Number Of Alveolar Macrophages Research Articles

Proteomic Analysis of Bronchoalveolar Lavage Fluid: Effect of Acute Exposure to Diesel Exhaust Particles in Rats

BackgroundInhalation of diesel exhaust particles (DEPs) is characterized by lung injury and inflammation, with significant increases in the numbers of polymorphonuclear leukocytes and alveolar macrophages. This influx of cellular infiltrates is associated with the activation of multiple genes, including cytokines and chemokines, and the production of reactive oxygen species.ObjectiveThe pathogenesis of the lung injury is not fully understood, but alterations in the presence or abundance of a number of proteins in the lung have been observed. Our objective in this study was to further characterize these changes and to ask whether additional changes could be discerned using modern proteomic techniques.MethodsThe present study investigates global alterations in the proteome of bronchoalveolar lavage fluid taken from rats 1, 7, or 30 days after exposure to 5, 35, or 50 mg/kg of animal weight of DEPs.ResultsAnalysis by surface-enhanced laser desorption/ionization–time of flight mass spectrometry identified two distinct peaks that appeared as an acute response postexposure at all doses in all animals. We identified these two peaks, with mass to charge ratios (m/z) of 9,100 and 10,100, as anaphylatoxin C3a and calgranulin A by additional mass spectral investigation using liquid chromatography coupled to mass spectrometry.ConclusionsWith this approach, we found a number of inflammatory response proteins that may be associated with the early phases of inflammation in response to DEP exposure. Further studies are warranted to determine whether serum levels of these proteins could be markers of diesel exhaust exposure in workers.

Road tunnel air pollution induces bronchoalveolar inflammation in healthy subjects

Traffic-related air pollution is associated with adverse respiratory effects. The aim of the present study was to investigate whether exposure to air pollution in a road tunnel causes airway inflammatory and blood coagulation responses. A total of 16 healthy subjects underwent bronchoscopy with bronchial mucosal biopsies and bronchoalveolar lavage (BAL) on two occasions, in random order: once at 14 h after a 2-h exposure to air pollution in a busy road tunnel, and once after a control day with subjects exposed to urban air during normal activities. Peripheral blood was sampled prior to bronchoscopy. The road tunnel exposures included particulate matter with a 50% cut-off aerodynamic diameter of 2.5 microm, particulate matter with a 50% cut-off aerodynamic diameter of 10 mum and nitrogen dioxide which had median concentrations of 64, 176 and 230 microg.m(-3), respectively. Significantly higher numbers of BAL fluid total cell number, lymphocytes and alveolar macrophages were present after road tunnel exposure versus control. Significantly higher nuclear expression of the transcription factor component c-Jun was found in the bronchial epithelium after exposure. No upregulation of adhesion molecules or cellular infiltration was present and blood coagulation factors were unaffected. In conclusion, exposure of healthy subjects to traffic-related air pollution resulted in a lower airway inflammatory response with cell migration, together with signs of an initiated signal transduction in the bronchial epithelium.

Increased Endothelin-1 Levels of BAL Fluid in Patients with Behçet's Disease

Objective and background. Pulmonary aneurysms and thrombosis constitute a significant cause of morbidity and mortality in Behçet's disease (BD). Various factors have been studied to explore the pathogenesis of vascular involvement in BD. As endothelin (ET) is known for its potent vasoconstrictor and proinflammatory properties, we supposed that it is involved during the inflammatory process of BD pulmonary vasculitis. Methods. To investigate the role of ET in BD, ET-1 concentrations were measured in bronchoalveolar lavage fluid (BALF) of 18 nonsmoking BD patients with pulmonary manifestations and 12 control subjects. Immunoreactivity of ET-1 was also evaluated in alveolar macrophages (AMs) cytoplasm. Results. ET-1 levels in BD-BALF were significantly higher than those of controls. ET-1 levels were correlated with the number of alveolar macrophages, but not with BAL-CD4/CD8 ratio. ET-1-immunoreactivity was found mainly in AM of BD-BAL. Conclusions. Increased ET-1 production from AM is associated with pulmonary BD manifestations.

