Unstimulated Alveolar Macrophages Research Articles

Characterization of alveolar macrophage eicosanoid production in a non-human primate model of mineral dust exposure

The relative activation of eicosanoid production which results from the exposure of the alveolar macrophage (AM) to mineral dusts is thought to be a key factor in the pathophysiology of occupational lung disease. We compared in vitro basal and silica-stimulated production of prostaglandin E 2 (PGE 2) and thromboxane A 2 (TXA 2) by AM from normal humans and non-human primates (Macaca nemistrina). In addition, we instilled mineral dusts directly into one lung of the non-human primate and evaluated AM eicosanoid production at two week intervals following dust instillation. Unstimulated AM from humans produce more PGE 2 and TXA 2 than do AM from M. nemistrina. However, in vitro exposure of AM from both species to silica dust produced a qualitatively similar increase in TXA 2 production accompanied by no change in PGE 2 production. Sequential analysis of AM eicosanoid production following a single bolus exposure to bituminous or anthracite coal dusts, titanium dioxide (TiO 2) dust or crystalline silica showed marked variability among individual non-human primates in qualitative and quantitative aspects of dust-induced eicosanoid production. However, the rank order of potency of the different dusts (silica > anthracite > bituminous) correlated with epidemiological evidence relating the type of dust mined to the incidence of pneumoconiosis. These studies suggest that the non-human primate may serve as a model for the study of both the role of eicosanoids in the etiology of dust-induced occupational lung disease and the biochemical basis for individual variability in the response of lung cells to mineral dust exposure.

Clinical Relevance of Cellular Mediators of Inflammation in Workers Exposed to Asbestos

To identify the clinical relevance of cellular mediators of inflammation in workers exposed to asbestos, we investigated the relationship between inflammatory products primarily released by alveolar macrophages and the clinical expression of asbestos-induced interstitial fibrosis. Our study population consisted of 93 white men who had been occupationally exposed to asbestos and were on average 60 yr of age. Pulmonary function tests, chest radiographs, high-resolution CT scans, and bronchoalveolar lavage (BAL) were performed on almost all study subjects; 11 (11.8%) had restrictive lung function, 22 (23.7%) had abnormal gas exchange, 30 (32.3%) had interstitial fibrosis on chest x-ray, and 24 (25.8%) had interstitial changes on high-resolution CT scan. The cellular markers of parenchymal inflammation that we examined included fibronectin in BAL fluid and alveolar macrophage release of prostaglandin E2 (PGE2), interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF-alpha) under unstimulated and endotoxin (LPS)-stimulated culture conditions. Significantly higher concentrations of fibronectin in BAL fluid were observed among those with restrictive lung function. In addition, higher concentrations of PGE2, released from cultured but otherwise unstimulated alveolar macrophages, were associated with restrictive lung function. However, the inverse relationship with PGE2 was observed among subjects with abnormal gas exchange. Interestingly, no consistent changes in these inflammatory mediators were observed in those with interstitial changes identified on either the chest radiograph or the high-resolution CT scan.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of Intracellular Candidacidal Activity Mediated by Calcium Ionophore in Human Alveolar Macrophages

In the present study, we investigated the effect of in vitro treatment with calcium ionophore (A23187) on candidacidal activity of human alveolar macrophages (AM) from normal subjects. In vitro incubation of AM with A23187 results in a significant dose-dependent enhancement of candidacidal activity. The availability of Ca2+ and Mg2+ ions in the culture medium is crucial for phagocytosis and killing to occur, but appears irrelevant for the binding between Candida albicans and AM. Enhancement of the killing effect mediated by A23187 does not correlate with increased phagocytic activity; in fact, the availability of ions is required for the phagocytic event, but an increase of cations does not correlate with enhancement of this activity. On the contrary, the augmentation of killing activity correlates with increased production of superoxide anion. Moreover, soluble material endowed with candidacidal activity has been extracted from cytoplasmic granules of AM both unstimulated and following A23187 treatment in vitro. Indeed, the granules extracted contain cationic proteases and, when isolated from stimulated cells, appear to be significantly more cytotoxic for C. albicans with respect to those obtained from unstimulated AM. In conclusion, the results reported here show that the phagocytic and killing events are ion dependent and the enhancement of intracellular candidacidal activity mediated by A23187 in AM is correlated with an augmented anti-Candida activity of cation-activated proteases.

