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Hjoberg, Josephine, Louis Le, Amy Imrich, Venkat Subramaniam, Sheeba I. Mathew, Joseph Vallone, Kathleen J. Haley, Francis H. Y. Green, Stephanie A. Shore, and Eric S. Silverman. Induction of early growth-response factor 1 by platelet-derived growth factor in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 286: L817-L825, 2004; 10.1152/ajplung.00190.2003.--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 he 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-l in airway remodeling and asthma remains to be established. hyperresponsiveness; remodeling; gene transcription; serum response factor

Polyclonal antibodies were generated against a segment from the amino terminus of the human parathyroid hormone receptor. The polyclonal antibodies specifically bound receptors expressed in a cell line. binding assay were conducted to determine the dynamics of receptor-ligand interactions. Results reveal that the domain of the human parathyroid hormone receptor from residue 88 to residue 97 is involved in agonist binding but not antagonist binding.

Abstract 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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The interaction of air particles and alveolar macrophages (AMs) may result in the release of proinflammatory cytokines. Normal mouse AMs were treated with concentrated air particle (CAPs) suspensions in vitro. After 5 h, cytokine release [macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor- a (TNF-a)] and phagocytosis of ambient air particles were measured. CAPs samples collected from urban air (Boston) on different days were used. The CAPs samples and their soluble and solid components caused significant MIP-2 and TNF- a production. Variability in the potency of samples collected on different days was observed. Trace endotoxin was measured in CAPs samples (EU/mg: 2.3 +/- 0.7, mean +/- SE, n = 10). A majority of biologic activity (cytokine induction) and endotoxin content was associated with the solid components. Neutralization of endotoxin by polymyxin B abrogated >80% of TNF- a induction by CAPs samples, but inhibited MIP-2 production by only 40%. The trace endotoxin present in CAPs caused much more MIP-2 production than predicted by concentration alone (28 +/- 8-fold increase, n = 9), indicating synergistic interaction with other AM-activating components of the particles. Data suggest that low levels of endotoxin may interact with air particles to activate lung macrophages. [ABSTRACT FROM AUTHOR]

Adverse health effects of urban air pollution particulates may be attributable to particle mediated oxidant stress and inflammation. Intracellular oxidant production in normal hamster alveolar macrophages (AMs) was measured upon exposure to concentrated ambient particulates (CAPs), residual oil fly ash (ROFA), and their water-soluble and particulate fractions. ROFA and CAPs caused increases in dichlorofluorescin (DCFH) oxidation, a fluorescent measure of intracellular reactive oxygen species (ROS) production, comparable to the positive control, phorbol myristate acetate (PMA). The water-soluble component of both CAPs and ROFA (CAPs, S and ROFA, S) significantly increased AM oxidant production over negative control. CAPs samples and components showed substantial day-to-day variability in their oxidant effects. Metal chelation by desferrioxamine (DF, 1 mM) caused significant inhibition of particulate-induced AM oxidant production. ROFA exposure resulted in increased macrophage inflammatory protein-2 (MIP-2) message in AMs and in increased tumor necrosis factor alpha(TNF-alpha) production by the monocyte-macrophage cell line, RAW 264.7. TNF- production was inhibitable by the antioxidant N -acetylcysteine (NAC). The data suggest that metal components adsorbed to urban air pollution particulates can significantly contribute to particulate ability to cause oxidant stress and cytokine production in AMs. [ABSTRACT FROM AUTHOR]

Interferon-gamma \((IFN-\gamma)\) 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 \(IFN-\gamma\) on F. tularensis infection. A primary screen identified ~200 replicated candidate genes. These were prioritized according to mRNA expression in \(IFN-\gamma-primed\) 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 \(IFN-\gamma\) activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis.

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.

Alveolar macrophages (AM) avidly ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs) SRA-I/II and MARCO. Studies to characterize the function of these SRAs have used AMs from SRA-I/II and MARCO knockout (KO) mice, but this approach is impeded by the relatively low yield of AMs. Hence, we sought to develop continuous cell lines from primary AMs from SRA-I/II and MARCO KO mice. We used in vitro infection with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor, we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. Their doubling time is approximately 12-16 hr grown in RPMI medium containing 10% FBS. All cell lines exhibit typical macrophage morphology upon microscopy after modified Wright's stain and express maerophage-associated cell surface antigens. Similar to freshly isolated AMs from the KO mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide, fluorescent latex beads and bacteria compared with the primary AMs from wild-type (Wt) mice. Unlike AMs from the Wt, only smaller 5-8-kDa dextran sulfate inhibited uptake of fluorescent beads. These cell lines will facilitate in vitro studies to further define SRA-I/II and MARCO function and may also be useful to identify other novel scavenger-type macrophage receptors. [ABSTRACT FROM AUTHOR]

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.