New aspects of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a leading cause of death and disability, but has only recently been extensively explored from a cellular and molecular perspective. In COPD there is a chronic inflammation that leads to fixed narrowing of small airways and alveolar wall destruction (emphysema). This is characterized by increased numbers of alveolar macrophages, neutrophils and cytotoxic T lymphocytes, and the release of multiple inflammatory mediators (lipids, chemokines, cytokines and growth factors). A high level of oxidative stress may amplify this inflammation. There is also increased elastolysis and evidence of the involvement of several elastolytic enzymes, including serine proteases, cathepsins and matrix metalloproteinases. The inflammation and proteolysis observed in COPD are an amplification of the normal inflammatory response to cigarette smoke. This inflammation, in marked contrast to asthma, appears to be resistant to corticosteroids, prompting a search for novel anti-inflammato...

Inflammatory Cells are Sources of Polyamines that Induce Alveolar Macrophage to Undergo Apoptosis During Pneumocystis Pneumonia

PNEUMOCYSTIS is an opportunistic fungal pathogen that causes pneumonia in immunocompromised individuals such as patients with AIDS. Alveolar macrophages are critical in the clearance of Pneumocystis organisms. However, the number of alveolar macrophages is decreased by approximately 60% during Pneumocystis pneumonia (PcP) (Lasbury, Durant, and Lee 2003). One mechanism that reduces the alveolar macrophage number is apoptosis (Lasbury et al. 2006). High concentrations of polyamines are known to induce apoptosis, and we have found that the levels of some polyamines (spermidine, acetylspermine, and acetylspermidine) are greatly increased in Pneumocystis-infected lungs and that these polyamines can induce normal alveolar macrophages to undergo apoptosis. We also have found that bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals can induce apoptosis in normal alveolar macrophages, but this apoptosis-inducing ability of the BAL fluid is lost when the polyamines are depleted. These results suggest that high polyamine levels induce apoptosis in alveolar macrophages during PcP. Pneumocystis organisms produce and excrete polyamines (Chin et al. 1996), but it is unknown whether host cells also produce polyamines during PcP. To identify cells that produce polyamines during Pneumocystis infection, we examined the differential expression of ornithine decarboxylase (ODC), a key enzyme on the production of polyamines. We also investigated the uptake of polyamines by alveolar macrophages from Pneumocystis-infected rats.

Survival Pathway Signal Transduction is Reduced in Alveolar Macrophages during Pneumocystis Pneumonia

PNEUMOCYSTIS pneumonia (PcP) causes increased alveolar macrophage apoptosis in humans, rats, and mice (Lasbury et al. 2006). The apoptosis of these cells proceeds via caspase-9 activation, as inhibition of caspase-9 activation increases alveolar macrophage numbers and prolongs survival of rats and mice with PcP (Lasbury et al. 2006). While a pro-apoptotic signal may be present in the lung during Pneumocystis infection, reduced alveolar macrophage resistance to apoptosis may also result in the reduction of alveolar macrophage numbers. Akt-1 is a kinase that acts to promote survival of cells. Akt-1, after it is activated by phosphorylation, is anti-apoptotic. Akt-1 can phosphorylate caspase-9 to prevent its activation (Cardone et al. 1998), inhibit forkhead transcription factor activity (Brunet et al. 1999), phosphorylate glycogen synthase kinase (GSK-3b) to inhibit its pro-apoptotic activities (Pap and Cooper 1998), and inhibit mitochondrial damage by promoting the release of the anti-apoptotic molecules Bcl-xl and Bcl-2 from Bad (del Peso et al. 1997). To begin to determine the role of Akt-1 in the apoptosis of alveolar macrophages during PcP, we assessed the expression and activation of Akt-1.