TUMOR-CYTOTOXICITY OF NITRIC-OXIDE PRODUCED FROM ALVEOLAR MACROPHAGES DIRECTLY STIMULATED WITH TUMOR-CELLS

Macrophages activated by lipopolysaccharide or interferon-gamma have been shown to be cytotoxic to tumor cells by releasing nitric oxide. Here, we report that unstimulated rat alveolar macrophages cultured with certain tumor cells produce nitric oxide and are cytotoxic to these tumor cells. Alveolar macrophages were taken from BUF/Mna rats, which were known to produce spontaneous thymoma, and cultured with syngeneic BUF/Mna-derived thymoma cells. They were killed by syngeneic or allogeneic alveolar macrophages and this killing was partially abolished by addition of N(G)-monomethyl-L-arginine. X-ray irradiated, mitomycin C-treated or membranous fragments of BUF/Mna-derived thymoma cells directly stimulated rat alveolar macrophages to produce nitric oxide.

Pneumocystis carinii stimulates tumor necrosis factor-alpha release from alveolar macrophages through a beta-glucan-mediated mechanism.

Recent studies suggest that TNF-alpha plays a central role in host defenses during Pneumocystis carinii pneumonia. To determine whether P. carinii directly stimulates TNF-alpha secretion, rat alveolar macrophages were cultured in the presence of purified P. carinii. Whereas unstimulated alveolar macrophages released only 13.0 +/- 2.7 pg/ml of TNF-alpha into the medium, macrophages stimulated with P. carinii released 108.2 +/- 20.4 pg/ml of TNF-alpha after overnight culture (p = 0.0001). Maximal TNF-alpha release was observed after 8 h of incubation and required a P. carinii: macrophage ratio of at least 2.5:1. Autoclaved P. carinii were also able to trigger TNF-alpha release from macrophages albeit at a reduced level. In view of recent evidence that P. carinii is phylogenetically related to the fungi and contains a beta-glucan-rich cell wall, we hypothesized that TNF-alpha release might in part be mediated by this cell wall component. Preincubation of macrophages with particulate beta-glucan derived from Baker's yeast resulted in complete inhibition of TNF-alpha release in response to P. carinii. In addition, digestion of P. carinii with zymolyase, a preparation containing predominantly beta-glucanase activity, substantially reduced the ability of P. carinii to cause TNF-alpha release from macrophages. In a similar manner, macrophages incubated with P. carinii in the presence of laminariheptaose, an oligosaccharide that binds to macrophage beta-glucan receptors, also displayed decreased TNF-alpha release. Interestingly, TNF-alpha release may not be completely linked to the adherence of the organism to macrophages. Particulate beta-glucan significantly reduced P. carinii adherence to macrophages and also impaired TNF-alpha release. However, yeast mannan also significantly reduced P. carinii adherence to macrophages and also impaired TNF-alpha release. However, yeast mannan also significantly reduced P. carinii adherence but had no effect on TNF-alpha release. These data demonstrate that P. carinii can directly stimulate the secretion of TNF-alpha from alveolar macrophages and that this effect is largely mediated by beta-glucan components of the P. carinii cell wall.

In vitro acute effects of tobacco smoke on tumor necrosis factor alpha and interleukin-6 production by alveolar macrophages.

