Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry.

BACKGROUND: Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized. METHODS: We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively. RESULTS: Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect. CONCLUSION: These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.

Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines.

BACKGROUND: Alveolar macrophages (AM) avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs) MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS-/-) mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS-/- mice. RESULTS: We used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF), we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b) and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS-/- mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO2), fluorescent latex beads and bacteria (Staphylococcus aureus) compared with the primary AMs from wild type (WT) C57BL/6 mice. CONCLUSION: Our results indicated that three contiguous murine alveolar macrophage cell lines with MS-/- (ZK1, ZK2 and ZK6) were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS-/- mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate in vitro studies to further define MARCO and SR-AI/II function, and may also be useful to identify other novel scavenger-type macrophage receptors and for additional studies of particle toxicology.

Negative regulatory role of mannose receptors on human alveolar macrophage proinflammatory cytokine release in vitro.

Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms 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)-kappaB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)-1beta, IL-6, or tumor necrosis factor (TNF)-alpha (important cytokines in the host response to Pneumocystis) and did not induce IL-1beta, IL-6, or TNF-alpha 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-kappaB-mediated IL-8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL-1beta, IL-6, and TNF-alpha release and mRNA induction were preserved in response to lipopolysaccharide or serum-opsonized Pneumocystis. The absence of IL-1beta, IL-6, and TNF-alpha release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF-alpha release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll-like receptor 4-mediated TNF-alpha 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.

Genome-wide RNAi screen in IFN-γ-treated human macrophages identifies genes mediating resistance to the intracellular pathogen Francisella tularensis.

Interferon-gamma (IFN-γ) inhibits intracellular replication of Francisella tularensis in human monocyte-derived macrophages (HMDM) and in mice, but the mechanisms of this protective effect are poorly characterized. We used genome-wide RNA interference (RNAi) screening in the human macrophage cell line THP-1 to identify genes that mediate the beneficial effects of IFN-γ on F. tularensis infection. A primary screen identified ∼200 replicated candidate genes. These were prioritized according to mRNA expression in IFN-γ-primed and F. tularensis-challenged macrophages. A panel of 20 top hits was further assessed by re-testing using individual shRNAs or siRNAs in THP-1 cells, HMDMs and primary human lung macrophages. Six of eight validated genes tested were also found to confer resistance to Listeria monocytogenes infection, suggesting a broadly shared host gene program for intracellular pathogens. The F. tularensis-validated hits included 'druggable' targets such as TNFRSF9, which encodes CD137. Treating HMDM with a blocking antibody to CD137 confirmed a beneficial role of CD137 in macrophage clearance of F. tularensis. These studies reveal a number of important mediators of IFN-γ activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis.

Heterogeneity in macrophage phagocytosis of Staphylococcus aureus strains: high-throughput scanning cytometry-based analysis.

Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.

Air pollution particles diminish bacterial clearance in the primed lungs of mice.

Epidemiological studies reveal increased incidence of lung infection when air pollution particle levels are increased. We postulate that one risk factor for bacterial pneumonia, prior viral infection, can prime the lung for greater deleterious effects of particles via the interferon-gamma (IFN-gamma) characteristic of successful host anti-viral responses. To test this postulate, we developed a mouse model in which mice were treated with gamma-interferon aerosol, followed by exposure to concentrated ambient particles (CAPs) collected from urban air. The mice were then infected with Streptococcus pneumoniae and the effect of these treatments on the lung's innate immune response was evaluated. The combination of IFN-gamma priming and CAPs exposure enhanced lung inflammation, manifest as increased polymorphonuclear granulocyte (PMN) recruitment to the lung, and elevated expression of pro-inflammatory cytokine mRNAs. Combined priming and CAPs exposure resulted in impaired pulmonary bacterial clearance, as well as increased oxidant production and diminished bacterial uptake by alveolar macrophages (AMs) and PMNs. The data suggest that priming and CAPs exposure lead to an inflamed alveolar milieu where oxidant stress causes loss of antibacterial functions in AMs and recruited PMNs. The model reported here will allow further analysis of priming and CAPs exposure on lung sensitivity to infection.

