratory mucosa in vitro, but their effects on bacterial clearance in the intact animal have not been determined. METHODS: Male Sprague Dawley rats were exposed twice daily for 12 weeks to either the smoke generated from 30 cigarettes (smoke-exposed) or room air (sham-exposed). For the last five weeks of smoke exposure, the rats were fed Lieber-DeCarli liquid diets containing 0%, 16%, 26%, or 36% EtOH calories. The rats then were infected intranasally with S. pneumoniae, and movement of the organisms into the lower respiratory tract was quantified by plate counts of the tracheas and lungs 4 hr later. Ciliary beat frequency (CBF) analysis was performed on tracheal ring explants from each animal before and after stimulation with the beta-agonist isoproterenol, and tracheal epithelial cell protein kinase C (PKC) activity was measured. RESULTS: Ingestion of any of the EtOH-containing diets resulted in a dose-dependent increase in movement of S. pneumoniae into the rats' lungs. This EtOH-induced defect was augmented further by concurrent smoke exposure, although smoke exposure alone had little effect on S. pneumoniae movement. Smoke, but not EtOH exposure, activated tracheal epithelial cell PKC. Increased movement of organisms into lungs correlated with a decrease in CBF and loss of the ciliary response to isoproterenol. CONCLUSION: EtOH ingestion in our model facilitated movement of S. pneumoniae into rats' lungs, a phenomenon exacerbated by concurrent smoke exposure. Furthermore, the organism's movement into the lungs correlated with a blunting of the rats' ciliary response to an established stimulus. Defects in mucociliary clearance thus may be one cause of the increased risk of pneumococcal infections in people who abuse alcohol, particularly if they also smoke.

Effect of cirrhosis on antibiotic efficacy in a rat model of pneumococcal pneumonia.

A rat model was used to study the effects of cirrhosis on antibiotic therapy of pneumococcal pneumonia. Cirrhotic and control male Sprague-Dawley rats were infected transtracheally with type 3 Streptococcus pneumoniae. Treatment began 18 h later with phosphate-buffered saline (PBS), azithromycin (50 mg/kg), trovafloxacin (50 mg/kg), or ceftriaxone (100 mg/kg) injected subcutaneously twice daily for 5 days. Antibiotic concentrations were measured by high-performance liquid chromatography. Azithromycin, trovafloxacin, and ceftriaxone were all equally effective at preventing mortality in both cirrhotic and normal rats. Free fraction area under the curve to minimum inhibitory concentration ratio (AUC/MIC) and maximum calculated serum concentration to MIC ratio (C(max)/MIC) and percent time that the serum concentration exceeded the MIC (%T > MIC) were greater for ceftriaxone compared with azithromycin or trovafloxacin. Azithromycin achieved higher concentrations in bronchoalveolar lavage fluid (BALF), epithelial lining fluid (ELF), and BAL white blood cells than ceftriaxone or trovafloxacin in cirrhotic rats. Macrolide, beta-lactam, or fluoroquinolone antibiotic efficacy in a pneumococcal pneumonia model does not appear to be affected by hepatic cirrhosis.

Desensitization of PKA-stimulated ciliary beat frequency in an ethanol-fed rat model of cigarette smoke exposure.