α-Tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse

Objective Short-term cigarette smoke (CS) exposure leads to acute lung inflammation through its influence over oxidants/antioxidants imbalance. Antioxidant vitamins such as ascorbic acid and α-tocopherol interact with oxidizing radicals. It is not clear if antioxidant supplementation can reduce inflammatory lung responses. Thus our aim was to analyze the effects of vitamin supplementation on the lungs of mice exposed to six cigarettes per day with histologic, cytological, and biochemical methods. Methods C57BL/6 mice were exposed to ambient air (control) or CS from 3, 6, 9, 12, or 15 cigarettes daily for up to 5 d. Mice alveolar macrophages and polymorphonuclear cells were counted in the bronchoalveolar lavage. Groups of CS animals received 50 mg/kg of ascorbic acid daily and/or 50 mg/kg of α-tocopherol daily as an oral supplementation (CS+C, CS+E, CS+C+E, respectively) 12 h before CS exposure. Thiobarbituric acid-reactive substances were detected and western blot to nuclear factor-κB were performed in lung extracts; metalloprotease-12 and tumor necrosis factor-α positive alveolar macrophages were quantified in the lungs processed for immunohistochemistry of the animals exposed to the smoke from six cigarettes daily for 5 d. Results The number of alveolar macrophages and polymorphonuclear cells in bronchoalveolar lavage (cells × 10 3/mL) in mice exposed to CS were increased and CS with vitamin supplementation groups presented bronchoalveolar lavage cells similar to those of control. Thiobarbituric acid-reactive substances values were reduced in vitamin supplementation groups when compared with CS and the lower value was found in the CS+C+E group. Metalloprotease-12 and tumor necrosis factor-α were more evident in CS as much as nuclear factor-κB activation when compared with control and vitamin supplementation groups. Conclusion Our results showed that CS induced acute lung inflammation. The inflammatory process after cigarette exposures was reduced by ascorbic acid, α-tocopherol, or more efficiently by both vitamin supplementations.

Protective effects of N-acetylcysteine and erdosteine on hemorrhagic shock-induced acute lung injury

The drugs N-acetylcysteine and erdosteine were used to evaluate their protective effects in hemorrhagic shock-induced acute lung injury in an animal model. Forty rats were used and randomly allocated into four groups (n=10). Animals in group III were fed with water containing 1 mg/dl erdosteine, and those in group IV were given 0.5 mg/dl N-acetylcysteine 3 days before the experiment. Group I was taken as the control and group II was taken as the hemorrhagic shock group. Hemorrhagic shock was initiated by blood withdrawal and reduction of the mean arterial pressure to 40 mmHg within 10 min via the right carotid artery. After a hypotensive period of 2 h, animals were resuscitated by transfusion of the shed blood and Ringer lactate in a volume equal to the shed blood. After a period of 1 h, blood samples were taken via the carotid artery. Bronchoalveolar lavage was performed to recover cells from the alveolar space with 40 ml of bronchoalveolar lavage fluid. Lung tissues were also resected to measure tissue malondialdehyde and L-gamma-glutamyl-L-cysteinyl-glycine levels with high performance liquid chromatography. The numbers of neutrophils and alveolar macrophages in bronchoalveolar lavage fluid were counted. Serum malondialdehyde levels were significantly higher in the shock groups (P<0.05), but there was no significant difference (P>0.05). Lung malondialdehyde levels were also significantly increased in the shock groups (P<0.05). In the erdosteine-applied group, tissue malondialdehyde levels were lower than in group II and the NAC-applied group (P<0.05). In the analyses of serum and lung tissue L-gamma-glutamyl-L-cysteinyl-glycine, the values of groups I, II and IV were found to be below the calibration graphics. The alveolar macrophage count was found to be the highest and the neutrophil count the lowest in group III when compared with the other groups in bronchoalveolar lavage fluid analyses (P<0.05). We may say that in the model of hemorrhagic shock-induced acute lung injury, it was found that erdosteine has a protective effect on lung tissue.