Alveolar macrophage cytokine response to air pollution particles: oxidant mechanisms.

Alveolar macrophages (AMs) primed with LPS and treated with concentrated ambient air particles (CAPs) showed enhanced release of tumor necrosis factor (TNF) and provide an in vitro model for the amplified effects of air pollution particles seen in people with preexisting lung disease. To investigate the mechanism(s) by which CAPs mediate TNF release in primed rat AMs, we first tested the effect of a panel of antioxidants. N-Acetyl-l-cysteine (20 mM), dimethyl thiourea (20 mM) and catalase (5 microM) significantly inhibited TNF release by primed AMs incubated with CAPs. Conversely, when LPS-primed AMs were treated with CAPs in the presence of exogenous oxidants (H(2)O(2) generated by glucose oxidase, 10 microM/h), TNF release and cell toxicity was significantly increased. The soluble fraction of CAPs suspensions caused most of the increased bioactivity in the presence of exogenous H(2)O(2). The metal chelator deferoxamine (DFO) strongly inhibited the interaction of the soluble fraction with H(2)O(2) but had no effect on the bioactivity of the insoluble CAPs fraction. We conclude that CAPs can mediate their effects in primed AMs by acting on oxidant-sensitive cytokine release in at least two distinct ways. In the primed cell, insoluble components of PM mediate enhanced TNF production that is H(2)O(2)-dependent (catalase-sensitive) yet independent of iron (DFO-insensitive). In the presence of exogenous H(2)O(2) released by AMs, PMNs, or other lung cells within an inflamed alveolar milieu, soluble iron released from air particles can also mediate cytokine release and cell toxicity.

Scavenger Receptors SR-AI/II and MARCO limit pulmonary dendritic cell migration and allergic airway inflammation.

The class A scavenger receptors (SR-A) MARCO and SR-AI/II are expressed on lung macrophages (MPhis) and dendritic cells (DCs) and function in innate defenses against inhaled pathogens and particles. Increased expression of SR-As in the lungs of mice in an OVA-asthma model suggested an additional role in modulating responses to an inhaled allergen. After OVA sensitization and aerosol challenge, SR-AI/II and MARCO-deficient mice exhibited greater eosinophilic airway inflammation and airway hyperresponsiveness compared with wild-type mice. A role for simple SR-A-mediated Ag clearance ("scavenging") by lung MPhis was excluded by the observation of a comparable uptake of fluorescent OVA by wild-type and SR-A-deficient lung MPhis and DCs. In contrast, airway instillation of fluorescent Ag revealed a significantly higher traffic of labeled DCs to thoracic lymph nodes in SR-A-deficient mice than in controls. The increased migration of SR-A-deficient DCs was accompanied by the enhanced proliferation in thoracic lymph nodes of adoptively transferred OVA-specific T cells after airway OVA challenge. The data identify a novel role for SR-As expressed on lung DCs in the down-regulation of specific immune responses to aeroallergens by the reduction of DC migration from the site of Ag uptake to the draining lymph nodes.

The macrophage scavenger receptor SR-AI/II and lung defense against pneumococci and particles.

The class A macrophage scavenger receptor SR-AI/II is implicated as a pattern recognition receptor for innate immunity, but its functional role in lung defense has not been studied. We used mice genetically deficient in SR-AI/II and their wild-type C57BL/6 counterparts to investigate the contribution of this receptor to defense against pneumococcal infection and inhaled particles. SR-AI/II deficiency caused impaired phagocytosis of fluorescent bacteria in vivo, diminished clearance of live bacteria from the lungs, and substantially increased pneumonic inflammation. Survival studies also showed increased mortality in SR-AI/II-deficient mice with pneumococcal lung infection. Similarly, after challenge of the airways with TiO(2) particles, SR-AI/II-deficient mice showed increased proinflammatory cytokine levels in lung lavage fluid and a more pronounced neutrophilic inflammation. The data indicate that the lung macrophage class A scavenger receptor SR-AI/II contributes to innate defense against bacteria and inhaled particles.

Effect of leptin on allergic airway responses in mice.

