Effect of IgE-stimulated alveolar macrophages on tracheal epithelial bioelectric properties in dogs

To investigate a possible interaction between pulmonary alveolar macrophages (AMs) and airway epithelial cells in patients with allergic conditions, we studied the effect of AMs on bioelectric properties of canine tracheal epithelium under short-circuited conditions in vitro. Mucosal addition of the supernatants from AMs stimulated with monoclonal antidinitrophenyl (DNP) IgE antibody and DNP-human serum albumin (DNP-HSA) increased short-circuit current (Isc) of cultured epithelium in a dose-dependent manner. The maximal increase from the baseline value and the EC₅₀ were 10.2±2.0μA/cm² (mean ± SE, p<0.01) and 3×10⁵ AMs/ml, respectively. This effect was accompanied by the release of prostaglandin E₂ and F_2α from AMs. In contrast, AMs incubated with anti-DNP IgE antibody alone or DNP-HSA alone had no effect. The AM-induced increase in Isc was attenuated by diphenylamine-2-carboxylate and Cl-free medium but not by amiloride. Pretreatment of AMs with indomethacin or piroxicam inhibited the effect of AMs on epithelial Isc. These results suggest that AMs may stimulate Cl secretion across the airway mucosa through an IgE-dependent release of prostaglandins.     

Opsonized zymosan decreases cytoplasmic motility of alveolar macrophages in dogs.

To examine the mechanisms of changes in alveolar macrophage (AM) activities caused by phagocytic stimulus, we studied the effect of opsonized zymosan (OZ) on cytoplasmic motility (CM) of AM from dog lungs in vitro. Four days after the instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the lower lobe bronchus, AM were harvested by broncho-alveolar lavage. AM were adhered to the bottom of plastic vials (10(6) cells of AM per each vial). Remanent field strength (RFS) from the AM containing Fe3O4 particles was measured immediately after magnetization. RFS decreased with time due to particle rotation (relaxation), which is related to cytoplasmic motility of AM. OZ (1-500 micrograms) decreased lambda 0 (the relaxation rate for the first min) in a concentration-dependent fashion. Neither BW755C (10(-5) M), indomethacin (10(-6) M), leupeptin (10(-5) M), bestatin (10(-5) M), nor superoxide dismutase (1000 U/ml) inhibited OZ (500 micrograms)-induced inhibitory effects on lambda 0, suggesting that cyclooxygenase and lipoxygenase products, serine, thiol enzymes, aminopeptidase and superoxide anion wer not responsible for OZ-induced effects. OZ (500 micrograms) significantly increased the intracellular concentration of Ca2+ (P less than 0.01). Likewise, OZ (500 micrograms)-induced effects on lambda 0 of AM were significantly inhibited by replacement of the medium with a Ca2+ free solution (P less than 0.01). These results imply that opsonized zymosan inhibits cytoplasmic motility of AM via external calcium influx.     

Toxic effects of oxygen on cultured alveolar epithelial cells, lung fibroblasts and alveolar macrophages.

Exposure to hyperoxia results in endothelial necrosis followed by type II cell proliferation. This suggests that type II cells are resistant to hyperoxia. Oxygen-induced lung injury may result from an overproduction of oxygen metabolites normally scavenged by antioxidants such as superoxide dismutase (SOD), glutathione peroxidase, catalase and reduced glutathione (GSH). Therefore, resistance of type II cells to hyperoxia may be linked to high antioxidant activities. To test this hypothesis we compared in vitro the effects of a 24 h exposure period to 95% O2 on cultured type II cells, lung fibroblasts and alveolar macrophages isolated from rats. We show that type II cells, when compared with other cell types, are highly sensitive to hyperoxia as shown by increased lactate dehydrogenase (LDH) release, decreased deoxyribose nucleic acid (DNA) and protein content of Petri dishes and decreased thymidine incorporation into DNA. Synthesis of dipalmitoylphosphatidylcholine was also significantly reduced. Antioxidant enzyme activities as well as glutathione content were not higher in type II cells than in other cell types. However, hyperoxia results in a decreased SOD activity and glutathione content in type II cells which was not observed in fibroblasts. We conclude that adaptative changes in SOD and glutathione metabolism could be important defence mechanisms in cells exposed to hyperoxia.     