Tobacco smoke is a usual form of oxidant aggression present in the domestic environment. In the present study, the in vitro acute effects of a 2-cigarette smoke gas phase were evaluated on cell viability and cytokine secretion by alveolar macrophages (AM) from guinea pigs and human healthy subjects. Cell injury was estimated immediately after smoke exposure by evaluation of ATP cell content (measured by bioluminescence) and lactic dehydrogenase (LDH) release in the culture medium. LDH release was also measured when the interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) activities were evaluated. No cytotoxic effect was found: The ATP cell content of both guinea pig AM and human AM did not significantly change after tobacco smoke exposure. Similarly, the LDH release in the culture medium was unchanged both immediately after tobacco smoke exposure and at the time of the cytokine evaluation (18-20 h later) compared to cells cultured in the air. The total protein synthesis by the guinea pig AM evaluated by 35S-L-methionine labeling was unaffected by tobacco smoke exposure. The production of IL-6 and TNF activities was evaluated 18-20 h after smoke exposure. The IL-6 activity was measured by the proliferation test of 7TD1 hybridoma cell line; the TNF activity was evaluated by the L929 mouse fibroblast cytotoxic test and by an immunoradiometric assay (for human AM). A 2-cigarette smoke exposure decreased both activities significantly. The exposure of the guinea pig AM reduced IL-6 activity by 24.3 +/- 6.7%, 42.4 +/- 7.8%, and 39.7 +/- 9.6% and TNF activity by 33.8 +/- 10.4%, 35.1 +/- 10.7%, and 38.8 +/- 9.9% (respectively unstimulated cells and AM activated by 0.1 and 10 micrograms LPS/mL). The decrease in monokine production by the human AM was, respectively, 57.8 +/- 8.8%, 59.7 +/- 11.4%, and 49.9 +/- 10.5% of IL-6 activity and 37.4 +/- 14.6%, 17.6 +/- 9.6%, and 37.2 +/- 6.3% of TNF activity. The possible release of cytokine inhibitors was also investigated. The inhibitory activity against recombinant TNF and IL-6 was evaluated in culture medium from unstimulated AM exposed to tobacco smoke and did not significantly differ from that of AM exposed to air, demonstrating that the decrease of monokine levels could not be explained by the release of inhibitory factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of GM-CSF on TNF-alpha and IL-1-beta production by alveolar macrophages and peripheral blood monocytes from patients with sarcoidosis.

Sarcoidosis is a systemic granulomatous disorder of unknown origin characterized by activated T-lymphocytes and macrophages. Macrophage/monocyte-derived cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) have been shown to be involved in the pathogenesis of sarcoidosis. Although the production of TNF-alpha and IL-1 by alveolar macrophages (AM) and peripheral monocytes (PM) in response to lipopolysaccharide (LPS) in sarcoidosis has been well demonstrated, the production of these cytokines in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) has not been delineated. The present studies were designed to examine the regulatory effect of GM-CSF on TNF-alpha and IL-1 beta production by AM and PM from patients with pulmonary sarcoidosis. Amounts of TNF-alpha and IL-1 beta in the culture supernatants of unstimulated AM from patients with sarcoidosis were significantly higher than those from normal subjects, whereas, there was no difference in the amounts of TNF-alpha and IL-1 beta in the culture supernatants of PM between patients with sarcoidosis and normal subjects. The amounts of TNF-alpha and IL-1 beta in the culture supernatants of GM-CSF or LPS-stimulated AM and PM were significantly higher than those of similarly stimulated AM and PM from normal subjects. This hyperresponsiveness of AM and PM to GM-CSF in patients with sarcoidosis might be relevant to the pathogenesis of sarcoidosis.

Differential effects of interleukin-4 on superoxide anion production by human alveolar macrophages stimulated with lipopolysaccharide and interferon-gamma.

