Exposure Of Alveolar Macrophages Research Articles

ATP citrate lyase links increases in glycolysis to diminished release of vesicular suppressor of cytokine signaling 3 by alveolar macrophages

Extracellular vesicles (EVs) are important vectors for intercellular communication. Lung-resident alveolar macrophages (AMs) tonically secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis in the distal lung via its actions in recipient neighboring epithelial cells. AMs are metabolically distinct and exhibit low levels of glycolysis at steady state. To our knowledge, whether cellular metabolism influences the packaging and release of an EV cargo molecule has never been explored in any cellular context. Here, we report that increases in glycolysis following in vitro exposure of AMs to the growth and activating factor granulocyte-macrophage colony-stimulating factor inhibit the release of vesicular SOCS3 by primary AMs. Glycolytically diminished SOCS3 secretion requires export of citrate from the mitochondria to the cytosol and its subsequent conversion to acetyl-CoA by ATP citrate lyase. Our data for the first time implicate perturbations in intracellular metabolites in the regulation of vesicular cargo packaging and secretion.

Oxidative Inactivation of the Proteasome Augments Alveolar Macrophage Secretion of Vesicular SOCS3.

Extracellular vesicles (EVs) contain a diverse array of molecular cargoes that alter cellular phenotype and function following internalization by recipient cells. In the lung, alveolar macrophages (AMs) secrete EVs containing suppressor of cytokine signaling 3 (SOCS3), a cytosolic protein that promotes homeostasis via vesicular transfer to neighboring alveolar epithelial cells. Although changes in the secretion of EV molecules—including but not limited to SOCS3—have been described in response to microenvironmental stimuli, the cellular and molecular machinery that control alterations in vesicular cargo packaging remain poorly understood. Furthermore, the use of quantitative methods to assess the sorting of cytosolic cargo molecules into EVs is lacking. Here, we utilized cigarette smoke extract (CSE) exposure of AMs as an in vitro model of oxidative stress to address these gaps in knowledge. We demonstrate that the accumulation of reactive oxygen species (ROS) in AMs was sufficient to augment vesicular SOCS3 release in this model. Using nanoparticle tracking analysis (NTA) in tandem with a new carboxyfluorescein succinimidyl ester (CFSE)-based intracellular protein packaging assay, we show that the stimulatory effects of CSE were at least in part attributable to elevated amounts of SOCS3 packaged per EV secreted by AMs. Furthermore, the use of a 20S proteasome activity assay alongside treatment of AMs with conventional proteasome inhibitors strongly suggest that ROS stimulated SOCS3 release via inactivation of the proteasome. These data demonstrate that tuning of AM proteasome function by microenvironmental oxidants is a critical determinant of the packaging and secretion of cytosolic SOCS3 protein within EVs.

Pulmonary surfactant phosphatidylcholines induce immunological adaptation of alveolar macrophages

Pulmonary surfactant plays an important role in lung surface tension, defense against invading pathogens, and immune response. Furthermore, alveolar macrophages (AM) that comprise the front line of immune defense against inhaled microorganisms are covered by a layer of pulmonary fluid. Phosphatidylcholines (PCs), including unsaturated lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), are the most prevalent phospholipids in pulmonary surfactant. POPC reacts with ozone to produce 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC), a soluble mediator that initiates an inflammatory reaction in the lungs. However, the modulatory effects of POPC and PONPC on biology and activity of AM remain inconclusive. The exposure of AM (cell line AMJ2-C11) to POPC and PONPC was not directly related to the production of inflammatory mediators. However, AM, pre-incubated with POPC or PONPC, showed enhanced response after lipopolysaccharide (LPS) stimulation, and increased the production of nitric oxide and cytokines. This phenomenon was also observed for classical-polarized macrophages (M1). This increment on the production of inflammatory mediators was not associated with macrophage polarization, but with up-regulation of Tlr4 and Myd88 gene expression, which was in accordance with the adaptation of immune cells. This observation was confirmed by the histone acetylation epigenetic pathway. In contrast to the priming effect of POPC on AM activity, a harmful immune response, induced on incubation with PONPC, improved prostaglandin E2 (PGE2) formation, resulting in diminished bacterial phagocytosis. Additionally, PONPC induced production of CXCL1/KC, which potentially mediates neutrophil recruitment and enhances tissue inflammation. These results disclosed another dynamic mechanism by which pulmonary surfactant lipids (natural or oxidized) primed macrophage activity, thus affecting lung host defense.

