Bronchoalveolar Lavage Fluid Supernatant Research Articles

Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.

Surfactant degradation activity in bronchoalveolar lavage fluid from guinea pigs challenged with antigen

Surfactant dysfunction is a characteristic of bronchial asthma, but mechanisms of dysfunction following antigen exposure are not understood. The aim of this study was to examine whether bronchoalveolar lavage fluid (BALF) has surfactant degradation activity after antigen challenge, using an animal model of asthma. BALF was collected 24 h after a challenge with aerosolized antigen solution in actively sensitized guinea pigs and from non-sensitized control guinea pigs. The surface tension of BALF was measured by pulsating bubble surfactometer. Surfactant activity was expressed as the minimum surface tension of BALF after 5 min of pulsation. BALF was separated into a cellular phospholipid fraction and supernatant, and reconstituted into 'pellet + supernatant' and 'pellet + saline' fractions. Surfactant activity of BALF from sensitized antigen-challenged animals was reduced after 4 h of incubation at 37 degrees C but a decrease was not observed in BALF from non-sensitized control animals. The decrease of surfactant activity in BALF from challenged animals was prevented by incubation at 4 degrees C. Disappearance of surfactant activity after incubation at 37 degrees C was observed in the 'pellet + supernatant', but not in the 'pellet + saline' fraction. The decrease of surfactant activity in BALF was also partially suppressed by the secretory phospholipase A2 inhibitor, indoxam, and by a cocktail of protease inhibitors. Surfactant-degrading activity was present in the supernatant of BALF from antigen-challenged guinea pigs. This activity may be attributed to secretory phospholipase A2 and to proteases present in the antigen-challenged airway.

Effect of choline chloride in allergen-induced mouse model of airway inflammation

The incidence of asthma has increased the world over, and current therapies for the disease suffer from potential side-effects. This has created an opportunity to develop novel therapeutic approaches. Here, the anti-inflammatory activity of choline was investigated in a mouse model of allergic airway inflammation. Choline (1 mg.kg(-1)) was administered via oral gavage or intranasally before and after ovalbumin (OVA) challenge in sensitised mice. Airway hyperresponsiveness (AHR) to methacholine was measured in the mice by whole-body plethysmography. Type-2 T-helper cell cytokine and leukotriene levels were estimated in bronchoalveolar lavage fluid (BALF) and spleen culture supernatant by ELISA. Eosinophil peroxidase activity was also determined in the BALF supernatant. Choline treatment in sensitised mice before OVA challenge via oral/intranasal routes significantly inhibited eosinophilic airway inflammation and eosinophil peroxidase activity. It also reduced immunoglobulin E and G1 production and inhibited the release of type-2 T-helper cell cytokines and leukotrienes. However, the development of AHR was prevented effectively by intranasal choline treatment. Most importantly, choline treatment after OVA challenge by both routes could reverse established asthmatic conditions in mice by inhibiting AHR, eosinophilic airway inflammation and other inflammatory parameters. This study provides a new therapeutic approach for controlling as well as preventing asthma exacerbations.

Mice Lacking Neutrophil Elastase Are Resistant to Bleomycin-Induced Pulmonary Fibrosis

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

A method to enable the investigation of murine bronchial immune cells, their cytokines and mediators

Innovative therapies for severe lung diseases (such as allergic and chronic asthma, chronic obstructive pulmonary disease or any type of lung cancer) require a detailed understanding of the cellular and immune processes in the lung. This protocol details a method to obtain the immune cells of the bronchi as well as the cytokines and mediators produced by these cells for further investigation. The broncho-alveolar lavage fluid (BALF) is taken by injecting physiological solution through the tracheal tube into the murine airways and carefully regained by winding up the connected syringe. After centrifugation, the resulting BALF supernatant can be stored for detection of cytokines or other mediators by enzyme-linked immunosorbent assay or other methods; the resuspended cell pellet can also be used for flow cytometric analyses, to check cell viability and the level of apoptosis, as well as other applications. In addition, CD4+ T cells isolated from wild-type and genetically modified mice alone or along with other immunologically important cells such as T regulatory cells, which can be used to reconstitute immunodeficient mice, may be retrieved from the airways with this method. This protocol can be completed within 35 min.

