Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages

Cigarette smoking can impair pulmonary immune function, and hence influences the development of lung diseases. Interleukin-8 (IL-8) is a proinflammatory peptide and a potent chemotactic factor for neutrophils, and is produced by both immune and non-immune cells including monocytes and alveolar macrophages (AM). We investigated the effect of cigarette smoking on the secretion of IL-8 by human AM. The IL-8 concentration in bronchoalveolar lavage fluid (BALF) was much higher in smokers than in non-smokers (18.4 +/- 3.9 vs 4.1 +/- 1.0 pg ml-1; P < 0.005). However, spontaneous IL-8 secretion by cultured AM was lower in smokers than in non-smokers (46.8 +/- 12.7 vs 124.1 +/- 24.0 ng ml-1; P < 0.01). When stimulated with lipopolysaccharide (LPS), AM from smokers secreted significantly less IL-8 than those from non-smokers at all tested concentrations of LPS. In contrast, the amount of IL-8 secreted by peripheral blood monocytes with or without LPS stimulation was comparable in smokers and non-smokers. These observations indicate that smoking decreases IL-8 secretion by AM, which may modify or decrease the inflammatory response in the lung.     

Thalidomide reduces IL-18, IL-8 and TNF-alpha release from alveolar macrophages in interstitial lung disease.

Thalidomide exhibits diverse actions of anti-inflammation, immunomodulation and anti-angiogenesis. The efficacy of thalidomide treatment in sarcoidosis with lupus pernio is thought to be due to inhibition of tumour necrosis factor (TNF)-alpha. The mechanisms that underlie the properties of thalidomide are still unclear in interstitial lung disease. The current authors investigated the potential inhibitory effects of thalidomide at concentrations of 0.1, 0.01 and 0.001 mM on the production of transforming growth factor-beta, TNF-alpha, interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, IL-12p40 and IL-18 by alveolar macrophages from bronchoalveolar lavage in patients with sarcoidosis (n = 8), hypersensitivity pneumonitis (HP; n = 8) and idiopathic pulmonary fibrosis (IPF; n = 12). In sarcoidosis and HP patients, thalidomide induced a dose-dependent, partial suppression of lipopolysacchride (LPS)-stimulated TNF-alpha, IL-12p40 and IL-18 release. At the highest thalidomide concentration (0.1 mM), LPS-stimulated IL-8 production was also suppressed. In IPF patients, although spontaneous production of TNF-alpha, IL-12p40, IL-18 and IL-8 was lower than in sarcoidosis and HP patients, with LPS stimulation the cytokines were significantly elevated and also partially inhibited by thalidomide. In conclusion, thalidomide has the potential to improve the therapeutic regimens for sarcoidosis, hypersensitivity pneumonitis and idiopathic pulmonary fibrosis by reducing tumour necrosis factor-alpha, interleukin-12p40, interleukin-18 and interleukin-8 production.     

Tumour necrosis factor-alpha production in human alveolar macrophages: modulation by inhaled corticosteroid.

Using an ex vivo alveolar macrophage model, the hypothesis that inhaled preparations of corticosteroids might have important anti-inflammatory effects on cells of the peripheral airway was tested. The tumour necrosis factor (TNF)-alpha-inducing potential of three glycolipid preparations from nonpathogenic (arabinofuranasyl lipoarabinomannan (LAM (Ara-LAM)) and virulent (mannase LAM (ManLAM)) mycobacteria and Gram-negative bacteria (lipopolysaccharide (LPS)), in primary alveolar macrophage preparations was investigated. A novel inhaled chlorofluorocarbon (CFC)-free preparation of beclomethasone dipropionate (hydrofluoroalkane 134a (HFA)-BDP) with increased peripheral lung deposition was investigated for its ability to modulate glycolipidinduced TNF-alpha production by human alveolar macrophages, in comparison with a CFC-containing preparation and placebo. Compared to the basal TNF-alpha bioactivity of 0.72 ng x mL-1 (geometric mean), the TNF-alpha bioactivity in the macrophage preparation increased following incubation with LPS (138 ng x mL-1, p<0.001), AraLAM (12.6 ng-mL-1, p<0.001) and ManLAM (1.42 ng x mL-1, p=0.02). HFA-BDP, administered in vivo, significantly reduced LPS- and ManLAM-induced TNF-alpha production by alveolar macrophages cultured ex vivo. No change in glycolipid-induced TNF-alpha production was observed following in vivo administration of CFC-BDP or HFA-placebo. This is the first demonstration of an immunomodulatory effect on alveolar cells of corticosteroid delivered via metered dose inhaler. The present findings suggest that alveolar deposition of beclomethasone dipropionate is capable of modulating the inflammatory potential of the alveolar macrophage population.     