Mechanisms of serum-enhanced adhesion of human alveolar macrophages to epithelial cells.

Adhesive interactions between macrophages and epithelial cells in the pulmonary alveoli may be important in the pathogenesis of inflammatory lung diseases, such as those induced by cigarette smoking. Potential mechanisms controlling the interactions between these cells were investigated using human alveolar macrophages (AM) and MDCK or A549 epithelial cells. Five percent human serum enhanced the adhesion of AM to MDCK cells by approximately 6-fold and to A549 cells by approximately 1.7-fold. This enhancement was reduced by heating the serum for 30 min at 55 degrees C. Treating normal human serum with methylamine to inactivate C3, substituting C3-deficient serum, or pretreating serum-exposed MDCK cells with anti-C3 F(ab')2 all significantly diminished the adhesion of AM, suggesting that complement is involved. With the use of flow cytometry to examine complement receptors on AM, both CD11b/CD18 and CD11c/CD18 were detected but CR1 was not evident. Preincubating AM with a monoclonal antibody to CD18 reduced the adhesion of AM to MDCK cells by 40% while a significant reduction could not be demonstrated after preincubation with antibodies to either CD11b or CD11c. These data suggest that in the presence of serum C3bi is deposited on the surface of MDCK cells and that AM may attach to these cells, at least in part, through interactions between C3bi and one or more receptors in the CD11/CD18 family, which are present on AM.     

Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages.

Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng x mL(-1)) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, alphavbeta3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease.     

Pulmonary microvascular and alveolar epithelial permeability characteristics in N-nitroso-N-methylurethane-injected dogs

The subcutaneous injection of 5 to 6 mg/kg of body weight of N-nitroso-N-methylurethane (NNNMU) has been reported to cause acute alveolar injury in animals. To determine the permeability characteristics of the alveolar epithelium, we employed the in vivo saline-filled dog lung model and determined the time to 50 percent equilibration in minutes of a specific tracer in the blood and the lung model and determined the time to 50 percent equilibration in minutes of a specific tracer in the blood and the lung liquid (T 1/2) for endogenous serum albumin (MW 69,000 daltons, molecular radius 35 A) and exogenously administered 500,000 MW polydispersed dextrans (molecular radius 200 A). Compared to control animals, T1/2 decreased (permeability increased) in NNNMU-injected dogs from 3,500 +/- 100 to 682 +/- 160 minutes for albumin and from 20,000 +/- 250 to 2,790 +/- 750 minutes for 500,000 MW dextran (P less than 0.001). To determine the permeability characteristics of the pulmonary microvasculature, we employed the right lymph duct cannulation dog model and measured lymph flow/30 minutes, lymph albumin and dextran concentration, and lymph/plasma albumin and dextran ratios in control and NNNMU-injected dogs. Compared to control animals, lymph flow was significantly greater in NNNMU dogs, 2.07 +/- 1.1 vs .71 +/- .50 ml/30 minutes (P less than 0.01), respectively. We conclude that NNNMU injection increases permeability in both the alveolar epithelium and the pulmonary microvasculature.     

Mechanisms of serum-enhanced adhesion of human alveolar macrophages to epithelial cells

Adhesive interactions between macrophages and epithelial cells in the pulmonary alveoli may be important in the pathogenesis of inflammatory lung diseases, such as those induced by cigarette smoking. Potential mechanisms controlling the interactions between these cells were investigated using human alveolar macrophages (AM) and MDCK or A549 epithelial cells. Five percent human serum enhanced the adhesion of AM to MDCK cells by approximately 6-fold and to A549 cells by approximately 1.7-fold. This enhancement was reduced by heating the serum for 30 min at 55°C. Treating normal human serum with methylamine to inactivate C3, substituting C3-deficient serum, or pretreating serum-exposed MDCK cells with anti-C3 F(ab′)₂ all significantly diminished the adhesion of AM, suggesting that complement is involved. With the use of flow cytometry to examine complement receptors on AM, both CD11b/CD18 and CD11c/CD18 were detected but CR1 was not evident. Preincubating AM with a monoclonal antibody to CD18 reduced the adhesion of AM to MDCK cells by 40% while a significant reduction could not be demonstrated after preincubation with antibodies to either CD11b or CD11c. These data suggest that in the presence of serum C3bi is deposited on the surface of MDCK cells and that AM may attach to these cells, at least in part, through interactions between C3bi and one or more receptors in the CD11/CD18 family, which are present on AM.     