BACKGROUND: Our previous studies have shown that the ciliary beat frequency (CBF) of cultured ciliated airway epithelial cells exposed to chronic ethanol fails to increase in response to beta-agonist stimulation. This loss of the ciliary "flight response" correlates with an ethanol-mediated desensitization of adenosine 3':5'-cyclic monophosphate-dependent protein kinase (PKA), a known regulatory component of CBF stimulation. We hypothesized that a similar ethanol-mediated desensitization of CBF would occur in vivo. METHODS: Sprague Dawley rats were fed a liquid diet containing various concentrations of ethanol for 1 or 5 weeks. Half were exposed to cigarette smoke for 12 weeks and half were sham exposed. Animals were killed and tracheal epithelial cells analyzed for CBF and PKA activity. RESULTS: Baseline CBF (approximately 6 Hz) was unchanged in tracheal epithelial cells of rats consuming diets containing 0-36% ethanol for 5 weeks. Isoproterenol stimulated CBF to 12 to 13 Hz in the tracheal epithelial cells of control rats not administered ethanol. However, isoproterenol stimulation of CBF was blunted to 7.5 Hz in rats eating a 26% ethanol diet, and there was no stimulation of CBF in rats fed a diet containing 36% ethanol. Similarly, isoproterenol stimulated a 2- to 3-fold increase in PKA activity in control rats, but this PKA response to isoproterenol was blunted in rats fed increasing concentrations of ethanol. No isoproterenol-stimulated PKA response was observed in rats fed 36% ethanol. No ethanol-induced changes in cyclic guanosine monophosphate-dependent protein kinase or protein kinase C were observed in the rats' tracheal epithelial cells. Cigarette smoke exposure slightly elevated baseline CBF and lowered the ethanol consumption level for isoproterenol-desensitization of CBF and PKA activation to 16%. No isoproterenol desensitization was observed after 1 week of alcohol feeding. Furthermore, 36% ethanol-feeding for 1 week stimulated rat tracheal CBF and PKA. CONCLUSION: These data demonstrate that in vivo administration of ethanol to rats results in decreased ciliary beating and the desensitization of PKA. This suggests a mechanism for mucociliary clearance dysfunction in alcoholics.

A rat model to determine the biomedical consequences of concurrent ethanol ingestion and cigarette smoke exposure.

BACKGROUND: Although scientists have used animal models for years to study the effects of ethanol (EtOH) ingestion on humans, the compounding effect of cigarette smoking has been virtually ignored. Because 80 to 95% of human alcoholics smoke, it is imperative to consider the added effects of smoking when trying to determine the consequences of excessive alcohol ingestion. We therefore have developed a rat model for studying the separate and combined results of smoking and drinking on human health. METHODS: Male Sprague-Dawley rats were exposed daily for 12 weeks in whole-body chambers to cigarette smoke (smoke-exposed) or room air (sham-exposed). During the final 5 weeks of exposure, the rats were fed liquid diets that contained 0, 16, 26, or 36% EtOH calories. Smoke exposure was quantified by measurement of carboxyhemoglobin, nicotine, and cotinine levels. Body weights, food consumption, blood EtOH concentrations, and various assessments of liver damage and function also were followed. RESULTS: Smoke exposure in this rat model approximates that of a moderate to heavy human smoker. Smoke-exposed rats weighed significantly less and ate less food than sham-exposed controls, but both groups ingested equivalent amounts of EtOH for their body weights and had comparable blood EtOH levels. Liver aspartate and alanine aminotransferase levels remained normal. There was an EtOH-induced decrease in asialoglycoprotein receptor binding, but it was not exacerbated by smoke exposure. Alterations in blood cholesterol levels reflected what has been reported for humans, rising with increasing EtOH ingestion and decreasing with smoke exposure. CONCLUSION: Our rat model is relevant to what transpires in the vast majority of alcoholics. Both ethanol ingestion and smoke exposure can be manipulated to mimic light to moderate to heavy levels, making it appropriate for studying the separate and combined biomedical consequences of alcohol abuse and cigarette smoking.

The impact of ethanol and tobacco smoke on intranasal epithelium in the rat.

BACKGROUND: Investigations have shown the influence of ethanol and tobacco smoke on olfaction, epithelial metaplasia, and cancer formation in the head and neck. Analysis of ethanol and tobacco smoke-induced histopathological mucosal changes in the upper respiratory tract may provide important insight into the pathophysiology of secondary olfactory dysfunction. METHODS: Three groups of laboratory rats were experimentally exposed to either ethanol, tobacco smoke, or both, with a control group having no such exposure. RESULTS: Compared with controls, histopathological analysis of nasal mucosa in exposed rats revealed a decrease in the length of olfactory epithelium, especially in the rats exposed to both ethanol and tobacco smoke. Structural changes included loss of cilia and metaplasia. CONCLUSION: The histological changes noted in rats after ethanol and tobacco smoke exposure, if relevant to human physiology, could explain the decreased olfactory ability seen in patients who use these products.