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.     

Tumor killing by human alveolar macrophages and blood monocytes. Decreased cytotoxicity of human alveolar macrophages

Tumor killing by human alveolar macrophages (AM) might be an important mechanism of pulmonary defense against neoplastic disease. We compared AM and blood monocytes (Mo) for the ability to kill 2 neoplastic targets, A549 human lung adenocarcinoma cells and P815 mastocytoma cells. Blood monocytes were able to kill both targets, whereas AM killed neither. Tumor killing by Mo was spontaneous and was not increased by incubation with lipopolysaccharide. Because the P815 target is highly sensitive to lysis by hydrogen peroxide (H2O2), it afforded the opportunity to compare AM and Mo for the ability to kill tumors by the production of toxic oxygen compounds. Comparable amounts of superoxide anion were produced by AM and Mo after stimulation with phorbol myristate acetate. However, luminol-enhanced chemiluminescence of AM was far less than that of Mo, suggesting that AM could not utilize the myeloperoxidase-H2O2-halide ion system for tumor killing. The addition of exogenous peroxidase to cultures of AM and P815 cells enabled AM to kill this tumor cell. Our results suggest that as Mo mature into AM, their ability to kill tumor cells declines and that AM may be unable to kill H2O2-sensitive tumors because of a loss of myeloperoxidase during maturation.     

Effects of N-acetylcysteine and ambroxol on the production of IL-12 and IL-10 in human alveolar macrophages

BACKGROUND: N-acetylcysteine (NAC) and ambroxol (AMB) have recently been proposed as possible therapeutic agents in the treatment of pulmonary disorders. IL-12 plays an important role in host resistance to infection and the development of Th-1 cells. In contrast, IL-10 is involved in anti-inflammatory and immunoregulatory mechanisms. OBJECTIVE: We investigated the effects of NAC and AMB on secretions of IL-12 and IL-10 from human alveolar macrophages. METHODS: Alveolar macrophages were obtained from 7 healthy nonsmokers by bronchoalveolar lavage. The cells were first incubated with either NAC or AMB for 2 h and then cultured in lipopolysaccharide (LPS) solution for 24 h. IL-12 and IL-10 secretions were measured by ELISA. RESULT: Both NAC and AMB enhanced LPS-induced secretion of IL-12. NAC also enhanced LPS-induced IL-10 secretion, while AMB did not. The ratio IL-12/IL-10 secretion was increased by AMB, but NAC did not affect it. CONCLUSIONS: The results suggest that NAC enhances inflammatory and immune responses and prevents excessive responses reciprocally, through keeping local balance of IL-12 and IL-10 production in alveolar macrophages at inflammatory sites of bacterial pneumonia. AMB appears to strengthen inflammatory responses and cell-mediated immunity, facilitating the development of Th-1 cells, through shifting the local balance to IL-12 dominance.     

CCK-8对LPS刺激大鼠肺泡巨噬细胞p38 MAPK、STAT3表达及TNF-α活性的影响

目的本实验在细胞水平,观察了八肽胆囊收缩素(CCK-8)及其受体非特异性抑制剂丙谷胺(proglumide,Pro)对LPS引起的大鼠肺泡巨噬细胞(AM)分泌TNF-α的影响,并进一步观察了p38MAPK和STAT3的表达。方法分离正常大鼠肺泡巨噬细胞,调节细胞浓度为1×106/ml,分组如下:①对照组;②LPS组:LPS终浓度5μg/ml;③CCK-8+LPS组:CCK-8终浓度10-10mol/L、10-8mol/L或10-6mol/L,LPS剂量同前;④CCK-8组:10-10、10-8和10-6mol/L;⑤Pro+LPS+CCK-8组:2μg/ml丙谷胺,10min后加入5μg/mlLPS及10-8mol/LCCK-8。收集刺激4h的细胞培养上清,应用L929细胞株进行TNF-α活性的生物学检测。刺激30min后,采用免疫细胞化学法观察p38MAPK和STAT3表达的变化。结果与对照组(6.76±1.39)pg/ml相比,LPS组TNF-α活性显著增加(1091.14±67.35)pg/ml(P<0.01)。10-10mol/L、10-8mol/L和10-6mol/L的CCK-8均显著抑制LPS诱导的AM释放TNF-α,且呈剂量依赖性,依次为(970.67±111.53)pg/ml、(583.48±62.03)pg/ml和(484.24±72.01)pg/ml(P均<0.01)。CCK-8本身不影响TNF-α产生。CCK受体非特异性拮抗剂丙谷胺可逆转CCK-8的抑制作用。免疫细胞化学结果显示CCK-8可增强LPS引起的AM中磷酸化的p38MAPK和STAT3的表达。结论p38MAPK为多种信号转导途径的交汇点。CCK-8可能通过激活p38MAPK途径及激活STAT3来调节AM的功能。     