The distribution of temafloxacin in bronchial epithelial lining fluid, alveolar macrophages and bronchial mucosa.

The concentrations of temafloxacin, a new fluoroquinolone antimicrobial, in the potential sites of pulmonary infection were assessed by fibreoptic bronchoscopy with bronchoalveolar lavage. Fourteen patients received a course of temafloxacin, 600 mg twice daily, for three days prior to sampling. The mean serum concentration was 9.6 (SEM 1.2) mg.l-1, compared with 14.9(SEM 1.8) mg.kg-1 for bronchial mucosa, 26.5 (SEM 3.6) mg.l-1 for epithelial lining fluid and 83.0 (SEM 11.5) mg.l-1 for alveolar macrophage. In the ten patients who completed the protocol, site concentrations correlated well with serum concentrations. Temafloxacin was concentrated in each of the potential sites of infection examined and is, therefore, a promising new agent for the treatment of respiratory tract infection.     

Effect of mabuterol on tracheal mucociliary clearance of magnetized iron particles in anesthetized dogs.

We studied the effect of mabuterol on the tracheal mucociliary clearance of magnetized iron particles in anesthetized dogs. A catheter was inserted directly into the trachea through the mouth and 0.05 ml of saline solution with 30 mg of Fe3O4 was instilled into the trachea. After magnetization from outside the chest wall, the remanent magnetic field (RMF) strength generated in the trachea was sequentially measured with a flux-gate magnetometer. The decay of RMF immediately after sequential magnetization showed a clearance of Fe3O4 particles. Mabuterol (10 micrograms/kg) increased clearance as much as isoproterenol (10 micrograms/kg) 30 min after intravenous injection relative to control measurement (p less than 0.01). These results suggest that mabuterol is useful to promote clearance.     

Iron content in human alveolar macrophages.

Intracellular iron can be estimated semi-quantitatively by histochemical determination using the ferrocyanide reagent's score. Particle-induced X-ray emission (PIXE) allows accurate determination of various elements including iron in cells and biological fluids. Both techniques have been used to measure iron in alveolar macrophages gathered by bronchoalveolar lavage. The purpose of this study was to investigate the clinical usefulness of the PIXE technique in occupational respiratory medicine and in various pulmonary diseases. Using the PIXE method, we measured the iron content of alveolar macrophages in healthy subjects, with and without occupational exposure to iron dust, and in patients with pulmonary diseases (chronic obstructive pulmonary disease (COPD), lung cancer, Goodpasture's syndrome). Our results were then compared with those obtained with the ferrocyanide reagent. Intramacrophagic iron was 0.33 +/- 0.21 micrograms.10(-6) (mean +/- SD) cells in healthy non-smoking subjects without occupational exposure. Intramacrophagic iron was increased in smokers, iron-steelworkers, and in patients with COPD or lung cancer even in the absence of pulmonary haemorrhage. The two patients with Goodpasture's syndrome had high intramacrophagic iron content. About 80% of the whole bronchoalveolar lavage fluid iron content was in the cells. Mean iron content of blood monocytes, lymphocytes and neutrophils of eight healthy subjects was significantly lower than that of alveolar macrophages. A significant correlation was found between iron determination by the PIXE method and the ferrocyanide reagent's score (r = 0.89). We conclude that intramacrophagic iron may be increased in steelworkers and subjects with pulmonary haemorrhage, but also in asymptomatic smokers, in COPD and lung cancer patients without occupational exposure to iron dust.     