Genetic and pharmacological blockade of interleukin-1 signaling attenuates neuropathic pain and spontaneous discharge following nerve injury

In order to elucidate the role played by alveolar cytokines in the pathogenesis of HIV-related lung damage, levels of interleukin (IL) 1ß, IL-2, IL-6, tumor necrosis factor (TNF)-$aL, and interferon (Ifn) were assessed on supernatant of bronchoalveolar lavage fluid from 30 consecutive HIV-1 seropositive (HIVAb+) patients with clinical and radiologic evidence of pneumonia, from 20 HIV— seronegative (HIVAb—) patients with pulmonary sarcoidosis, and from 10 HIVAb— healthy control subjects. Cytokine levels were expressed as picogram (IL-1, TNF), nanogram (IL-6), and international unit (IL-2, Ifn) per milligram of albumin per deciliter. Total and differential cell counts, cytofluorimetric enumeration of CD3+, CD3+/DR+, CD4+, CD8+, and CD8+/CD16+ cells, as well as microbiologic investigations for opportunistic agents were performed on lavage pellets. HIV-related pneumonia was characterized by higher mean alveolar level of IL-2 (12 ±5 IU), and by more elevated mean counts of T cells (109 ± 16), activated T cells (60 ± 12), and CD8+ cells (90 ± 13)/µ l if compared with both active sarcoidosis and control subjects, where respective values of 0.2±0.1 and 0.3±0.2 IU IL-2/mgAlb/dl, of 52±11 and 7±2 T cells, of 20±5 and 1.2±0.3 activated T cells, and of 11 ±2 and 3 ±0.6 CD8+ cells per microliter were found. HIV-infected patients with opportunistic lung infections (OIs) showed the highest mean IL-2 level (21 ±4 IU), and higher counts of both CD8+ (117 ±20) and CD8+/CD16+ (36 ±7) cells per microliter if compared with patients without evidence of OIs (respectively, 62 ±13 CD8+ and 18 ±3 CD8+/CD16+ cells per microliter). By contrast, extremely high IL-1 levels (1,463 ±760 pg), and IL-2 levels similar to control subjects (3.4 ± 1.2 IU), were found in the absence of OIs. Different mechanisms depending respectively on IL-2-mediated cytotoxic cell recruitment and activation, or IL-1-mediated tissue injury may account for HIV-related lung damage, depending on the presence or absence of opportunistic agents.

Rapid, Multiwell Colorimetric Assay for Measuring Neutrophil Chemoattractant Activity in Bronchoalveolar Lavage Fluid of Horses with Recurrent Airway Obstruction

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.

Cysteine cathepsins in human silicotic bronchoalveolar lavage fluids

Mature, active cysteine cathepsins (CPs) were identified in human inflammatory bronchoalveolar lavage fluid (BALF) supernatants from patients suffering from silicosis by both western blot and surface plasmon resonance analyses. BALFs are not a reservoir of activatable proforms, since no autocatalytic maturation at acidic pH occurs. Cathepsin H is the most profuse among studied CPs (median value: 36.5 nM), while cathepsins B and L are the two most abundant thiol-dependent endoproteases. The overall concentration of active cathepsins B, H, K, L, and S is approximately 10-fold lower than their concentration in BALF supernatants from patients suffering from inflammatory acute lung injuries (962+/-347 nM).The cathepsins (approximately 70 nM)/cystatin-like inhibitors (approximately 9 nM) ratio is unbalanced in favor of enzymes ( approximately 8-fold). This presence of uncontrolled CPs suggests that they may contribute, in addition to matrix metalloproteases, to the lung tissue breakdown/remodeling occurring during silicosis, although their exact contribution to interstitial inflammation remains to be evaluated.

Effects of CpG oligodeoxynucleotide on transcription factors GATA-3 and T-bet mRNA expression in asthmatic mice