Measurement of angiotensin-converting enzyme activity in intact human alveolar macrophages and effect of smoking

Serum angiotensin-converting enzyme (ACE) activity is known to be elevated in various granulomatous conditions such as sarcoidosis, whose characteristic epithelioid cells are thought to belong to the macrophage series. It was recently shown that alveolar macrophages had ACE activity in their sonicated and homogenate forms. We investigated ACE activity of human alveolar macrophages in the intact form using a sensitive radioimmunoassay of generated angiotensin II. We also investigated the effect of smoking on ACE activity of alveolar macrophages using this method. Alveolar macrophages generated angiotensin II (218 +/- 106 pg/20 min/10(5) cells). More angiotensin II was generated in smokers (246 +/- 119 pg/20 min/10(5) cells) than in nonsmokers (160 +/- 50 pg/20 min/10(5) cells) (p less than 0.05). These data showed that ACE activity of macrophages is higher in smokers than nonsmokers.     

Acute effect of smoking on superoxide production by pulmonary alveolar macrophages

We determined the acute effect of smoking on superoxide (O ₂ ⁻ ) production by pulmonary alveolar macrophages (AM) in 32 smokers who had bronchoalveolar lavage both before and after smoking. In 18 subjects, AM were obtained either 10, 30, or 60 minutes after the subjects had smoked 2 cigarettes, while in 14, AM were recovered either at 2, 10, or 60 minutes after they had smoked 4 cigarettes. Prior to smoking, O ₂ ⁻ production by AM was significantly greater in smokers than in 8 control nonsmokers for unstimulated AM, as well as for AM stimulated by zymosan and phorbol myristate acetate. Superoxide production by unstimulated AM increased significantly 1 h after subjects had smoked 2 cigarettes, to 140.8 ± SD 14.7% of levels prior to smoking, but there was no increase in 4 control smokers tested before and 1 h after sham smoking. Superoxide production by unstimulated AM decreased significantly 2 minutes after smoking 4 cigarettes but O ₂ ⁻ production by stimulated AM was not affected. Smoking had no effect on O ₂ ⁻ production by AM under the other conditions tested. We conclude that, in addition to the chronic effect of smoking that increases O ₂ ⁻ production by AM, there may be a stimulating effect of acute smoking, depending on the amount smoked and the time after smoking. The increased O ₂ ⁻ production by AM following smoking may be a factor contributing to inactivation of alpha₁-protease inhibitor. Presented in part at the American Thoracic Society Meetings, Miami, May 1984.     

Dexamethasone modulation of tumour necrosis factor-alpha (cachectin) release by activated normal human alveolar macrophages.

Recurrent infections of the lower respiratory tract are a frequent and serious side-effect of chronic corticosteroid treatment. Since alveolar macrophages (AM) are currently thought to play a central role in the protection of the lower respiratory tract against infectious agents, it is likely that a steroid-induced deficiency of AM is involved in this process. In this respect, when activated, AM are major producers of tumour necrosis factor-alpha (TNF or cachectin), a versatile cytokine with several biological properties including antiviral and anti-infectious activities. A deficit of TNF production induced by corticosteroid may be one mechanism of the sensitivity to infections. Thus normal human AM obtained by bronchoalveolar lavage were pretreated with dexamethasone (DXM) before activation with lipopolysaccharides (LPS) and the amounts of TNF released in culture were quantified. Pretreatment with DXM resulted in a marked decrease of TNF release in a dose-dependent fashion. In contrast, when AM were activated with LPS before DXM treatment, TNF release by AM was suppressed in a more limited fashion. Thus DXM suppression of LPS-activated AM ability to release TNF may play a role in the susceptibility to infections of patients chronically treated with corticosteroids.     

