Human Bronchoalveolar Macrophages Research Articles

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.

Envelope glycoprotein (gp120) from HIV-1 enhances Mycobacterium avium growth in human bronchoalveolar macrophages; an effect mediated by enhanced prostaglandin synthesis.

Human bronchoalveolar lavage (BAL) macrophages were obtained from normal human volunteers and infected with an AIDS-associated strain of Mycobacterium avium. Infected cells were exposed to purified envelope glycoprotein (gp120) from HIV-1 or to the recombinant non-glycosylated gp120 fragments PBI-RF and PBI-IIIB. Native gp120 increased Myco. avium growth in human cells from six separate donors, whereas the non-glycosylated fragments of gp120 had no such effect. Moreover, gp120 induced a substantial secretion of prostaglandin E2 (PGE2) from macrophages; inclusion of indomethacin blocked the enhanced permissiveness of infected cells treated with gp120. Soluble CD4 also neutralized the effect of gp120. Overall, these results indicate a role for gp120 in the susceptibility of AIDS patients to Myco. avium infections, mediated by an enhanced PGE2 release.

CYTOKINE PROFILE OF HUMAN BRONCHOALVEOLAR MACROPHAGES AND BRONCHIAL EPITHELIAL CELLS IN RESPONSE TO INHALATION PARTICLES OF THE CYCLOSPORINE DERIVATIVE IMM 125

Administration of antiasthmatic drugs in the form of inhalation particles may alter the cytokine network in the airways, independently of their pharmacological actions. Changes induced by drugs not well tolerated may potentially contribute to the immunopathology of the disease, a strongly undesirable effect. In this study, cell viability assays and characterization of the cellular profile of cytokines and chemokines were performed in order to investigate the response of human bronchoalveolar macrophages and bronchial epithelial cells in culture to inhalation particles of the cyclosporine derivative IMM 125. Interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-8 were assayed by enzyme-linked immunosorbent assay (ELISA) in the supernatants of bronchoalveolar macrophages, and RANTES, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-8 in those of bronchial epithelial cells. Cells were studied both under basal and stimulated conditions (lipopolysaccharide and TNFalpha were used for activating macrophages and epithelial cells, respectively). The immunosuppressant FK 506 and the glucocorticoid Budesonide served as comparison. IMM 125 did not affect cell viability (except at high concentrations and long time periods). Moreover, IMM 125 did not induce an increase in the secretion of any of the cytokines and chemokines measured with respect to nontreated cells, except for a slight increase in IL-8, an effect that was also observed for FK 506, Budesonide, and inert latex particles, and was therefore regarded as nonspecific. Furthermore, IMM 125 significantly decreased the secretion of TNFalpha, IL-1beta by macrophages, and GM-CSF by epithelial cells, suggesting an antiinflammatory potential. In conclusion, the present in vitro results point to a good tolerance of human airways to IMM 125 inhalation particles.

Bronchoalveolar macrophage CD14 expression: shift between membrane-associated and soluble pools.

The bacterial endotoxin [lipopolysaccharide (LPS)]-binding protein CD14 modulates the host response to LPS, but membrane-associated and soluble forms of the molecule exert different biological effects. CD14 anchored to the mononuclear phagocyte membrane (mCD14) enhances response to LPS. Soluble CD14 (sCD14) may block LPS stimulation of CD14-bearing cells while supporting LPS presentation to non-CD14-bearing cells. We analyzed cell mCD14 and sCD14 expression in simultaneously collected human bronchoalveolar macrophages (BAM) and peripheral blood monocytes (PBM). Expression of mCD14 in freshly isolated BAM was only 9% as high as in PBM. Levels of sCD14 in 48 h in BAM culture supernatants were 19% as high as in PBM cultures. Interleukin (IL)-6 increased CD14 expression in both BAM and PBM but exerted different effects on CD14 distribution in these cell types. IL-6 increased only sCD14 release (2.5-fold) in BAM while increasing only mCD14 expression (2.5-fold) in PBM. IL-4 reduced both mCD14 (> 40%) and sCD14 (> 60%) expression in both cell types. We speculate that the balance between sCD14 and mCD14 expression influences the response to aspirated or inhaled LPS in the bronchoalveolar compartment. Cytokine expression and monocyte recruitment may influence this process by modulating CD14 expression.

Interactions between human bronchoalveolar macrophages and Blastomyces dermatitidis conidia: demonstration of fungicidal and fungistatic effects.

The effects of human bronchoalveolar macrophages (BAMs) on Blastomyces dermatitidis conidia were investigated. Macrophage monolayers were incubated for 5 days with or without interferon (IFN)-gamma or macrophage colony-stimulating factor (M-CSF) before challenge with conidia for 48 h at 37 degrees C. Hyphal growth (germination) was inhibited 12% by resident BAMs (P < .05). In contrast, resident BAMs blocked conidial phase transition by 88% (P < .05). Intermediate forms, aggregates of large cells in clums (not conidia or yeasts by morphologic criteria), appeared after incubation with BAMs and grew into typical yeasts once removed from the BAMs. IFN-gamma and M-CSF inhibited the ability of BAMs to block phase transition but not germination. Human BAMs demonstrated fungicidal and fungistatic activity against B. dermatitidis conidia independent of reactive oxygen intermediates and had greater effects on phase transition than on germination. The phase transition-associated fungicidal and fungistatic activities of BAMs were inhibited by IFN-gamma and M-CSF.