In vitro toxicity screening of polyglycerol esters of fatty acids as excipients for pulmonary formulations

One of the main problems for the development of pulmonary formulations is the low availability of approved excipients. Polyglycerol esters of fatty acids (PGFA) are promising molecules for acting as excipient for formulation development and drug delivery to the lung. However, their biocompatibility in the deep lung has not been studied so far. Main exposed cells include alveolar epithelial cells and alveolar macrophages. Due to the poor water-solubility of PGFAs, the exposure of alveolar macrophages is expected to be much higher than that of epithelial cells. In this study, two PGFAs and their mixture were tested regarding cytotoxicity to epithelial cells and cytotoxicity and functional impairment of macrophages. Cytotoxicity was assessed by dehydrogenase activity and lactate dehydrogenase release. Lysosome function, phospholipid accumulation, phagocytosis, nitric oxide production, and cytokine release were used to evaluate macrophage function. Cytotoxicity was increased with the increased polarity of PGFA molecules. At concentrations above 1 mg/ml accumulation in lysosomes, impairment of phagocytosis, secretion of nitric oxide, and increased release of cytokines were noted. The investigated PGFAs in concentrations up to 1 mg/ml can be considered as uncritical and are promising for advanced pulmonary delivery of high powder doses and drug targeting to alveolar macrophages.

Urban airborne particle exposure impairs human lung and blood Mycobacterium tuberculosis immunity

RationaleAssociations between urban (outdoor) airborne particulate matter (PM) exposure and TB and potential biological mechanisms are poorly explored.ObjectivesTo examine whether in vivo exposure to urban outdoor PM in Mexico City...

Pro-inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages

ObjectiveVaping may increase the cytotoxic effects of e-cigarette liquid (ECL). We compared the effect of unvaped ECL to e-cigarette vapour condensate (ECVC) on alveolar macrophage (AM) function.MethodsAMs were treated with...

Human Alveolar Macrophages May Not Be Susceptible to Direct Infection by a Human Influenza Virus.

The current studies were undertaken to determine the susceptibility of human alveolar macrophages (AMs) to influenza A virus (IAV) infection in comparison with autologous peripheral blood-derived monocytes-macrophages (PBMs). AMs and PBMs were exposed to IAV in vitro and examined for their ability to bind and internalize IAV, and synthesize viral proteins and RNA. PBMs but not AMs demonstrated binding and internalization of the virus, synthesizing viral proteins and RNA. Exposure of AMs in the presence of a sialidase inhibitor or anti-IAV antibody resulted in viral protein synthesis by the cells. Exposure of AMs to fluorescein isothiocyanate-labeled IAV in the presence of anti-fluorescein isothiocyanate antibody also resulted in viral protein synthesis. Thus, human AMs are apparently not susceptible to direct infection by a human IAV but are likely to be infected indirectly in the setting of exposure in the presence of antibody that binds the challenging strain of IAV.

Fatty acid ethyl esters disrupt neonatal alveolar macrophage mitochondria and derange cellular functioning.