Evaluation of alveolar clearance by Tc-99m DTPA radioaerosol inhalation scintigraphy in welders

The welding process produces metal fumes and gases which may affect respiratory health. Technetium-99m diethylenetriaminepentaacetic acid (Tc-99m DTPA) dynamic lung scanning is an easy, noninvasive method to assess disorders of alveolar-capillary barrier permeability secondary to epithelial damage. We aimed to investigate the alveolar clearance by Tc-99m DTPA radioaerosol inhalation scintigraphy in welders, to assess additive effects of exposure to welding fumes and cigarette smoking on clearance rate of alveolar epithelium and to determine the correlation between Tc-99m DTPA aerosol lung scintigraphy and spirometric measurements. Nine nonsmoking welders, 9 smoking welders, and a control group of 6 nonsmokers and 6 smokers were accepted to the study. Tc-99m DTPA radioaerosol inhalation scintigraphy was performed in all subjects. Clearance half time (Tl/2) was calculated by placing a monoexponential fit on the curves. Penetration index (PI) was also calculated on the first minute image. Pulmonary function tests of welders and control group were compared. The mean T1/2 values of Tc-99m DTPA of the nonsmoking welders were significantly higher than those of the nonsmoking control group (82.1+/-24.3 min and 48.1+/-9.7 min, respectively; p = 0.003). The mean T1/2 values of Tc-99m DTPA of the smoking welders were higher than those of the smoking control group (53.3+/-24.5 min and 44.5+/-9.7 min, respectively; p = 0.510). PI of the nonsmoking welders was significantly higher than that of the nonsmoking control group (0.46+/-0.38 and 0.39+/-0.46 respectively; p = 0.004). PI of the smoking welders was significantly higher than that of smoking control group (0.43+/-0.38 and 0.37+/-0.45, respectively; p = 0.019). There was a negative correlation between T1/2 value and FEV1% (r = -0.468, p = 0.016), FVC% (r = -0.442, p = 0.024) and FEF25-75% (r = -0.391, p = 0.048) in the welders and control group. No statistically significant differences were found in the values of the standard pulmonary function tests of any of the subjects. Welding seems to decrease alveolar clearance which causes an increase in the penetration index. This was considered to be due to fibrotic changes and increased number of alveolar macrophages induced by welding fumes.

Different inflammatory cell pattern and macrophage phenotype in chronic obstructive pulmonary disease patients, smokers and non-smokers

Smokers exhibit airway inflammation and increased number of alveolar macrophages (AM), but not all develop chronic obstructive pulmonary disease (COPD). We hypothesized that AMs in COPD patients have an altered functional capacity mirrored in a different phenotype. Sixteen steroid-naive COPD patients [forced expiratory volume in 1 s (FEV(1)) < 70% of predicted] underwent bronchoalveolar lavage (BAL). Age- and smoking-matched non-obstructive smokers (n = 10) and healthy non-smokers (n = 9) served as controls. Nine COPD patients had a BAL cell yield sufficient for flow cytometry analysis, where expression of AM cell surface markers reflecting various functions was determined. AMs from COPD patients showed decreased expression of CD86 (co-stimulation) and CD11a (adhesion) compared to smokers' AMs (P < 0.05). Furthermore, smokers' AMs showed lower (P < 0.05) expression of CD11a compared to non-smokers. AM expression of CD11c was higher in the COPD and smokers groups compared to non-smokers (P < 0.05). The expression of CD54 (adhesion) was lower in smokers' AMs compared to non-smokers (P < 0.05), whereas CD16 was lower (P < 0.05) in COPD patients compared to non-smokers. The AM expression of CD11b, CD14, CD58, CD71, CD80 and human leucocyte antigen (HLA) Class II did not differ between the three groups. The AM phenotype is altered in COPD and further research may develop disease markers. The lower AM expression of CD86 and CD11a in COPD implies a reduced antigen-presenting function. Some alterations were found in smokers compared to non-smokers, thus indicating that changes in AM phenotype may be associated with smoking per se. The functional relevance of our findings remains to be elucidated.