Airway epithelial beta 3-adrenergic receptor--effect on bioelectric properties and its mechanism of action]

To characterize the "atypical" beta-adrenergic receptor (beta 3-adrenergic receptor) and its action on ion transport across airway mucosa, we measured the bioelectric properties of canine cultured tracheal epithelium under short-circuited conditions in vitro. Submucosal but mucosal addition of BRL37344, a selective beta 3-adrenergic agonist, increased short-circuit current (Isc) in a dose-dependent fashion, the EC50 value being 30 fold higher than that of isoproterenol. This effect on Isc was accompanied by the accumulation of intracellular cyclic AMP, and it was abolished by diphenylamine-2-carboxylate, bumetanide, and Cl-free medium, but not by amiloride. Pretreatment of cell with beta 1- and beta 2-adrenergic antagonists greatly reduced the Isc response to isoproterenol, whereas it had little effect on the BRL37344-induced response. In addition, the increase in Isc produced by BRL37344 was competitively antagonized by cyanopindolol, but pA2 was significantly different from the case of isoproterenol. These results suggest that beta 3-adrenergic receptors exist on airway epithelium, and may stimulate Cl secretion across the airway mucosa via accumulation of intracellular cyclic AMP.     

Pulmonary alveolar proteinosis. Further evaluation of abnormal alveolar macrophages

To investigate the function of alveolar macrophages (AM) and the mechanisms of impairment in pulmonary alveolar proteinosis, we established in culture AM from three patients and from eight normal nonsmokers and assessed phagocytosis and phagolysosome fusion by the acridine orange assay with live yeast as the phagocytic challenge. Alveolar macrophages from the patients with pulmonary alveolar proteinosis ingested fewer yeasts per cell than did normal AM (mean +/- SE, 2.3 +/- 0.3 vs 3.3 +/- 0.2; p less than 0.05) and had decreased phagolysosome fusion (33 +/- 6 percent vs 64 +/- 1 percent; p less than 0.001). Alveolar macrophages from three normal subjects were incubated with cell-free fractions isolated by centrifugation of lavage fluid from the patients at 250 g (P1) or centrifugation of P1 supernatant at 20,000 g (P2). The P1 fraction did not decrease the number of AM ingesting yeast or the number of yeast cells ingested per cell, but the P2 fraction decreased both phagocytic indices. Conversely, phagolysosome fusion was depressed by the P1 fraction (48 +/- 3 percent vs 66 +/- 2 percent for untreated AM from the same subject; p less than 0.02) but not by the P2 fraction. Significant morphologic changes were noted in AM cocultured with both P1 and P2. Comparable concentrations of pooled P2 fractions from normal subjects did not decrease phagocytic indices in normal AM. These data confirm that AM in pulmonary alveolar proteinosis are dysfunctional, and, in particular, the finding of decreased phagolysosome fusion may be related to the high incidence of uncommon infections in these patients. We have shown that different fractions of alveolar filling material from patients with pulmonary alveolar proteinosis have unique effects on the phagocytic process in the normal AM, and the induced defects may be associated with apparent uptake of this material. These observations further support the hypothesis that in patients with pulmonary alveolar proteinosis, locally produced "toxic" substances may lead to impaired alveolar clearance and contribute to the pathogenesis of this disease.     

Pulmonary alveolar proteinosis: abnormal in vitro function of alveolar macrophages.

Pulmonary alveolar proteinosis is characterized by the accumulation of granular proteinaceous material within the alveoli of the lung. It is well established that patients with pulmonary alveolar proteinosis have a high incidence of complicating pulmonary infections, which suggests that the function of the alveolar macrophages is abnormal. To investigate the function of these cells, they were obtained from two patients by pulmonary lavage with physiologic saline solution and were incubated with Staphylococcus aureus in vitro. The decline in viable organisms from the culture was measured and compared with results obtained with normal alveolar macrophages. On the basis of decreased phagocytosis, results indicate that pulmonary alveolar macrophages from these patients had defective antibacterial function.     