Receptors for human IgG subclasses on human alveolar macrophages

The biology of individual heavy chain subclasses of human IgG (IgG1-4) in lung host defenses has become important now that specific deficiencies of certain subclasses (IgG2 and IgG4) can be associated with chronic sinopulmonary infections and that IgG4 can be increased in forms of hypersensitivity lung disease. Because IgG is an important opsonic antibody that promotes attachment of bacteria or particles to phagocytes, the relative binding of IgG subclasses to membrane receptors on human alveolar macrophages might predict the efficacy of specific opsonin-mediated phagocytosis. With in vitro cultured normal alveolar macrophages, various IgG complexes were assessed for receptor binding with a rosetting assay. For respiratory cells in culture for 24 h, about 25% of the macrophages bound IgG3 and about 10% bound IgG1; binding with IgG2 and IgG4 complexes was minimal. In macrophage cultures maintained for as long as 6 days, this pattern of binding persisted. However, in very short-term cultures, 30 min and 105 min after cell adherence had occurred, binding was much greater for IgG3 complexes (about 60%); likewise IgG1 and IgG4 bound to about 20% of the cells. The IgM erythrocyte complexes, usually showing no binding at later time points in culture, bound to 20% of the cells, acutely. Therefore, our studies found that IgG3 consistently bound to more alveolar macrophages than the other subclasses, including IgG1. Also, the duration in culture of adherent cells may significantly affect the pattern of binding.     

Cytochrome b-245 in human alveolar macrophages

To kill microorganisms, phagocytes exhibit an oxidative burst with, in particular, a NADPH-dependent, superoxide-generating system that consists, in polymorphonuclear leukocytes (PMN), of a flavin enzyme and cytochrome b-245 (cyt b-245). We investigated the existence of this cytochrome in human alveolar macrophages (AM) because its presence would support its wide-spread occurrence in phagocytes and would raise the possibility of similarities in the oxygen-dependent killing mechanisms in AM and PMN. Moreover, we compared the amount of cyt b-245 in AM from patients with lung disorders with that from healthy subjects, by a differential spectroscopic measurement of its 558 to 559 nm characteristic band. This spectrum showed that cyt b-245 was present in AM. In AM of healthy subjects, the amount was similar to that found in PMN of blood. In AM of patients with miscellaneous lung diseases and in Am of infected lungs, the data were not modified.     

The mechanism and specificity of IL-1 inhibitory factor released from human alveolar macrophages]

We have previously reported the presence of interleukin 1 (IL-1) inhibitory factor in the culture supernatants of alveolar macrophages. The activity is decreased in healthy smokers and patients with interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis), compared with healthy non-smokers. In this study, we further examined the mechanism and specificity of IL-1 inhibitory factor. The inhibitory factor exhibited specific inhibition of augmentation by IL-1 of PHA induced murine thymocyte proliferation, whereas there was no inhibition of the augmentation by IL-2, IL-4, IL-6 or TNF. This IL-1 inhibitory factor competitively blocked the binding of IL-1 to the IL-1 receptor on PHA-stimulated murine thymocytes. These results indicate that alveolar macrophages produce a specific IL-1 inhibitory factor which functions as a IL-1 receptor antagonist.     

Lymphocyte macrophage interactions: peripolesis of human alveolar macrophages.

Peripolesis is a phenomenon in which a lymphocyte attaches itself to another cell, usually a macrophage or veiled cell, and proceeds to circle around it. In emperipolesis, a related phenomenon, the lymphocyte invaginates the target cell so deeply that it appears to be intracytoplasmic. Lung cells in bronchoalveolar lavage fluids from 20 patients were observed in the living state and filmed. Peripolesis of the alveolar macrophages was recorded in six cases. These patients included one case each of carcinoma of the bronchus, tuberculosis, sarcoidosis and asthma, while two patients had no detectable lung disease. Five out of the six positive cases were females. In every instance there was a high number of lymphocytes in the washing. The peripolesed macrophages were not injured, but temporary alteration of the cell membrane was noted in a minority of film sequences. The peripolesing cells were also examined by transmission and scanning electron microscopy. The lymphocyte was found to be closely attached to the surface of the macrophage, with no invagination and its ultrastructure was that of a small lymphocyte. Peripolesis is probably a physiological mechanism concerned with regulation of the immune response in the lung.     

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 +/- 2.0 microA/cm2 (mean +/- SE, p less than 0.01) and 3 x 10(5) AMs/ml, respectively. This effect was accompanied by the release of prostaglandin E2 and F2 alpha 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.