To investigate effects of CpG oligodeoxynucleotide (CpG ODN) on the mRNA expression of transcription factors GATA binding protein 3 (GATA-3) and T-box expressed in T cells (T-bet) in asthmatic mice. An asthmatic mouse model was established and treated with CpG ODN. Total inflammatory cells and eosinophils in bronchoalveolar lavage fluid (BALF) were counted and inflammatory cell infiltration in lung tissue was evaluated. Interferon-gamma and interleukin-4 concentrations in BALF and splenocyte culture supernatants were detected using an enzyme-linked immunosorbent assay. Transcription factor GATA-3 and T-bet mRNA expression in splenocytes and lung tissue were detected by reverse transcription-polymerase chain reaction. Total inflammatory cells and eosinophils in BALF were reduced in the CpG ODN-treated group compared with the asthma group, and inflammatory cell infiltration in lung tissue was also significantly alleviated. CpG ODN treatment increased the interferon-gamma concentration but decreased the interleukin-4 concentration in both BALF and splenocyte culture supernatants. GATA-3 mRNA expression was reduced in both lung tissue and splenocytes in the CpG ODN-treated group, while the mRNA ratio of T-bet to GATA-3 in splenocytes was increased. CpG ODN treatment inhibits airway inflammatory cell infiltration and regulates interferon-gamma/interleukin-4 synthesis in asthmatic mice, possibly through a mechanism of downregulation of GATA-3 mRNA expression in both lung tissue and splenocytes.

Liquid chromatographic–tandem mass spectrometric assay for the simultaneous determination of didanosine and stavudine in human plasma, bronchoalveolar lavage fluid, alveolar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue

We have developed a sensitive, high-pressure liquid chromatographic–tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of didanosine (ddI) and stavudine (d4T) in human plasma, bronchoalveolar lavage fluid (BALF), alveolar cells (AC), peripheral blood mononuclear cells (PBMC), seminal plasma, cerebrospinal fluid (CSF), and tonsil tissue. Plasma, AC, PBMC and CSF were run with an isocratic HPLC method, while BALF supernatant, semen, and tonsil tissue utilized a gradient elution. Samples were prepared by solid phase extraction. Detection was by electrospray positive ionization with multiple reaction monitoring mode. The lower limits of quantitation for both ddI and d4T were 2.0 ng/ml in plasma; 0.5 ng/ml in CSF; 0.4 ng/ml in AC, PBMC, and BALF; 1.0 ng/ml in seminal plasma; and 0.01 ng/mg in tonsil tissue.

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors.

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.

Tumor-Derived Granulocyte-Macrophage Colony-Stimulating Factor and Granulocyte Colony-Stimulating Factor Prolong the Survival of Neutrophils Infiltrating Bronchoalveolar Subtype Pulmonary Adenocarcinoma

We evaluated the role of the tumor environment in the regulation of apoptosis of tumor-infiltrating neutrophils, the number of which correlates negatively with outcome, in patients with adenocarcinoma of the bronchioloalveolar (BAC) subtype. We examined three different parameters of apoptosis, namely morphological aspect, annexin-V expression, and DNA fragmentation. Bronchoalveolar lavage fluid (BALF) supernatants from patients with BAC significantly inhibited the 24-hour spontaneous apoptosis of normal peripheral blood neutrophils in vitro compared to BALF supernatants from control patients (64 +/- 4% versus 90 +/- 2% measured by annexin-V flow cytometry, P = 0.04). The alveolar neutrophil count correlated positively with the granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations in the patient's BALF. Furthermore, neutralizing antibodies (Abs) against GM-CSF and G-CSF significantly inhibited BALF anti-apoptotic activity (15 to 40% and 34 to 63% inhibition, respectively), whereas neutralizing Abs against interleukin (IL)-8, IL-6, IL-1beta and tumor necrosis factor-alpha had no significant effect. In an attempt to identify the cell origin of anti-apoptotic cytokines, we tested in vitro the effect of BAC cells (A549 cell line and primary culture derived from a patient's BAC tumor) on the apoptosis of peripheral blood neutrophils. Cell-free supernatants from tumor cells did not inhibit neutrophil apoptosis. In contrast, cell-free supernatants from tumor cells previously exposed to conditioned media from peripheral blood mononuclear cells and alveolar macrophages significantly inhibited spontaneous neutrophil apoptosis. This inhibition was partially lifted when conditioned media from mononuclear cells were previously treated with Abs against IL-1beta and tumor necrosis factor-alpha. As in vivo, neutralizing Abs against GM-CSF significantly inhibited the anti-apoptotic activity of cell culture supernatants, and combination with Abs against G-CSF had an additive effect. In vivo, GM-CSF and G-CSF were strongly expressed by tumor cells and moderately or not expressed by the normal epithelium, as assessed by immunohistochemical studies. These findings demonstrate that the tumor environment generates local conditions that prolong alveolar neutrophil survival through the production of soluble factors, thereby contributing to the persistence of the neutrophil alveolitis observed in BAC.