BACKGROUND: Epidemiologic data indicate that the incidence of asthma is increased in obese patients. OBJECTIVE: Because the serum levels of the satiety hormone and proinflammatory cytokine leptin are increased in obese individuals, we sought to determine whether leptin can augment allergic airway responses. METHODS: We sensitized and challenged BALB/cJ mice with ovalbumin. Alzet micro-osmotic pumps were implanted in the mice to deliver a continuous infusion of either saline or leptin (1.75 mug/g/d). Two days later, the mice were challenged with either aerosolized saline or ovalbumin once per day for 3 days. We measured airway responsiveness, performed bronchoalveolar lavage, and obtained blood to measure serum leptin and IgE 24 or 48 hours after the last challenge. RESULTS: Leptin infusion increased serum leptin concentrations, which were increased further after ovalbumin sensitization and challenge. Ovalbumin challenge increased bronchoalveolar lavage fluid cells and cytokines, serum IgE, lung cytokine mRNA expression, and responses to inhaled, aerosolized methacholine. It is important to note that the changes in methacholine responsiveness and IgE were augmented in leptin- versus saline-infused mice. CONCLUSIONS: These results indicate that serum leptin is increased during allergic reactions in the airways and may play a role in the relationship between obesity and asthma.

Oncostatin M causes eotaxin-1 release from airway smooth muscle: synergy with IL-4 and IL-13.

BACKGROUND: Eotaxin is implicated in asthmatic eosinophilia. Oncostatin M (OSM) causes eotaxin release from fibroblasts. OBJECTIVE: We sought to examine the effects and mechanism of action of OSM and other IL-6 family cytokines on eotaxin release from human airway smooth muscle cells. METHODS: Eotaxin 1 release was measured by means of ELISA. Western blotting was used to examine mitogen-activated protein kinase and signal transducer and activator of transcription 3 (STAT-3) phosphorylation. Eotaxin promoter activity was analyzed in cells transfected with wild-type STAT-3, a mutant form of STAT-3 that cannot be phosphorylated, and a constitutively active form of STAT-3. The mRNA and protein expression of IL-4R alpha, the signaling receptor for IL-4 and IL-13, was evaluated by means of real-time PCR and flow cytometry, respectively. RESULTS: OSM increased eotaxin 1 release and augmented IL-4- or IL-13-induced eotaxin release, whereas other IL-6 family cytokines did not. OSM caused a greater increase in STAT-3 phosphorylation and STAT-3-mediated gene transcription than other IL-6 family cytokines. OSM increased eotaxin promoter activity and augmented IL-13- and IL-4-induced increases in promoter activity. The constitutively active form of STAT-3 increased eotaxin promoter activity, whereas the mutant form of STAT-3 that cannot be phosphorylated significantly reduced eotaxin promoter activity induced by OSM or IL-4 plus OSM. OSM increased IL-4R alpha mRNA and protein levels. CONCLUSIONS: OSM induces eotaxin 1 expression in human airway smooth muscle cells by a mechanism involving STAT-3. OSM synergizes with IL-13 and IL-4 to increase eotaxin 1 expression, possibly as a result of effects on IL-4R alpha expression.

MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages.

Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.

Pneumocystis activates human alveolar macrophage NF-kappaB signaling through mannose receptors.

Alveolar 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 Pneumocystis organisms stimulate AM NF-kappaB 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-kappaB nuclear translocation is associated with I-kappaB 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-kappaB 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-kappaB nuclear translocation and is associated with reduced I-kappaB phosphorylation and reduced interleukin-8 (IL-8) release. In contrast, NF-kappaB 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-kappaB pathway. Thus, in addition to phagocytic and endocytic effector functions, the present study identifies mannose receptors as pattern recognition receptors capable of NF-kappaB activation in response to infectious non-self challenge. AM mannose receptor-mediated NF-kappaB activation may represent an important mechanism of the host cell response to Pneumocystis, and altered NF-kappaB activation in the context of HIV infection may impair a critical innate immune signaling response and may contribute to pathogenesis of opportunistic lung infections.