Interleukin 13 and Interleukin 4 Protect Bronchoalveolar Macrophages from Productive Infection with Human Immunodeficiency Virus Type 1

In this study, we examined the impact of the predominantly Th2-type lymphokines interleukin 13 (IL-13) and interleukin 4 (IL-4) on acute infection of human bronchoalveolar macrophages with a macrophage-tropic isolate of human immunodeficiency virus type 1 (HIV-1). Addition of 0.01-10 ng of IL-4 or IL-13 per milliliters significantly blocked HIV-1 replication in infected cells, judging from levels of reverse transcriptase and p24 antigen in the supernatants of infected cells. Both IL-4 and IL-13 were almost as efficient as interferon-gamma (IFN-gamma) in preventing HIV-1 replication, when given in equivalent amounts. Moreover, neither IL-13 nor IL-4 interfered with the IFN-gamma-mediated enhancement of anti-HIV-1 activity in alveolar macrophages. Both IL-4 and IL-13 interfered with enhanced replication of HIV-1 in macrophages pulsed with the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). Interleukin 13 also prevented HIV-1 release from peripheral blood mononuclear cells in a cocultivation experiment with feeder cells from a seronegative subject. These data suggest that Th2-derived lymphokines have significant anti-HIV-1 activity in cells of the macrophage lineage, although they may enhance the susceptibility of HIV-1-infected subjects to some opportunistic pathogens.

Interaction of VIP, PACAP and related peptides in normal and leukemic human monocytes and macrophages

The activation of the cAMP signaling pathway by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP) and related peptides was studied (i) in normal peripheral human monocytes and THP-1 leukemic human monocytes, (ii) in their derived macrophage counterparts respectively obtained after spontaneous differentiation or retinoic acid (RA) treatment, and (iii) in human bronchoalveolar macrophages. In THP-1 monocytes, PACAP increased basal adenylate cyclase activity 5.3-fold, with an affinity 50-times greater than that of VIP or helodermin ( K a = 3.2 × 10 −11 M VIP), whereas in normal peripheral monocytes, PACAP and VIP exhibited similar affinities and only increased eAMP generation 2-fold (EC 50 = 10 −9 M). Spontaneous and RA-induced differentiation into normal and leukemic macrophages induced a progressive loss of cAMP production and regulation of superoxide anion production by VIP and related peptides. The neoplastic transformation in THP-1 monocytes and the deficiencies in the cAMP cascade observed during the terminal differentiation of normal and leukemic human macrophages may relate to a differential genetic expression of the VIP/PACAP receptor subtypes, and alterations in the functional activity of the stimulatory and inhibitory G s/G i subunits of adenylate cyclase.

HIV-1 envelope protein (gp120) inhibits the activity of human bronchoalveolar macrophages against Cryptococcus neoformans.

Cryptococcus neoformans infections are a major cause of morbidity and mortality for HIV-infected persons. Containment of the initial respiratory inoculation to the lung appears defective in patients with AIDS despite the low burden of HIV in bronchoalveolar macrophages. We have studied the fungistatic activity of human bronchoalveolar macrophages (BAM) cultured with an encapsulated strain of C. neoformans in the presence of pooled human serum. We observed 51.6% fungistasis after 24 h of culture. Fungistasis was diminished if the pooled human serum was heat-inactivated but was not affected by anticryptococcal capsular IgG. HIV envelope protein (gp120) has been shown to interfere with lymphocyte activation in vitro. We studied the effects of gp120 on BAM function and found that fungistatic activity was inhibited 25% (p < 0.001). Although binding of yeasts was not affected, gp120 inhibited the internalization of bound yeasts by 46% (p = 0.025). These experiments indicate that gp120 decreases the internalization and fungistasis of C. neoformans by human BAM, and they suggest a mechanism to explain how a small number of HIV-1-infected cells in the lung could impair the containment of C. neoformans.

Binding of unopsonized Cryptococcus neoformans by human bronchoalveolar macrophages: inhibition by a large-molecular-size serum component.

Infection with Cryptococcus neoformans usually begins after inhalation of airborne organisms. Since levels of opsonins in the alveolar space may be low, the ability of human bronchoalveolar macrophages to bind C. neoformans in the presence and absence of opsonins was studied. Bronchoalveolar macrophages bound unopsonized C. neoformans. Surprisingly, component(s) in pooled human serum (PHS) inhibited binding, as evidenced by 26% and 71% inhibition of binding when 20% PHS and heat-inactivated PHS (HI-PHS), respectively, were added to the system. Separation of PHS by molecular size revealed that the inhibitory component had an apparent molecular weight greater than 10(6) and was inhibitory at nanomolar concentrations. PHS stimulated and HI-PHS had no effect on binding of acapsular C. neoformans and zymosan particles to bronchoalveolar macrophages. These data demonstrate that bronchoalveolar macrophages can bind unopsonized, encapsulated C. neoformans, but that serum component(s) inhibits binding.