The effect of interleukin-4 (IL-4) on the activation state of human alveolar macrophages (AMs) and blood monocytes induced by lipopolysaccharide (LPS) or recombinant interferon-gamma (IFN-gamma) was investigated on the basis of their ability to produce superoxide anion (O2-). AMs were obtained from healthy donors by bronchoalveolar lavage, and O2- productions of these cells were assayed by a cytochrome c reduction method after incubation with stimulants for 24 h. AMs produced more O2- than autologous blood monocytes when stimulated with LPS. IL-4 alone had little effect on O2- production by unstimulated AMs but down-regulated O2- production by LPS-stimulated AMs in a dose-dependent manner. IL-4 also suppressed O2- production by AMs induced by the synergistic actions of muramyl dipeptide (norMDP) and IFN-gamma. Maximum suppression by IL-4 of O2- production by AMs was observed when IL-4 was added within 1 h after initiation of LPS stimulation. AMs also showed high O2- production when stimulated with IFN-gamma alone. In contrast to its suppression of O2- production by LPS-stimulated AMs, IL-4 enhanced O2- production by AMs stimulated with IFN-gamma. These data suggest that IL-4 is an important regulator of O2- production by macrophages through different pathways depending on the stimulus.

Zinc hydroxide stimulates superoxide production by rat alveolar macrophages

The effect of zinc hydroxide on superoxide (O 2 −) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH 2), a neutralized form of zinc ions, increased the chemiluminescence level and O 2 − release. Increased O 2 − release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimulate the O 2 − production by alveolar macrophages by receptor-mediated and Ca 2+-dependent process.

Formation of LTB4 by fMLP-stimulated alveolar macrophages accounts for eosinophil migration in vitro.

Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 +/- 11.9, number of migrating eosinophils [mean +/- SEM], n = 17) as compared to unstimulated macrophages (4.8 +/- 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 microM), a specific PAF antagonist, and with indomethacin (10 microM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 microM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 +/- 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 +/- 5.4% of desensitization toward LTB4 and 83.1 +/- 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.

Oxidative inactivation of alpha 1-proteinase inhibitor by alveolar macrophages from healthy smokers requires the presence of myeloperoxidase.

The aim of this work was to study the ability of human alveolar macrophages (AM) of 10 healthy smokers to inactivate alpha 1-proteinase inhibitor (alpha 1PI). Purified alpha 1PI was incubated for 45 min, with human alveolar macrophages before and after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. As a positive control, the same experiments were performed in parallel with blood human neutrophils (PMN). Results are expressed as percentage of inactivation of alpha 1PI as evaluated from its inhibitory activity against porcine pancreatic elastase. A strong correlation (r = 0.99) was shown when inhibitory activity of alpha 1PI was evaluated against porcine pancreatic elastase or human neutrophil elastase. Unstimulated AM (1.57 +/- 0.9%) as well as stimulated AM (PMA: 1 +/- 0.4%; zymosan: 3 +/- 0.6%) were unable to inactivate alpha 1PI. Gel electrophoresis of alpha 1PI demonstrated that AM before or after stimulation induced a slight proteolysis of alpha 1PI, whereas both cleaved and complexed alpha 1PI were found when alpha 1PI was incubated with activated PMN. Both unstimulated (22 +/- 2.6%) and activated PMN (PMA: 91.7 +/- 4.7%; zymosan: 90 +/- 5.5%) were responsible for a significant inactivation of alpha 1PI. Catalase, in contrast to superoxide dismutase, was responsible for a near complete protection of alpha 1PI inactivation by PMN. To better determine the role of PMN secretory products, especially myeloperoxidase (MPO), we also investigated the effect of zymosan-activated PMN supernatants or of purified MPO on the alpha 1PI-AM reaction. MPO assay in PMN supernatants demonstrated that activated neutrophils released significant amounts of MPO (16.8 +/- 4.1 U/ml), whereas MPO was undetectable in activated AM supernatants.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Macrophages from Patients with AIDS and AIDS-related Complex Constitutively Synthesize and Release Tumor Necrosis Factor Alpha