Efficacy of liposome-encapsulated ciprofloxacin compared with ciprofloxacin and ceftriaxone in a rat model of pneumococcal pneumonia.

Encapsulation of ciprofloxacin in sterically stabilized liposomes results in a prolonged circulation time and improved pharmacokinetics. Liposome-encapsulated ciprofloxacin was compared with conventional ciprofloxacin and ceftriaxone in a rat model of pneumococcal pneumonia. Male Sprague-Dawley rats were infected transtracheally with type 3 Streptococcus pneumoniae and then treated with intravenous ceftriaxone (100 mg/kg), ciprofloxacin (40 or 80 mg/kg) or liposomal ciprofloxacin (40 or 80 mg/kg) administered once or twice daily for 3 days. White blood counts, development of bacteraemia and mortality were measured for 10 days. Antibiotic concentrations in serum, lung lavage fluid and white blood cells recovered from lung lavage fluid were determined. Liposomal ciprofloxacin concentrations were significantly higher in serum and lavage fluid compared with conventional ciprofloxacin, resulting in greater area under the serum concentration-time curve and maximum serum concentration. Despite these higher concentrations, survival rates were similar between groups treated with equivalent doses of liposomal ciprofloxacin versus ciprofloxacin. When antibiotics were given once daily, ceftriaxone was more effective than either form of ciprofloxacin.

Pharmacodynamic activity and efficacy of linezolid in a rat model of pneumococcal pneumonia.

Linezolid is a new oxazolidinone antibiotic with potent activity against gram-positive bacteria, including Streptococcus pneumoniae. The pharmacodynamic activity and in vivo efficacy of linezolid were compared to those of ceftriaxone in an immunocompetent rat model of pneumococcal pneumonia. Rats infected intratracheally with 8 x 10(7) CFU of a penicillin-sensitive (MIC, 0.032 microg/ml) strain of S. pneumoniae were treated for 5 days beginning 18 h postinfection. Groups of rats were sham treated with oral phosphate-buffered saline or received oral liquid linezolid at 25 or 50 mg/kg of body weight twice a day (b.i.d.) or subcutaneous ceftriaxone at 100 mg/kg once daily. Mortality was monitored for 10 days postinfection; blood culturing was performed on day 1 (pretreatment) and on days 3, 5, and 10 postinfection for the determination of bacteremia. Serum also was collected for the determination of pharmacokinetic and pharmacodynamic parameters at 30 min and at 3, 5, and 12 h (linezolid) or 3, 5, and 24 h (ceftriaxone) postdose. The cumulative mortality rates were 100% for the sham-treated group, 58.3% for the low-dose linezolid group, 8.3% for the high-dose linezolid group, and 0% for the ceftriaxone group. Rats in each of the antibiotic treatment groups had significantly fewer bacteria (P < 0.00001) in their bronchoalveolar lavage fluid (BALF) on day 3 postinfection than sham-treated rats. There also were significantly fewer organisms in the BALF of rats treated with ceftriaxone than in the BALF of rats treated with either dose of linezolid. Oral linezolid at 50 mg/kg b.i.d. therefore was as effective as ceftriaxone in experimental pneumococcal pneumonia, whereas the 25-mg/kg b.i.d. dose was significantly less effective. All pharmacodynamic parameters reflected efficacy and were significantly different for the two dosage regimens of linezolid (P < 0.01). However, the free-fraction pharmacodynamic parameters predictive of outcome were a value of >39% for the percentage of time in the experimental dosing interval during which the linezolid concentration exceeded the MIC and a value of >147 for the ratio of the area under the serum concentration-time curve to the MIC.