Expression of the Ym2 lectin-binding protein is dependent on interleukin (IL)-4 and IL-13 signal transduction: identification of a novel allergy-associated protein.

Asthma pathophysiology is intimately regulated by CD4(+) Th2 lymphocytes and the cytokines interleukin (IL)-4 and IL-13. However, the mechanisms by which these cytokines promote disease have not been fully elucidated. In order to identify novel molecular mediators of allergy, a comparison was made of the bronchoalveolar lavage, which demonstrated that the Ym2 protein was abundantly up-regulated in the lung during the development of allergy. Low levels of the Ym1 isomer were also detected. Importantly, neither Ym1 nor Ym2 has been characterized previously in the context of allergic pulmonary inflammation. Western immunoblot showed that enhanced expression of these proteins was dependent on CD4(+) T cells and IL-4 or IL-13 signaling via the IL-4Ralpha subunit. In addition, intratracheal instillation of IL-13 into naive mice was sufficient to induce expression. Ym1 is homologous to eosinophil chemotactic factor L. However, only weak eosinophil chemotaxis was observed in response to Ym protein in both in vitro and in vivo assays. By contrast, the homology of Ym1 and Ym2 to proteins associated with tissue remodeling, together with the previous findings that Ym1 is homologous to chitinase and binds heparin sulfate and GlcN oligomers (chitobiose, chitotriose, and chitotetraose), strongly suggests these proteins play an important role in airway wall remodeling in the allergic lung.

Relevance of Tissue Factor and Tissue Factor Pathway Inhibitor for Hypercoagulable State in the Lungs of Patients with Idiopathic Pulmonary Fibrosis

We investigated the role of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Bronchoalveolar lavage (BAL) fluid was obtained from 22 patients with IPF, and the levels of TF and TFPI antigen were measured by ELISA. The TF and TFPI levels in BAL fluid supernatant were significantly higher in IPF patients than in normal controls. In addition, both levels were significantly higher in advanced cases than in nonadvanced cases. There was a significant correlation between the TF and TFPI levels Rs=0.541, p<0.05 . Localization of TF and TFPI antigens was investigated by immunohistochemical staining. Both antigens were mainly localized in hyperplastic cuboidal epithelial cells, suggesting that the widespread distribution of these cells contributed to the increase of TF and TFPI antigen levels in the lungs of IPF patients. To assess whether TF activity is counterbalanced by TFPI in the lungs of IPF patients, we examined procoagulant activity and TF activity. It was found, however, that both procoagulant and TF activities were significantly higher in the BAL fluid supernatant of IPF patients than in that of normal controls, which suggested that TFPI was actually increased, but the increase was insufficient to counterbalance TF, leading to the development of a hypercoagulable state in the lungs of IPF patients.

Lysosomal activity of pulmonary alveolar macro phages in acute experimental pancreatitis in rats with reference to positive PAF-antagonist (BN 52021) effect

The activation of pulmonary alveolar macrophages (PAM's), might play an important role in severe complications of acute pancreatitis. The aim of our study was to assess the labilization of macrophage lysosomal membranes and release of lysosomal cathepsin B (CB) and N-acetyl-beta-D-hexosaminidase (NAH) into bronchoalveolar lavage fluid (BALF) during taurocholate acute pancreatitis (AP) in rats treated with PAF-antagonist--BN 52021. Total activity of CB increased by 374% after 6 h and by 237% after 12 h of AP in lysosomal enriched fraction of PAM's. Fractional free activity of CB increased to 40% after 6 h and to 38% after 12 h of AP. Free activity of CB was increased 5 fold in the supernatant of macrophage homogenate, and 10 fold in the supernatant of BALF after 6 h of AP. The values of NAH activity roughly paralleled that of CB. Treatment with BN 52021 (5 mg x kg(-1) every 6 h i.v.) partially normalized the measured parameters. Our results indicate that the PAF-antagonist BN 52021 reduced the increase of total and free activity of lysosomal hydrolases of PAM's and partly prevented the labilization of their lysosomal membranes. Therefore, an important mechanism of BN 52021 beneficial effect in pulmonary complications of acute pancreatitis could be dependent on the stabilization of PAM's lysosomes.