Suppression of Alveolar Macrophage Apoptosis Prolongs Survival of Rats and Mice withPneumocystisPneumonia

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40-60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.

Role of lung surfactant in phagocytic clearance of apoptotic cells by macrophages in rats

Two of the common features of inflammatory lung diseases are the increased production of pulmonary surfactant and the induction of lung cell apoptosis. However, the relationship between these two events has not been addressed. To investigate the role of surfactant in pulmonary inflammation and apoptosis, we instilled natural lung surfactant (Survanta) (1.6–12.5 mg) into the rat lungs and determined the number of alveolar macrophages (AMs) and apoptotic lung cells. High-dose treatments of Survanta (>6.25 mg/rat) caused an increase in macrophage cell influx and lung cell apoptosis at 4 weeks post-treatment. In vitro studies using lavaged macrophages showed Survanta did not cause apoptosis. We then examined the role of Survanta on ability of macrophages phagocytizing apoptotic cells. This study demonstrated that macrophages were able to eliminate apoptotic cells more efficiently in the absence of surfactant than in its presence. In vivo, high doses of Survanta decreased the ability to clear exogenously instilled apoptotic cells or bacteria. Taken together, our results suggest that excessive accumulation of lung surfactant by Survanta treatment can impair or overwhelm the phagocytic clearance function of AMs and that this impairment may lead to increased presence of apoptotic cells in the lung and bacterial survival.

Endotoxin induced peritonitis elicits monocyte immigration into the lung: implications on alveolar space inflammatory responsiveness

BackgroundAcute peritonitis developing in response to gram-negative bacterial infection is known to act as a trigger for the development of acute lung injury which is often complicated by the development of nosocomial pneumonia. We hypothesized that endotoxin-induced peritonitis provokes recruitment of monocytes into the lungs, which amplifies lung inflammatory responses to a second hit intra-alveolar challenge with endotoxin.MethodsSerum and lavage cytokines as well as bronchoalveolar lavage fluid cells were analyzed at different time points after intraperitoneal or intratracheal application of LPS.ResultsWe observed that mice challenged with intraperitoneal endotoxin developed rapidly increasing serum and bronchoalveolar lavage fluid (BALF) cytokine and chemokine levels (TNFα, MIP-2, CCL2) and a nearly two-fold expansion of the alveolar macrophage population by 96 h, but this was not associated with the development of neutrophilic alveolitis. In contrast, expansion of the alveolar macrophage pool was not observed in CCR2-deficient mice and in wild-type mice systemically pretreated with the anti-CD18 antibody GAME-46. An intentional two-fold expansion of alveolar macrophage numbers by intratracheal CCL2 following intraperitoneal endotoxin did not exacerbate the development of acute lung inflammation in response to intratracheal endotoxin compared to mice challenged only with intratracheal endotoxin.ConclusionThese data, taken together, show that intraperitoneal endotoxin triggers a CCR2-dependent de novo recruitment of monocytes into the lungs of mice but this does not result in an accentuation of neutrophilic lung inflammation. This finding represents a previously unrecognized novel inflammatory component of lung inflammation that results from endotoxin-induced peritonitis.

Detection of cytokine protein expression in mouse lung homogenates using suspension bead array

BackgroundThe objective for this present study was to determine whether or not suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates. Here, we report on suspension bead array as a feasible method for detection of lipopolysaccharide (LPS)-evoked changes in cytokine protein expression in mouse lung tissue homogenates.Materials and methodsMice were treated (0.2 ml, intraperitoneal, i.p.) with phosphate buffered saline (PBS) or LPS (0.25 mg/ml) and sacrificed at either 2- or 24-hours post treatment. Formalin-fixed and paraffin-embedded lung sections were evaluated by light microscopy. Flash frozen lung tissues were homogenized for measurement of various cytokine protein expression levels using suspension bead array, antibody array and ELISA. Comparison between groups was performed using a Wilcoxon signed rank test.ResultsPulmonary perivascular edema and an accumulation of mixed cell infiltrates within blood and lymphatic vessels, as well as in the adjacent interstitium, were present at both 2- and 24-hours following LPS treatment. A minimal increase in the number of alveolar macrophages was also observed in the 24-hour LPS-treated mice only. The suspension bead array assay revealed statistically significant increases in mouse lung tissue homogenate levels of interleukin-6 (IL-6) and granulocyte/macrophage colony-stimulating factor (GM-CSF) proteins and a decrease in IL-2 protein at 24-hours post LPS-treatment only. Similar cytokine protein expression patterns were observed using antibody array. Significantly increased IL-6 protein expression levels were also detected using ELISA, which correlated with the suspension bead array data.ConclusionThe present study shows that suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates.

Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.

A plant polyphenol-rich extract restores the suppressed functions of phagocytes in influenza virus-infected mice

Influenza infection was induced in white ICR mice by intranasal (i.n.) inoculation of the virus A/Aichi/2/68 (H3N2). The number, migration and phagocyte indices of alveolar and peritoneal macrophages (pMØ) and of blood polymorphonuclear leukocytes (PMNs), as well as the inhibition of the PMN adherence in the presence of a specific antigen were followed for 9 days after infection. The effect of the i.n. application of a polyphenol-rich extract, designated as polyphenolic complex (PC), isolated from the medicinal plant Geranium sanguineum L., on the inspected immune parameters was studied in parallel with the virological parameters of the infection, e.g. rate of mortality, mean survival time (MST), infectious lung virus titre and consolidation of the lungs. It was found that the application of PC induced a continuous 2- to 2.5-fold rise in the number of both peritoneal and alveolar macrophages (aMØ) in the infected and healthy controls. The migration of both peritoneal and aMØ increased 1.5- to 2-fold in the group of infected PC-treated animals and four to fivefold in the control group, the maximum being on day 9. PC stimulated phagocyte activities of blood PMNs in both infected and healthy mice. The leukocyte adherence inhibition (LAI) index decreased in the infected and PC-treated animals. The restoration of the suppressed functions of phagocytes in influenza virus-infected mice (VIM) was consistent with a prolongation of MST and reduction in mortality rate, infectious virus titre and lung consolidation. The immunoenhancing properties of PC apparently contribute to the overall protective effect of the plant preparation in the lethal murine experimental influenza A/Aichi infection.

Influence of phosphatidylglycerol on the uptake of liposomes by alveolar cells and on lung function

The effect of phosphatidylglycerol on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages as well as the effect on endogenous surfactant function was studied in vivo. Healthy ventilated rats were intratracheally instilled with fluorescent labeled liposomes with different concentrations of phosphatidylglycerol. Lung function was determined by monitoring arterial oxygenation and, at the end of the experiment, by recording static pressure-volume curves. In addition, alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that, in the presence of cofactors (Ca(2+), Mg(2+)), phosphatidylglycerol stimulates the uptake by alveolar macrophages but hardly affects the uptake by alveolar type II cells. High concentrations of phosphatidylglycerol reduce the number of alveolar macrophages in the alveolar space and deteriorate lung function. On the other hand, the presence of cofactors protects the lung against the negative effects of phosphatidylglycerol on endogenous surfactant and alveolar macrophages. This study indicates that the phosphatidylglycerol concentration may play a fundamental role in the surfactant function and metabolism depending on the presence of so-called cofactors like calcium and magnesium; further study is needed to clarify the mechanisms involved.