To verify the hypothesis that alveolar macrophages (AMs) from patients infected with HIV-1 could synthesize and release TNF alpha, AMs recovered from the BAL fluid of 11 patients with seropositive HIV-1 (six with AIDS and five with ARC) were tested in vitro for their ability to destroy TNF alpha-susceptible targets. Furthermore, the presence of TNF alpha was assessed in AM-conditioned supernatants on the basis of their cytotoxic activity and by using an immunoenzymatic test and immunoblotting. Transcription of the TNF alpha gene in AMs was also studied by means of the Northern blot analysis. AMs freshly recovered from patients infected with HIV-1 exhibited high levels of cell-mediated cytotoxicity against U937 targets, and the addition of a polyclonal anti-TNF alpha antibody resulted in a significant inhibition of the target lysis. Cell-free supernatants conditioned by unstimulated AMs exerted high levels of cytotoxic activity against TNF alpha-sensitive targets, whereas duplicate, neutralization experiments performed in the presence of an anti-TNF alpha antibody proved that the observed cytotoxic activity was mostly mediated by TNF alpha. The presence of high amounts of TNF alpha in the conditioned media was confirmed by the immunoenzymatic test. In addition, the immunoblot analysis showed that the TNF alpha released by AMs has a Mr 17,000 band, identical to a standard preparation of recombinant TNF alpha. The Northern blot demonstrated that unstimulated AMs express detectable levels of mRNA transcripts for TNF alpha. Taken together, our data support the concept that AMs from patients with HIV-1 infection constitutively release TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective Candidacidal Activity of Alveolar Macrophages and Peripheral Blood Monocytes from Patients with Chronic Obstructive Pulmonary Disease

We investigated the in vitro candidacidal activity of alveolar macrophages (AM) and peripheral blood monocytes (PBM) from normal subjects or from patients with chronic obstructive pulmonary disease (COPD) displaying defective skin test delayed-type hypersensitivity (DTH) reactivity to seven antigens including Candida albicans. The results showed that cells from patients with COPD were significantly less effective than cells from control subjects in the killing of C. albicans. To explore whether the observed functional impairment could be reversed, interferon-gamma (IFN-gamma) was added to AM and PBM from patients with COPD, alone or in the presence of lipopolysaccharide (LPS) as a suboptimal stimulus. The cells were cultured for 24 h and then assayed for anti-Candida activity. After IFN-gamma treatment, the fungicidal activity of cells from patients with COPD was comparable to that of unstimulated AM or PBM from healthy donors. Treatment with IFN-gamma plus LPS resulted in a further enhancement in the killing of C. albicans. To gain more insight into the mechanisms involved in the modulation of killing, we evaluated the possible stimulating activity of IFN-gamma plus LPS treatment on the secretion of tumor necrosis factor (TNF) and interleukin-1 (IL-1), two cytokines produced by activated macrophages and capable of stimulating natural effectors. The results showed that IFN-gamma plus LPS can indeed stimulate TNF and IL-1 secretion by AM and PBM from patients with COPD. Therefore, a precise role can reasonably be ascribed to these soluble factors in the observed augmentation of candidacidal activity as ascertained by treatment with IFN-gamma plus LPS.

Production of Tumor Necrosis Factor‐α by Alveolar Macrophages of Lung Cancer Patients

The abilities of human alveolar macrophages (AM) obtained from healthy donors and patients with lung cancer to produce tumor necrosis factor (TNF) were compared with those of their blood monocytes after activation with lipopolysaccharide (LPS). TNF activity was assayed by measuring cytotoxicity against actinomycin D‐treated L929 cells and TNF was determined quantitatively by sandwich enzyme‐linked immnnosorbent assay (ELISA) with polyclonal and monoclonal antibodies against TNF‐α. Unstimulated AM from healthy donors released variable amounts of TNF spontaneously, whereas blood monocytes did not. When treated with LPS for 24 h, AM and monocytes produced TNF dose‐dependently, but TNF production by AM was significantly more than that by blood monocytes. This TNF activity was inhibited completely by monoclonal anti‐TNF‐α antibody. Macrophages generated by in vitro maturation of monocytes induced by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) produced more TNF than freshly isolated monocytes. No difference was found in the abilities of AM from healthy donors and patients with lung cancer to produce TNF after activation stimuli. These observations suggest that human AM may be important in in vivo antitumor defense of the lung through TNF‐α production.