Effects of Inhaled Low Molecular Weight Heparin on Airway Allergic Inflammation in Aerosol-Ovalbumin-Sensitized Guinea Pigs

Low molecular weigh, heparin (LMWH) possesses multiple nonanticoagulant properties. In the present study, we observed its anti-airway allergic inflammatory effects by bronchoalveolar lavage in guinea pigs. Guinea pigs were sensitized by repeatedly inhaling aerosolized ovalbumin. LMWH (400 u/1, 800 u/1), dexamethasone (1.2 mg/1) or vehicle (normal saline) was inhaled for 7 days. Then the animals were sacrificed under anesthesia and then lavaged with ice-cold Hank’s buffer immediately; bronchoalveolar lavage fluid (BALF) was prepared 24 h after the animals were challenged by antigen exposure. The effects of LMWH on total cell counts, absolute eosinophil counts and cell catalogues in BALF were studied; effects on the activity of eosinophil peroxidase (EPO) and the contents of histamine and eosinophil cationic protein (ECP) in BALF supernatant were detected. Our results showed that compared with the vehicle group, LMWH at 400 u/1 and 800 u/1 could significantly reduce total cell counts, absolute eosinophil counts and percentage of eosinophils in BALF (P<0.05 and P<0.01, respectively); LMWH at 800 u/1 markedly inhibited the activity of EPO in BALF supernatant (P<0.05); LMWH at 400 u/1 and 800 u/1 remarkably reduced the content of histamine in BALF supernatant (P<0.05 and P<0.01, respectively), LMWH at 800 u/1 decreased the content of ECP (P<0.05) significantly. It suggested that LMWH exerted anti-airway allergic inflammatory action by inhibiting infiltration of inflammatory cells and reducing release of inflammatory mediators, as well as antagonizing their activities, and that LMWH could be developed as a potential anti-bronchial asthmatic drug.

Monocyte inflammation augments acrolein-induced Muc5ac expression in mouse lung.

Acrolein, an unsaturated aldehyde found in smog and tobacco smoke, can induce airway hyperreactivity, inflammation, and mucus hypersecretion. To determine whether changes in steady-state mucin gene expression (Muc2 and Muc5ac) are associated with inflammatory cell accumulation and neutrophil elastase activity, FVB/N mice were exposed to acrolein (3.0 parts/million; 6 h/day, 5 days/wk for 3 wk). The levels of Muc2 and Muc5ac mRNA were determined by RT-PCR, and the presence of Muc5ac protein was detected by immunohistochemistry. Total and differential cell counts were determined from bronchoalveolar lavage (BAL) fluid, and neutrophil elastase activity was measured in the BAL fluid supernatant. Lung Muc5ac mRNA was increased on days 12 and 19, and Muc5ac protein was detected in mucous granules and on the surface of the epithelium on day 19. Lung Muc2 mRNA was not detected at measurable levels in either control or exposed mice. Acrolein exposure caused a significant and persistent increase in macrophages and a rapid but transient increase in neutrophils in BAL fluid. Recoverable neutrophil elastase activity was not significantly altered at any time after acrolein exposure. To further examine the role of macrophage accumulation in mucin gene expression, additional strains of mice (including a strain genetically deficient in macrophage metalloelastase) were exposed to acrolein for 3 wk, and Muc5ac mRNA levels and macrophage accumulation were measured. The magnitude of macrophage accumulation coincided with increased Muc5ac mRNA levels, indicating that excessive macrophage accumulation augments acrolein-induced Muc5ac synthesis and secretion after repeated exposure. These findings support a role for chronic monocytic inflammation in the pathogenesis of mucus hypersecretion observed in chronic bronchitis.

Low neutral endopeptidase levels in bronchoalveolar lavage fluid of lung cancer patients.

Neutral endopeptidase (NEP) is a cell surface enzyme found in normal human lung and which hydrolyzes small bioactive peptides, some of which act as growth factors for normal and malignant airway epithelial cells. Expression of NEP varies widely in human lung tissue from different individuals. NEP is often expressed at low or undetectable levels in both small-cell and non-small-cell lung cancer, and inhibits the growth of lung cancer cell lines. Variation in the expression of NEP could be a factor in susceptibility to lung cancer. We hypothesized that NEP could be measured in bronchoalveolar lavage fluid (BALF) and that airway levels of NEP would be low in lung cancer patients as compared with normal controls. We measured NEP and total protein in cell-free BALF supernatant, and expressed the respective concentrations as a ratio. NEP levels showed wide variation in BALF of healthy volunteers. Most patients with lung cancer had no NEP detectable in BALF. The mean NEP/total protein ratio was significantly lower in patients with lung cancer (0.87 +/- 0.7 ng NEP/mg protein) than in normal healthy subjects (14.0 +/- 4.3, p < 0.0003). We conclude that NEP levels are highly variable in BALF of normal volunteers, and are low or undetectable in most BALF specimens from patients with lung cancer. Low NEP levels in the airways may be a factor in the pathogenesis of carcinoma of the lung.

