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Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystispneumonia. Recognition of unopsonized Pneumocystisorganisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)‐κB. However, the AM host defense genes activated by Pneumocystishave not been defined. In the present study, incubation of AM with unopsonized Pneumocystisorganisms was not associated with release of interleukin (IL)‐1β, IL‐6, or tumor necrosis factor (TNF)‐α (important cytokines in the host response to Pneumocystis) and did not induce IL‐1β, IL‐6, or TNF‐α mRNA transcripts. These findings were not attributed to Pneumocystis‐induced cytopathic changes, as these same AM released IL‐8 and matrix metalloproteinase‐9 in response to Pneumocystis. NF‐κB‐mediated IL‐8 release was independent of Pneumocystisphagocytosis. The observed response was specific, as IL‐1β, IL‐6, and TNF‐α release and mRNA induction were preserved in response to lipopolysaccharide or serum‐opsonized Pneumocystis. The absence of IL‐1β, IL‐6, and TNF‐α release in response to Pneumocystiswas predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF‐α release in response to unopsonized Pneumocystisorganisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystisorganisms reduced Toll‐like receptor 4‐mediated TNF‐α release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.

ABSTRACTAlveolar macrophages (AM) represent important effector cells in the innate immune response to the AIDS-related pathogen Pneumocystis, but the early AM host defense signaling events are poorly defined. Using AM from healthy individuals, we showed in the present study that Pneumocystisorganisms stimulate AM NF-κB p50 and p65 nuclear translocation in a time-dependent and multiplicity-of-infection-dependent manner as determined by electrophoretic mobility shift assay and immunofluorescence microscopy and that NF-κB nuclear translocation is associated with I-κB phosphorylation. Importantly, competitive inhibition of mannose receptor and targeted short interfering RNA-mediated gene suppression of mannose receptor mRNA and protein is associated with complete elimination of NF-κB nuclear translocation in response to Pneumocystis. Furthermore, human immunodeficiency virus (HIV) infection of AM (as a model human disease state of reduced AM mannose receptor expression and function) inhibits Pneumocystis-mediated NF-κB nuclear translocation and is associated with reduced I-κB phosphorylation and reduced interleukin-8 (IL-8) release. In contrast, NF-κB nuclear translocation and IL-8 release in response to lipopolysaccharide are intact in AM from both healthy and HIV-infected individuals, indicating that the observed impairment is not a global disturbance of the NF-κB pathway. Thus, in addition to phagocytic and endocytic effector functions, the present study identifies mannose receptors as pattern recognition receptors capable of NF-κB activation in response to infectious non-self challenge. AM mannose receptor-mediated NF-κB activation may represent an important mechanism of the host cell response to Pneumocystis, and altered NF-κB activation in the context of HIV infection may impair a critical innate immune signaling response and may contribute to pathogenesis of opportunistic lung infections.

We sought to characterize the bioactive constituents of concentrated ambient air particles (CAPs) through correlation of alveolar macrophage (AM) biological responses (production of TNF, macrophage inflammatory protein (MIP-2), nitrite; cell viability) to components of particle samples. CAPs samples collected on different days showed a range of bioactivity and a strong correlation was found between AM cytokine release and increased AM light scatter, a flow cytometric measure of relative particle load. Evaluation of soluble and insoluble fractions of CAPs suspensions indicate that 1) most biological effects on AMs are mediated by insoluble components and certain particle adsorbed factors such as endotoxin; 2) the variable bioactivity of CAPs collected on different days arises primarily from differences in the relative proportion of insoluble and soluble mass present in particle suspensions; and 3) the activation state of the AM influences which insoluble components are most bioactive. Use of endotoxin neutralizing agents (e.g., polymyxin-B) showed particle-adsorbed endotoxin in CAPs suspensions causes activation of normal (control) AMs while other (nonendotoxin) components are predominantly responsible for the enhanced cytokine release observed by primed AMs incubated with CAPs. The AM biological response did not correlate with any of a panel of elements quantified within insoluble CAPs samples (Al, Cd, Cr, Cu, Fe, Mg, Mn, Ni, S, Ti, V). These data demonstrate an important role for cell activation and phagocytosis of insoluble particulate matter in the response of AMs to CAPs suspensions.