BackgroundChronic alcohol exposure alters the function of alveolar macrophages (AM), impairing immune defenses in both adult and neonatal lungs. Fatty acid ethyl esters (FAEEs) are biological markers of prenatal alcohol exposure in newborns. FAEEs contribute to alcohol-induced mitochondrial (MT) damage in multiple organs. We hypothesized that in utero ethanol exposure would increase FAEEs in the neonatal lung and that direct exposure of neonatal AM to FAEEs would contribute to MT injury and cellular dysfunction.MethodsFAEEs were measured in neonatal guinea pig lungs after ± in utero ethanol exposure via gas chromatography/mass spectrometry. The NR8383 cell line and freshly isolated neonatal guinea pig AM were exposed to ethyl oleate (EO) in vitro. MT membrane potential, MT reactive oxygen species generation (mROS), phagocytosis, and apoptosis were evaluated after exposure to EO ± the MT-specific antioxidant mito-TEMPO (mitoT) or ± the pan-caspase inhibitor Z-VAD-FMK. Whole lung FAEEs were compared using the Mann–Whitney U-test. Cellular results were analyzed using 1-way analysis of variance, followed by the Student–Newman–Keuls Method for post hoc comparisons.ResultsIn utero ethanol significantly increased ethyl linoleate and the combinations of ethyl oleate + linoleate + linolenate (OLL), and OLL + stearate in the neonatal lung. In vitro EO caused significant MT dysfunction in both NR8383 and primary neonatal AM, as indicated by increased mROS and loss of MT membrane potential. Impaired phagocytosis and apoptosis were significantly increased in both the cell line and primary AM after EO exposure. MitoT conferred significant but only partial protection against EO-induced MT injury, as did caspase inhibition with Z-VAD-FMK.ConclusionsIn utero ethanol exposure increased FAEEs in the neonatal guinea pig lung. Direct exposure to the FAEE EO significantly contributed to AM dysfunction, in part via oxidant injury to the MT and in part via secondary apoptosis.

Metabolic Activation of the Organic Fraction Coated-Onto Air Pollution PM<sub>2.5</sub> and its Genotoxicity in a Co-Culture Model of Human Lung Cells

Air pollution Particulate Matter (PM2.5) is described as one of the major risk factors affecting human health. Hence, the objective of our research project was to evaluate the lung toxicity of PM2.5collected in Dunkerque (France), through the study of the metabolic activation of its organic fraction (e.g. Polycyclic Aromatic Hydrocarbons, PAHs; Volatile Organic Compounds, VOCs) and its genotoxicity in two human cell models: embryonic lung epithelial L132 cells and Alveolar Macrophages (AM) isolated from bronchiolo-alveolar lavages of healthy outpatients, in mono- and/or coculture. The coculture system we used allowed the direct exposure of AM to PM2.5, and the interaction between the two cell types only through soluble factor diffusion. Exposure to Dunkerque City’s PM2.5induced the gene expression of phase I and phase II enzymes (e.g. CYP1A1, CYP2E1, CYP2F1, NQO1, GST∏1, GSTμ3) involved in the metabolic activation of PAHS and/or VOCS, in AM, in mono- and coculture, and in L132 cells, only in monoculture. Taken together, these results reinforced the key role of AM in lung defenses, and indicated that particles, as physical vector of the penetration and retention of coated-PAHS and/or VOCS within cells, enabled them to exert a durable toxicity. DNA bulky adduct formation was also reported not only in Dunkerque City’s PM2.5-exposed AM, in mono- and coculture, but also in L132 cells from PAH-exposed coculture. Loss of Heterozygosity (LOH) and/or MicroSatellite Instability (MSI) of some MicroSatellites (MS) located in multiple critical regions of chromosome 3 were reported in L132 cells from Dunkerque City’s PM2.5-exposed mono- or cocultures.

Role of cysteinyl leukotrienes in human allergen-specific Th2 responses induced by granulocyte macrophage-colony stimulating factor

The pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF), which is elevated in the lungs of atopic asthmatic patients, has been shown to enhance major histocompatibility class II expression of alveolar macrophages (AM). We hypothesized that exposure of AM and monocytes from atopic asthmatic patients to GM-CSF would enhance their antigen presenting function, and investigated putative mechanisms for this effect. Alveolar macrophages were purified from bronchoalveolar lavage by plastic adherence. Monocytes and CD4(+) T cells were purified from peripheral blood by magnetic bead separation. Antigen-presenting cell (APC) were pretreated with GM-CSF, pulsed with allergen and cocultured with autologous T cells. T-cell proliferation was determined by tritiated thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay. Exposure of allergen-pulsed AM and peripheral blood monocytes to GM-CSF significantly increased allergen-specific T-cell proliferation and T helper 2 (Th2) cytokine production. The enhanced response was dependent on costimulation by CD86, but not CD80. Inhibition of the 5-lipoxygenase pathway abrogated GM-CSF-mediated upregulation by monocytes of allergen-specific interleukin-5 (IL-5) and IL-13 cytokine production. Blocking of the cysteinyl leukotriene receptor 1 (cysLT(1)) receptor by a specific receptor antagonist inhibited allergen-specific IL-5 production in response to GM-CSF pretreatment. Exposure to GM-CSF enhanced the capacity of human APC from atopic asthmatic patients to induce allergen-specific Th2 responses by a mechanism involving cysLT. Novel immunotherapies, targeting production of GM-CSF or its actions on APC have the potential, therefore, to prove beneficial in treatment of patients with inflammatory airway disease.

Tobacco Smoking Inhibits Expression of Proinflammatory Cytokines and Activation of IL-1R-Associated Kinase, p38, and NF-κB in Alveolar Macrophages Stimulated with TLR2 and TLR4 Agonists

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers' alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers' AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam(3)Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers' AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-kappaB activation was suppressed in smokers' AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-kappaB, resulting in suppressed expression of proinflammatory mediators.

Antigen-Presenting Cell Population Dynamics during Murine Silicosis

Silicosis is an occupational lung disease resulting from the inhalation of silica particles over prolonged periods of time, which causes chronic inflammation and progressive pulmonary fibrosis. Alveolar macrophages (AM) are critical effector cells, while less is known about the role and function of pulmonary dendritic cells (DC) in silicosis. We hypothesize that a balance exists between the suppressive nature of AM and the stimulatory capacity of DC to regulate lung immunity, and that this equilibrium may be overcome by silica exposure in vivo. Our results demonstrate that in response to silica exposure, both the percent and absolute number of AM significantly decreased over time, with a concomitant significant increase in DC. Both AM and DC exhibited cellular activation in response to silica, indicated by increased expression of cell surface markers. In the absence of silica-induced AM apoptosis (TNFR 1/2-null and Gld mice), no change was observed in the percent or absolute number of either cell type. Furthermore, bone marrow-derived DC, but not bone marrow-derived macrophages, migrated from the alveoli into the lung parenchyma in response to silica, resulting in significantly increased numbers of activated T lymphocytes. Collectively, the results demonstrate that AM and DC are distinct antigen-presenting cells within the respiratory tract that respond to silica exposure in vivo in unique ways, with significant implications for immune reactivity of the lung in response to environmental pathogens.

In Vitro Pro-inflammatory Regulatory role of Substance P in Alveolar Macrophages and Type II Pneumocytes after JP-8 Exposure

The effects of JP-8 on pro-inflammatory cytokine interleukin (IL)-1α,β and nitric oxide (NO) secretion as well as the role of substance P (SP) in these processes were examined in cultured alveolar macrophages (AM), type II epithelial cells (AIIE), and AM/AIIE co-cultures. Exposure of AM to JP-8 for 24 hr exhibited release of IL-1α,β, whereas exposure to AIIE showed a concentration-dependent NO overproduction. Data indicate that there are cell-dependent inflammatory mechanisms responsible for the actual level of JP-8 exposure in alveoli. However, treatment with substance P significantly attenuated JP-8 induced the IL-1α,β secretion. This finding was confirmed by using [Sar9 Met (O2)11] SP (10− 10 M), an agonist of substance P, suggesting that substance P may have signal pathway(s) to AM in the inflammatory response mediated by IL-1. Moreover, AM/AIIE co-culture obviously reduced NO overproduction observed in AIIE alone, suggesting that there may be cell interactions or communications between AM and AIIE in response to the JP-8 exposure.

Lung-Restricted Macrophage Activation in the Pearl Mouse Model of Hermansky-Pudlak Syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.