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats.

Several methodologies have been developed to assess alveolocapillary membrane permeability in acute lung injury. The purpose of this study was to determine the reliability of FITC-dextran compared with radioactive tracers to assess lung permeability alterations. After intraperitoneal administration of alpha-naphthylthiourea (ANTU, 50 mg/kg) or DMSO-ANTU vehicle, the animals were euthanized and their lungs were studied in an isolated-lung preparation. FITC-dextran or radiolabeled tracers were added to the perfusate. At 2 h the bronchoalveolar lavage (BAL) fluid from the ANTU group showed a significantly greater amount of fluorescence in the supernatant after centrifugation of BAL fluid compared with the DMSO group. Consistent results were observed with the radioactive tracers: there was an increase in extravascular albumin space and extravascular lung water compared with the control group. No cleavage of the FITC from the dextran molecule was evident by chromatography comparing samples recovered from the BAL fluid to the pure FITC-dextran molecule. In conclusion, measurement of FITC-dextran in the supernatant of BAL fluid after intravascular administration is a reliable method of assessing lung permeability changes in vivo and ex vivo.

Molecular species of acidic phospholipids in human lung surfactant.

Lung surfactant is a complex mixture of phospholipid (PL) and apoproteins that opposes surface tension forces within the lungs, and so prevents alveolar collapse on expiration. The major surfactant component responsible for this reduction in surface tension, dipalmitoyl phosphatidylcholine (PC16:0/16:0), is a solid at body temperature (T,=4 1'C). Consequently, other components of surfactant, including acidic PLs and hydrophobic proteins, have been proposed to facilitate adsorption of PC16:0/16:0 to its site of activity at the air-liquid interface in the lungs [l]. The major acidic PL responsible is thought to be phosphatidylglycerol (PG), which is uniquely enriched in lung surfactant (1 5% total PL) compared with its minor contribution to mammalian cellular PL. Additionally, phosphatidylinositol (PI), present at 7% total PL in human surfactant, has been reported to replace PG in various animal species without altering normal lung function [2]. Limitations of sensitivity inherent in available HPLC methodologies have restricted previous studies of the roles PG and PI composition in surfactant function to animal studies [3,4] and diseases such as alveolar proteinosis (AP) [5] where large sample volumes are available. Consequently, we have developed a sensitive and rapid method for the routine analysis of the acidic phospholipids PG and PI in lung surfactant, based on electrospray ionisation mass spectrometry (ESI-MS). Bronchoalveolar lavage (BAL) (1201111 instilled volume) was collected by fibreoptic bronchoscopy from 12 control adult volunteers who had not exhibited evidence of upper respiratory tract infection within the preceding month. After filtration of mucus and centrifugation at 400 x g x 10 min, the PL concentration of the cell-free BAL fluid supernatant was determined from its phosphate content [6]. Total lipid was extracted from an aliquot of BAL fluid containing 25nmoles of PL using chlorofon/methanol[7] after adding lnmole of dimyristoyl PG (PG14:0/14:0) as an internal standard. The total lipid extract was dried under N2 gas and re-dissolved in 2Opl methano1:chloroform 2:l (v:v) containing 5mM NaOH. An aliquot (5pI) was introduced by rheodyne injection into a methano1:chloroform:water (80: 10: 10, v:v) mobile phase pumped at 1 Opl /min into the electrospray interface of a Quattro II quadrapole mass spectrometer. Spectra were collected at MSl under negative ionisation conditions, and M-IT ions of PG and PI were quantified after correction for the I3C isotope effect. This method was rapid ( ~ 5 midsample) and routinely analysed sample volumes containing about 6 nrnoles acidic PL (0.5-1 ml BAL fluid). Molecular species present at 4 0 0 pmoles could not be resolved. While there was considerable compositional variation between individual subjects, surfactant PG and PI were composed ofthe same three major species, 16:0/18:1, 18:0/18:1 and 18:1/18:1 (+18:0/18:2) (table 1). However, the relative