香烟烟雾对小鼠肺泡巨噬细胞 RIG-I 样受体功能影响的研究

目的：探讨吸烟对肺泡巨噬细胞 RIG-I 样受体介导的信号传导功能。方法制备香烟烟雾提取物(cigarette smoke extract,CSE),用病毒模拟物 Poly(I∶C)及不同浓度 CSE 单独或者联合刺激小鼠肺泡巨噬细胞(MH-S)。收集细胞并抽提 RNA,用实时荧光定量 PCR 检测 RIG-I、MDA-5、干扰素β(IFN-β)、IL-1及 IL-6 mRNA 表达水平。结果单独的 CSE 刺激能引起 IL-1、IL-6 mRNA 等炎症细胞因子的水平升高,而对 RIG-I、MDA-5及 IFN-βmRNA 几乎无影响;CSE 能够抑制 Poly(I∶C)刺激引起的 RIG-I、MDA-5受体及分子 IFN-β、IL-1、IL-6增高现象,尤其对 IFN-β mRNA 的抑制最为明显,并且CSE 的这种抑制作用与其浓度呈正相关。结论单独 CSE 刺激 MH-S 细胞可以引起炎症因子的表达水平升高,Poly(I∶C)刺激引起的 RIG-I 样受体抗病毒信号通路中相关受体、IFN 及炎症因子表达水平增高现象可以被 CSE 抑制,巨噬细胞对病毒防御功能的减低可能与吸烟抑制 RIG-I 样受体信号通路功能有关。     

沙利度胺对肺泡上皮细胞上皮-间质转分化的作用

目的探讨沙利度胺对A549上皮-间质转分化的作用,阐明其抗肺纤维化的机理。方法 A549细胞分为3组,A:对照组;B:TGF-β1+DMSO组;C:TGF-β1+沙利度胺组。通过免疫印迹法检测A549细胞磷酸化p38 MAPK和JNK的蛋白,以及间质表型蛋白和上皮表型蛋白的表达;实时荧光定量PCR方法检测A549细胞p38 MAPK和JNK mRNA的表达。结果沙利度胺显著抑制(P0.05)TGF-β1诱导的A549细胞间质表型蛋白表达增加,可阻止(P0.05)A549细胞上皮表型蛋白表达的减少,显著减少活性MAPK蛋白及mRNA的表达(P0.05)。结论沙利度胺可能通过p38MAPK和JNK信号通道,从而抑制肺泡上皮细胞的上皮-间质转分化过程而发挥抗纤维化作用。     

Spectrum of immunoregulatory functions and properties of human alveolar macrophages

In health, pulmonary alveoli are maintained free of inflammatory responses to inhaled foreign antigens. The specific role of alveolar macrophages (AM) in modulating the local cellular immune response to antigens is controversial. Immunoregulatory function and properties of AM and blood monocytes (MN) were compared. The AM were obtained by bronchoalveolar lavage of healthy volunteers, MN by adherence of peripheral blood mononuclear cells to plastic. These accessory cells were added in increasing ratios to a responder population rendered rigorously accessory cell-dependent by nylon wool adherence and depletion of cells bearing the surface Class II MHC determinant, HLA-DR. At low ratios of mononuclear phagocytes to lymphocytes (less than or equal to 1:10), MN and AM supported significant and comparable blastogenic responses to tetanus toxoid (3H-thymidine incorporation at a 1:10 ratio was 9,697 +/- 2,508 for MN and 8,969 +/- 1,454 for AM, mean cpm +/- SE, n = 9, p = NS) and other antigens. Interleukin-1 (IL-1) activity in supernatants of MN stimulated with lipopolysaccharide (LPS), 10 micrograms/ml, was 115 +/- 28 versus 67 +/- 21 U/ml in supernatants of AM (n = 9, p greater than 0.2). At suboptimal concentrations of LPS, however, MN expressed more IL-1 activity than did AM. The specific mean fluorescence intensity of surface expression of HLA-DR determinants as assessed by flow cytometry was similar for MN and AM. At the higher ratio of 1:2, MN supported 32% increased responses to tetanus toxoid compared with that at 1:5 (p less than 0.05). In contrast, AM at a ratio of 1:2 suppressed lymphocyte response by 69% (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)