Particle-epithelial interaction: effect of priming and bystander neutrophils on interleukin-8 release.

Exposure to ambient air pollution particles causes greater health effects in individuals with preexisting inflammatory lung diseases. To model inflammatory priming in vitro, HTB54 lung epithelial cells were pretreated with tumor necrosis factor-alpha (TNF-alpha) and then exposed to a panel of environmental particles, including concentrated ambient particles (CAPs). TNF-alpha priming significantly enhanced interleukin (IL)-8 secretion in response to CAPs and other urban air particles in HTB54 cells. Enhancement was seen with whole CAP suspensions as well as with its separate water-soluble and -insoluble components. Treating CAP suspensions with 20 microM deferoxamine or 2 mM dimethylthiourea attenuated the enhancement, indicating that transition metals and oxidative stress participate in the CAPs-dependent IL-8 response of primed cells. Because activated neutrophils are also present in diseased lungs and are sources of additional oxidative stress on epithelial cells, primed HTB54 cells were cocultured with activated neutrophils. Wild-type neutrophils markedly enhanced IL-8 release to CAPs in primed HTB54 cells, an effect substantially diminished when neutrophils from NADPH knockout mice were used. Cytokine priming and interactions with activated neutrophils can amplify lung epithelial inflammatory responses to ambient air particles.

Combined air pollution particle and ozone exposure increases airway responsiveness in mice.

We investigated whether coexposure to inhaled ambient particles and ozone affects airway responsiveness (AR, measured as enhanced pause, Penh) and allergic inflammation (AI) in a murine model of asthma. Ovalbumin-sensitized mice were challenged with either ovalbumin ("asthmatic") or phosphate-buffered saline (PBS) aerosols for 3 successive days. Immediately after daily challenge, mice were exposed for 5 h to concentrated ambient particles (CAPs), or 0.3 ppm ozone, or both, or neither (n > or = 61/group, 12 experiments). Exposure to CAPs alone or coexposure to CAPs + O(3) caused an increase in Penh in both normal and "asthmatic" mice. These responses were transient and small, increasing approximately 0.9% per 100-microg/m(3) increase in CAPs. Analysis of the effects of particle composition on AR revealed an association between the AlSi particle fraction and increased AR in "asthmatic" mice exposed to ozone and particles. No effects of pollutants on AI were noted. We conclude that (1) particle exposure causes an immediate, short-lived (<24 h) increase in AR in mice; (2) these responses are small; and (3) changes in AR may be correlated with specific elements within the particle mixture.

Induction of early growth-response factor 1 by platelet-derived growth factor in human airway smooth muscle.

Platelet-derived growth factors (PDGF) may contribute to the activation and growth of smooth muscle that is characteristic of airway remodeling in asthmatic patients. Early growth response 1 (EGR-1) is a transcription factor that is induced in several cell types by PDGF and may mediate some of the effects of PDGF. We show that human airway smooth muscle cells in cell culture express EGR-1 1 h after addition of PDGF. Analysis of the EGR-1 promoter indicates that a serum response element located between 663 and 654 bp 5' to the ATG start site is essential for this induction. Serum response factor, E26 transcription factor-like protein 1, and serum protein 1 bind to this region. PDGF causes phosphorylation of ERK1/2 and is temporally associated with E26 transcription factor-like protein 1 phosphorylation. Finally, the specific ERK1/2 inhibitor U-0126 abolishes PDGF-induced expression of EGR-1 in these cells. On the basis of these data, we speculated that EGR-1 would be increased in airway smooth muscle of asthmatic patients compared with nonasthmatic controls. Using immunohistochemistry, we found that EGR-1 protein was expressed in airway smooth muscle cells and epithelial cells of asthmatic patients and nonasthmatic controls; however, there was no significant difference in the intensity of staining between groups. EGR-1 was similarly expressed in the lungs of mice with and without ovalbumin-induced airway inflammation; however, there was no difference between groups by immunohistochemistry and quantitative PCR. Although EGR-1 is induced by PDGF in human airway smooth muscle cells in cell culture, the role of EGR-1 in airway remodeling and asthma remains to be established.