Effects of diesel exhaust particles on human alveolar macrophage ability to secrete inflammatory mediators in response to lipopolysaccharide

Ambient particulate matter (PM) has been shown to be associated with mortality and morbidity. Diesel exhaust particles (DEP) contribute to ambient PM. Alveolar macrophages (AM) are important targets for PM effects in the lung. The effects of DEP exposure on human AM response to lipopolysachharide (LPS; from gram-negative bacteria) challenge in vitro were determined by monitoring the production of interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and prostaglandin E 2 (PGE 2). The roles of organic compounds and carbonaceous core of DEP in response to LPS were evaluated by comparing the DEPs effect to that of carbon black (CB), a carbonaceous particle with few adsorbed organic compounds. AMs were exposed in vitro to Standard Reference Material (SRM) DEP 2975, SRM DEP 1650, SRM 1975 (a dichloromethane extract of SRM DEP 2975) and CB particles for 24 h. DEPs induced a decreased secretion of IL-8, TNF-α and PGE 2 in response to a subsequent LPS stimulation. DEPs also show suppressive effect on the release of inflammatory mediators when stimulated with lipoteichoic acid, a product of gram positive bacteria. In summary, in vitro exposure of human AM to DEPs significantly suppress AM responsiveness to gram-negative and positive bacterial products, which may be a contributing factor to the impairment of pulmonary defense.

Prostanoids Secreted by Alveolar Macrophages Enhance Ionic Currents in Swine Tracheal Submucosal Gland Cells

We examined the effect of substances released by swine alveolar macrophages (AMs) on ionic currents in airway submucosal gland cells (SGCs). AMs obtained by lavage were activated by 24-h zymosan exposure (0.1 mg/ml). Supernatant was collected and used to stimulate short-circuit current changes (DeltaI(SC)) in SGC monolayers in Ussing chambers. Dexamethasone (1 microM) or indomethacin (5 muM) during zymosan exposure of AMs reduced or abolished the supernatant-induced DeltaI(SC). Zymosan exposure induced a 5-fold increase in cyclooxygenase (COX)-2 but not COX-1 protein levels in AMs. Prostaglandin E(2) (PGE(2)) concentration in the supernatant from zymosan-activated AMs was 550 +/- 10 nM (n = 3) compared with 28 +/- 3 nM for unstimulated AMs (n = 3). PGE(2), applied serosally, induced DeltaI(SC) with an EC(50) of 15.5 +/- 1.3 nM (n = 4) and 3.6 +/- 1.8 microM (n = 3) when applied apically. Four types of endoprostanoid receptors (EP(1-4)) were detected in SGCs using Western blot. PGE(2)-induced DeltaI(SC) were inhibited by AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) but not by SC19220 (8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide), suggesting that endoprostanoid (EP)(2) but not EP(1) receptors were activated by PGE(2). Pretreatment of SGCs with supernatant from zymosan-activated AMs, PGE(2), or forskolin enhanced the sensitivity to acetylcholine (ACh)-induced DeltaI(SC). PGE(2)-induced DeltaI(SC) were blocked by charybdotoxin (ChTX), chromanol 293B, or glibenclamide. ACh-induced DeltaI(SC) were only blocked by ChTX or glibenclamide. None of these blockers altered PGE(2) pretreatment-induced sensitization of ACh-induced DeltaI(SC). These results demonstrate that prostanoids released from activated AMs directly increase cystic fibrosis transmembrane conductance regulator and K(+) channel activity. ACh-induced DeltaI(SC) are also enhanced due to enhanced activation of Ca(2+)-activated K(+) channels (K(Ca)).