Human and rabbit newborn lung macrophages have reduced anti-Candida activity.

Bronchoalveolar macrophages (BAM) protect the adult lung from low level microbial contamination. The antimicrobial activity of human newborn BAM is unknown. BAM were isolated from effluents of suctioned, intubated newborns and from bronchoalveolar lavage of healthy, nonsmoking adult volunteers. An in vitro cytologic slide assay was developed and used to ascertain: 1) inhibition of intracellular filamentation of Candida albicans (active yeast growth) and 2) killing + digestion of ingested C. albicans. The ability to restrict intracellular yeast filamentation was markedly different for human newborn versus adult BAM. Adult BAM were five times more effective in restricting intracellular filamentation of Candida compared to newborn cells (p less than 0.01). Nonfilamented ingested yeast were also handled differently by newborn and adult macrophages. Nonfilamented yeast were killed and digested by adult BAM at a rate that was 2.5 times above that noted in neonatal lung macrophages (p less than 0.005). However, no differences were found in the total number of killed + digested Candida within human newborn and adult BAM [adult = 32.4 +/- 10.5% (n = 5), newborn less than 1200 g = 39.6 +/- 16.8% (n = 8), and newborn greater than 1200 g = 30.2 +/- 11.1% (n = 16), mean +/- S.D.]. Neonatal BAM were able to destroy C. albicans at a level equivalent to adult cells because these newborn phagocytes allowed intracellular Candida to enter a state of active growth, thereby rendering the yeast more susceptible to killing and digestion. The anti-Candida activity noted in lung macrophages recovered from normal 1-day-old and adult rabbits was similar to that seen in human BAM.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement biosynthesis by human bronchoalveolar macrophages

Complement production by bronchoalveolar macrophages recovered from 8 normal volunteers and 15 patients with a variety of lung diseases was measured functionally and immunochemically. While macrophages from all eight normals demonstrated the capacity to secrete hemolytically active C2 and factor B within 48 hr of culture at consistent rates, bronchoalveolar macrophages from patients secreted C2 and factor B in widely differing amounts, and in some cases, not at all. No functional, secreted C3 was detected from normal macrophage monolayers, although apparently native C3 protein was synthesized and secreted. In contrast, functional C3 was produced by macrophage monolayers from 3 of 15 patients. These findings suggest that complement production by the normal human bronchoalveolar macrophage differs from its progenitor cell, the blood monocyte, and that complement production by bronchoalveolar macrophages may be altered in different pulmonary diseases.

Human bronchoalveolar macrophage cytotoxicity for cultured human lung-tumour cells.

Human bronchoalveolar macrophages were separated from other free lung cells by density sedimentation on Percoll gradients. They were then tested for cytotoxicity against the human lung adenocarcinoma cell line A549, using a Selenomethionine-75 post-labelling assay. The cytotoxicity of the macrophages increased as the effector:target cell ratio was increased, approaching 100% at 20:1. There was no significant difference in the cytotoxicity of macrophages isolated from the lungs of bronchial-carcinoma or non-carcinoma patients. The highly cytotoxic nature of the macrophages was not due to selection of a more potent cytotoxic subpopulation of macrophages on the Percoll gradient, nor to a generally elevated activation of the macrophages due to the pathological conditions in the patients' lungs. An attempt to determine whether low concentrations of macrophages could potentiate target-cell growth proved negative. Cytotoxicity of macrophages for cultured lung target cells was not restricted to A549 cells and is not in accordance with the view that defective bronchoalveolar macrophage cytotoxicity contributes to the emergence of bronchial neoplasia.

Inhibition of natural killer activity by human bronchoalveolar macrophages.

Mononuclear phagocytes were isolated by adherence from peripheral blood, peritoneal exudates, early lactation milk, ovarian carcinomatous ascites and bronchoalveolar lavages. Their capacity to modulate natural killer (NK) activity was assessed by mixing them with blood lymphocytes and by measuring lysis of 51Cr-labeled K562 cells. Unlike other mononuclear phagocyte populations, alveolar macrophages caused a marked dose-dependent inhibition of NK activity. Significant inhibition (40%) of the expression of cytotoxicity was evident at a ratio of alveolar macrophages to lymphoid cells of 0.12:1, and more than 80% suppression was usually observed at a ratio of 0.5:1. Blood monocytes, peritoneal and milk macrophages were consistently inactive up to the highest ratio tested, 2:1. Inhibition of the expression of NK activity by alveolar macrophages was observed at lymphocyte to K562 ratios ranging from 6:1 to 100:1 and over a 4 h or 20 h 51Cr release assay. Alveolar macrophages also inhibited interferon-stimulated cytotoxicity. Alveolar macrophages are unique among the mononuclear phagocyte populations studied in their capacity to inhibit the expression of NK activity effectively, and they could play a role in determining the low levels of NK activity associated with human pulmonary tissue.