Dosimetric Comparisons of Particle Deposition and Retention in Rats and Humans

Much of the information on the toxicity of particulate matter (PM) comes from studies in which laboratory rats were exposed to PM by inhalation or instillation. Optimal use of these toxicologic data requires extrapolation to the human scenario. Assuming that comparable doses should cause comparable effects across species and that species respond similarly to a given dose at a target site, extrapolations only require that dose be defined and then characterized. Dose may be defined in terms of a PM indicator (e.g., particle number or mass), a respiratory region, and the time over which the dose is integrated (i.e., deposited versus retained dose and incremental versus accumulated dose). Dose must also be normalized: for example, unit of dose per body mass, respiratory region surface area, or number of alveolar macrophages. The parameters chosen to define a normalized dose can drastically affect the rat exposure concentration required to provide a normalized dose equivalent to that occurring in a human. The publicly available multiple path particle dosimetry model developed by CIIT Centers for Health Research was used to predict particle deposition and retention in rats and humans. Estimates of particle concentration and exposure duration required for a rat to receive the same dose as received by a human were obtained with consideration of daily activity levels and ambient PM size distributions. These techniques were also used to compare dose and response between rats and humans in several published studies. Results indicate that the relationship between PM dose and response may differ between rats and humans. For acute PM exposures, rats may be less susceptible to inflammatory responses than humans. For chronic exposures to high levels of PM, however, an overload of alveolar clearance in rats may cause them to become more susceptible than humans to adverse pulmonary effects. The dosimetric calculations indicate that to achieve nominally similar acute doses per surface area in rats, relative to humans undergoing moderate to high exertion, PM exposure concentrations for rats would need to be somewhat higher than for humans. Since the clearance of PM is faster from the lung of rats than humans, much higher exposure concentrations are required for the rat to simulate retained burdens. In other cases, rats will require lower exposures than humans to have comparable doses, illustrating the complexity of such analyses. To make accurate estimates of dose, it is essential to have accurate and complete information regarding exposure conditions—that is, not only concentration and duration of exposure, but also the aerosol size distribution. Establishing a firm linkage between exposure and dose requires that consideration be given to particle characteristics, definitions of dose metrics, and biological normalizing factors.

IMMUNOHISTOCHEMICAL STUDY OF TUMOR NECROSIS FACTOR-α, MATRIX METALLOPROTEINASE-12, AND TISSUE INHIBITOR OF METALLOPROTEINASE-2 ON ALVEOLAR MACROPHAGES OF BALB/c MICE EXPOSED TO SHORT-TERM CIGARETTE SMOKE

Alveolar cells were evaluated in BALB/c mice exposed to smoke from 9 cigarettes per day. The mice were sacrificed at 1, 5, and 10 days and examined by lung morphometry and immunohistochemical staining of alveolar macrophages for tumor necrosis factor (TNF)-α, matrix metalloproteinase (MMP)-12, and tissue inhibitor of metalloproteinase (TIMP)-2. Cigarette smoke (CS)-exposed mice showed a progressive increase in numbers of alveolar macrophages (AMs) up to 10 days. MMP-12 was increased in AMs compared to the control group. The number of AMs with TNF-α and TIMP-2 labeling was higher at 5 days but decreased at 10 days. Short-term CS exposure induced pulmonary inflammation with major participation of MMP-12–positive AMs.

Acute stress reduces intraparenchymal lung natural killer cells via beta-adrenergic stimulation

There are lines of evidence that natural killer (NK) cells are sensitive to physical and psychological stress. Alterations in the immune system including NK cells are known to differ among tissues and organs. The effect of stress on the lung immune system, however, has not been well documented in spite of the fact that the lungs always confront viral or bacterial attacks as well as tumour cell metastasis. In this study, we intended to investigate the effect of restraint stress on lung lymphocytes including NK cells. C57BL/6 mice were exposed to 2 h restraint stress. The concentration of plasma epinephrine significantly rose immediately after the release from restraint as compared to home-cage control mice. Flow cytometric analysis revealed that the numbers of most lymphocyte subsets including NK cells were decreased in the lungs and blood but not in the spleen, immediately after restraint stress. Immunohistochemical examination revealed that the number of NK cells was decreased in the intraparenchymal region of the lungs, while the number of alveolar macrophages did not change. The decrease in the number of NK cells in the lungs and blood was reversed by the administration of propranolol, a nonselective beta adrenergic antagonist. Taken together, our findings suggest that acute stress reduces the number of intraparenchymal lung NK cells via activation of beta adrenergic receptors.