Similarity of Gene Expression Patterns in Human Alveolar Macrophages in Response to Pseudomonas aeruginosa and Burkholderia cepacia

To determine if differences in the severity of pulmonary infection in cystic fibrosis seen with late isolates of Pseudomonas aeruginosa and Burkholderia cepacia are associated with differences in the initial response of alveolar macrophages (AM) to these pathogens, we assessed gene expression changes in human AM in response to infection with a laboratory strain, early and late clinical isolates of P. aeruginosa, and B. cepacia. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays, following exposure to laboratory P. aeruginosa strain PAK, showed significant (P < 0.01) >2.5-fold upregulation of 42 genes and >2.5-fold downregulation of 45 genes. The majority of the changes in gene expression involved genes as part of inflammatory pathways and signaling systems. Interestingly, similar responses were observed following exposure of AM to early and late clinical isolates of P. aeruginosa, as well as with B. cepacia, suggesting that the more severe clinical outcome of infections with late clinical isolates of P. aeruginosa or with B. cepacia cannot be explained by differences in the early interactions of these organisms with the human AM, as reflected by the similarity of gene expression changes in response to exposure of AM to these pathogens.

Neisseria meningitidis pili induce type-IIA phospholipase A 2 expression in alveolar macrophages

Induction of type-IIA secreted phospholipase A 2 (sPLA 2-IIA) expression by bacterial components other than lipopolysaccharide has not been previously investigated. Here, we show that exposure of alveolar macrophages (AM) to Neisseria meningitidis or its lipooligosaccharide (LOS) induced sPLA 2-IIA synthesis. However, N. meningitidis mutant devoid of LOS did not abolish this effect. In addition, a pili-defective mutant exhibited significantly lower capacity to stimulate sPLA 2-IIA synthesis than the wild-type strain. Moreover, pili isolated from a LOS-defective strain induced sPLA 2-IIA expression and nuclear factor kappa B (NF-κB) activation. These data suggest that pili are potent inducers of sPLA 2-IIA expression by AM, through a NF-κB-dependent process.

Meconium Inhibits Phagocytosis and Stimulates Respiratory Burst in Alveolar Macrophages

The meconium aspiration syndrome is an important cause of respiratory distress in newborn infants. Alveolar macrophages (AMs) provide a first line of defense in the lower respiratory tract against inhaled pathogens and particles such as meconium. In this study, we examined the effect of meconium on two primary macrophage functions: phagocytosis and respiratory burst. Short-term exposure of rat NR8383 AMs to sterile meconium from human or equine neonates (1.2-24 mg/mL) produced a dose-dependent decrease in phagocytosis of fluorescent latex beads. This effect was not due to decreased cell viability or to an elevation of intracellular cAMP. The effect of short-term exposure to meconium on the respiratory burst response in AMs was quantified using flow cytometry to measure oxidation of dichlorofluorescin diacetate. A robust respiratory burst was triggered when AMs were exposed to 12 or 24 mg/mL meconium. This effect was attenuated but not eliminated by filtration of the meconium. However, subsequent to meconium exposure, AMs had a reduced respiratory burst in response to stimulation with phorbol myristate acetate. In addition, AMs that were exposed to meconium for an extended period (24 h) showed DNA fragmentation indicative of apoptosis. Meconium therefore may interfere with AM function by inducing oxidative stress and apoptosis. Tissue injury from release of reactive oxygen species by AMs may be important in the pathophysiology of the meconium aspiration syndrome.

The Sources of Inflammatory Mediators in the Lung after Silica Exposure

The expression of 10 genes implicated in regulation of the inflammatory processes in the lung was studied after exposure of alveolar macrophages (AMs) to silica in vitro or in vivo. Exposure of AMs to silica in vitro up-regulated the messenger RNA (mRNA) levels of three genes [interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2)] without a concomitant increase in the protein levels. AMs isolated after intratracheal instillation of silica up-regulated mRNA levels of four additional genes [granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-1β, IL-10, and inducible nitric oxide synthase]. IL-6, MCP-1, and MIP-2 protein levels were elevated in bronchoalveolar lavage fluid. Fibroblasts under basal culture conditions express much higher levels of IL-6 and GM-CSF compared with AMs. Coculture of AMs and alveolar type II cells, or coculture of AMs and lung fibroblasts, in contact cultures or Transwell chambers, revealed no synergistic effect. Therefore, such interaction does not explain the effects seen in vivo. Identification of the intercellular communication in vivo is still unresolved. However, fibroblasts appear to be an important source of inflammatory mediators in the lung.