Elevated concentrations of ambient air particles can result in increased mortality and morbidity, especially in people with preexisting pulmonary disease. We postulate that in the inflammatory milieu of diseased lungs, alveolar macrophages (AMs) may be primed for enhanced responses to stimuli such as inhaled air particles. To test this hypothesis in vitro,we first cultured normal AMs with or without lipopolysaccharide (LPS). We then incubated the cells with particle suspensions (urban air particles (UAP, Washington, D.C.), residual oil fly ash (ROFA), concentrated respirable-size (PM2.5) air particles (CAPs), and inert TiO2.) and compared rat and human AM production of the critical proinflammatory mediator, tumor necrosis factor (TNF). LPS priming amplified TNF production by both rat and human AMs in response to UAP and CAPs but not inert TiO2. There were also differences observed between rat and human AM responses to particle suspensions. Striking changes seen only in rat were cytotoxic effects of ROFA and diminished particle uptake in response to LPS priming. The potency of CAPs samples (which are collected on separate days) varied when comparing one day's sample with another. When centrifuged, the majority of bioactivity seen in particle suspensions (TNF release) remained within the pelleted fraction while the supernatant showed minimal bioactivity. The data suggest that AMs activated by extant pulmonary inflammation may promote further inflammation by an enhanced cytokine response to inhaled ambient air particles.

The fluorescent probes dichlorofluorescin (DCFH), dihydrorhodamine (DHR), and hydro‐ ethidine (HE) allow convenient assay of alveolar macrophage (AM) oxidant responses to environmental particulates and pathogens. We sought to more precisely define the relationship of these measures of oxidant stress to production of pro‐inflammatory cytokines. Normal AMs were challenged in vitrowith a panel of soluble or particulate stimuli in the presence of DCFH, HE, or DHR. Flow cytometry measured cell‐associated fluorescence and relative particle uptake. Tumor necrosis factor α and macrophage inflammatory protein 2 expression were quantitated in the same experiments. We observed variable and complex correlations between intracellular oxidant production as reported by these probes and subsequent cytokine response, including examples of striking discordance (e.g., lipopolysaccharide induced large cytokine responses with minimal probe oxidation, whereas fly ash particles caused marked oxidation of DCFH but trivial TNF release; TiO2caused oxidation of DHR and HE, but not DCFH, and also did not increase cytokine production). Although fluorescent probes offer many advantages in analysis of intracellular oxidant responses, the data indicate that they cannot be used reliably as quantitative predictors of AM cytokine responses to environmental particulates or other stimuli. J. Leukoc. Biol.65: 499–507; 1999.

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR−/−) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 ± 5, 77 ± 2, and 85 ± 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of ∼70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1–reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 ± 5 and 47 ± 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected ∼70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 ∼40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.

We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-γ (IFN-γ, 25 U/ml). Using both microplate-based fluorometry and flow cytometric analysis, AMs treated withl(but notd)-arginine showed a dose- and time-dependent increase in DCFH oxidation above buffer control (e.g., DCF production (nM): control,d-arginine 100 uM,l-arginine 100 μM respectively; 91 ± 9, 76 ± 11, 396 ± 45, mean ± SE,n= 6, 120 min). Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of thel-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (μM): 2.2, 3.6, 7.1; for 5, 10, 50 μMl-arginine, respectively). In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated withl-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 μM). We conclude that DCFH is a useful probe for quantitation of NOS-2 activity in activated rat lung macrophages.

The A549 cell line was used to model in vitro the interaction of alveolar epithelium with environmental particulates. Confocal and electron microscopy demonstrated A549 binding and internalization of titanium dioxide (TiO2), iron oxide (Fe2O3), concentrated ambient air particulates (CAPs), and the fibrogenic particle α-quartz. Flow cytometry allowed quantitation of particle binding by measuring increased right angle light scatter (RAS) (TiO2 [40 μg/mL], Fe2O3 [100 μg/mL], a-quartz [200 μg/mL], or CAPs [40 μg/mL] fold increase RAS: 8.1 ± 0.9, 4.3 ± 0.4, 2 ± 0.1, 1.6 ± 0.1, respectively). With this quantitative assay, binding of particles was found to be calcium-dependent for TiO2 and Fe2O3 (% inhibition, 61.0 ± 1.9, 40.0 ± 5.6, respectively), while α-quartz binding was calcium-independent. A panel of polyanionic ligands known to inhibit scavenger-type receptors was used to identify binding mechanisms for environmental particulates. Both heparin and polyinosinic acid (polyl), but not the control polyanion chondroitin sulfate, caused marked inhibition of particulate binding by A549 cells (e. g., TiO2 [40 μg/mL] binding; polyl, heparin, and chondroitin sulfate: 73.8 ± 3.5, 75.5 ± 6.0, 7.5 ± 6.7% inhibition, respectively; mean ± SE, n ± 4), indicating that scavenger receptor(s), albeit those distinct from the heparin-insensitive acetylated-LDL receptor, mediate particulate binding. The particulates ability to stimulate interleukin (IL-8) production in A549 cells was also tested, a-quartz, but not TiO2 or CAPs, caused a dose-dependent production of IL-8 (range 1-6 ng/mL), demonstrating a particle-specific spectrum of epithelial cell cytokine (IL-8) response. The results suggest that lung epithelial cell interaction with environmental particles is mediated by distinct receptors and can lead to particle-dependent cytokine responses.