Interleukin-1 Release by Alveolar Macrophages in Asthmatic Patients and Healthy Subjects

A thymocyte proliferative response assay was used to compare spontaneous and lipopolysaccharide (LPS)-induced interleukin-1 (IL-1) release by alveolar macrophage (AM) in asthmatic patients and normal subjects. Twelve asthmatic patients and seven nonsmoking healthy subjects underwent a bronchoalveolar lavage (BAL). All asthmatic patients had a reversible airway obstruction and 7/12 were allergic. BAL AM were separated by adherence on tissue culture plates in medium RPMI-1640 supplemented with antibiotics and fetal calf serum, and were incubated with or without 10 micrograms/ml LPS for 20 h. Free-cell supernatants were tested by C3H/HeJ mice thymocyte proliferative assay. Unstimulated AM supernatant IL-1 activity was significantly higher in asthmatic patients (mean +/- SEM: 47.8 +/- 11.9 units/10(6) AM) in comparison with healthy subjects (4.8 +/- 2.3 units/10(6) AM; p less than 0.05, Mann-Whitney U test) but did not significantly differ between allergic (42.2 +/- 15.5 units/10(6) AM) and intrinsic asthmatic patients (55.8 +/- 20.7 units/10(6) AM). For healthy subjects, IL-1 activity was significantly higher in LPS-stimulated AM supernatants (85 +/- 20 units/10(6) AM, p less than 0.05; Mann-Whitney U test) in comparison with unstimulated ones; for asthmatic patients, unstimulated and LPS-stimulated AM supernatant IL-1 activity did not significantly differ. This finding is in accordance with previous work suggesting that AM from asthmatic patients have a weak suppressive activity upon lymphocyte proliferation and emphasize the enhanced AM releasability in asthma.

Synthesis of Complement by Alveolar Macrophages from Patients with Sarcoidosis

Sarcoidosis is a granulomatous disorder of unknown aetiology. Alveolar macrophages (AM) in sarcoidosis release a variety of mediators important to the pathogenesis of the disease. Complement is essential for the inflammatory response and we investigated whether there were any major defects in the potential for sarcoidosis AM to synthesize complement in vitro. AM from 11 patients with active sarcoidosis and three healthy controls were cultured under serum-free conditions. There was a significant binding of polyclonal (anti-C5, -C6, -C7, -C8) and monoclonal anti-complement antibodies (anti-C3c and anti-C9 neoepitope (aE11] to agarose beads incubated with unstimulated AM for 24, 48, or 72 h. A significant and inhibitable production of soluble C3c, C5, C9, and S-protein was found in the harvested medium as detected by enzyme immunoassays. Activated C3 and C9 were also detected based on neoepitope expression. Presence of co-cultured agarose beads reduced the amount of soluble S-protein due to deposition on the agarose. We argue that the C9 neoepitope is an integral part of the terminal complement complex (TCC), both in the fluid and solid phase when bound to the agarose. In the fluid phase, SC5b-9 was generated, whereas the agarose-bound S-protein is assumed not to be associated with TCC on the beads. The results demonstrate for the first time that AM from sarcoidosis patients synthesize the functional alternative and terminal pathway of complement.

Asbestos-Induced Release of a Human Alveolar Macrophage-Derived Neutrophil Chemotactic Factor

Neutrophils accumulate in the alveoli of asbestos-exposed individuals. In determining whether asbestos fibers induce the release of neutrophil chemotactic factor (NCF) from human alveolar macrophages, alveolar macrophages (10(6) cell/mL) obtained by bronchoalveolar lavage from six non-asbestos-exposed control subjects were exposed to crocidolite (0.1 and 1 mg/mL), chrysotile (1 mg/mL), or medium alone for 4 h, and NCF activity was measured in the supernatants in a 48-well microchemotaxis chamber with polycarbonate membrane filters (pore size, 3 microns) and purified human neutrophils. Alveolar macrophages in medium alone released negligible amounts of NCF (4 +/- 1 neutrophils per high-power field [N/HPF]). When macrophages were exposed to crocidolite (0.1 and 1 mg/mL), significant NCF release occurred (43 +/- 9 and 105 +/- 32 N/HPF, respectively; p less than 0.01 for each amount compare to alveolar macrophages cultured in medium alone). Chrysotile (1 mg/mL) induced similar NCF release (96 +/- 14 N/HPF; p less than 0.01 compared to unstimulated alveolar macrophages). Partial characterization of the NCF by Sephadex G-25 fine gel filtration demonstrated a molecular size of less than 1,000 daltons. These results show that human alveolar macrophages release NCF after exposure to asbestos. Release of NCF by alveolar macrophages in asbestos-exposed individuals may play a central role in the pathogenesis of asbestosis.

Feline hybridoma growth factor/interleukin-6 activity.

An assay system was developed to measure feline hybridoma growth factor (HGF)/interleukin‐6 (IL‐6) activity in biological samples containing many kinds of cytokines by using the proliferation of the newly established mouse‐rat hybridoma clone. B3B1. The proliferative response of this B3B1 clone was IL‐6‐specific, and could not be promoted by other cytokines including IL‐1, IL‐2, IL‐3, and granulocyte‐colony‐stimulating factor (G‐CSF), The anti‐human B‐cell stimulatory factor 2 (BSF‐2)/IL‐6 antiserum did not neutralize feline HGF/IL‐6 activity in conditioned media prepared from feline con A‐stimulated splenocytes and unstimulated alveolar macrophages, indicating antigenic differences between species. Feline HGF/IL‐6 was eluted into the fractions corresponding to a molecular weight of 30,000–40,000 in gel filtration, and into the fractions at a salt concentration of 0.2–0.3 M NaCI in anion exchange chromatography. The physicochemical properties of feline HGF/IL‐6 were slightly different from those of murine and human IL‐6.

Lucigenin chemiluminescence and its relationship to mitochondrial respiration in phagocytic cells

Unstimulated alveolar macrophages, but not polymorphonuclear leukocytes, elicit chemiluminescence from lucigenin which cannot be entirely accounted for by the resting level of superoxide generation. This chemiluminescence was inhibited by both superoxide scavengers and inhibitors of mitochondrial respiration. Although 12-0-tetradecanoyl phorbol-13-acetate addition resulted in a significant increase in cellular superoxide generation, an unexpected decrease in lucigenin chemiluminescence was noted. These results suggest that mitochondria in alveolar macrophages may be a site of lucigenin accumulation and dioxygenation and that 12-0-tetradecanoyl phorbol-13-acetate may modulate this activity.

Evidence for the presence of angiotensins in normal, unstimulated alveolar macrophages and monocytes.

The presence of angiotensins was demonstrated in normal unstimulated alveolar macrophages and monocytes from both mice and rats. These peptides were partially purified from cell homogenates by ion exchange chromatography and identified as being [Ile5] angiotensin I (Ang I), [Ile5] angiotensin II (Ang II) and to a lesser extent [Ile4] angiotensin III (Ang III) using high performance liquid chromatography (HPLC). Based on the present data both alveolar macrophages and monocytes expressed Ang I as quantified by a specific and sensitive radio-immunoassay (RIA) from the HPLC eluates. In contrast to this, alveolar macrophages from both mice and rats exhibited a fairly low, if detectable Ang II content. It seems reasonable to suggest that, in contrast to monocytes, macrophages do not generate and/or incorporate Ang II appreciably, at least in their resting stage. Although it is still not obvious whether these mononuclear phagocytes generate or simply capture angiotensin(s) from the blood pool or from the tissues; they must serve as in vivo target cells for the angiotensin system, at least for the plasma or tissue clearance of these molecules.