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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>We used <it>in vitro </it>infection of the primary AMs with the J2 retrovirus carrying the <it>v-raf </it>and <it>v-myc </it>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 (<it>Staphylococcus aureus</it>) compared with the primary AMs from wild type (WT) C57BL/6 mice.</p> <p>Conclusion</p> <p>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 <it>in vitro </it>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.</p

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

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

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

Fc-receptor on ZK1 cells mediated phagocytosis of opsonized sheep red blood cells (SRBC)

ZK1 cells were plated at 1 × 10cells/well in a 6-well plate containing a sterile micro cover glass per well in RPMI complete medium for overnight at 37°C. Unopsonized (as negative control) or preopsonized SRBC were plated on monolayer of ZK1 cells at a ratio of 20:1 and incubated at 37°C for 1 h. After removal of free SRBC by medium exchange and lysis by osmotic shock, the cells on the cover glass were fixed and stained with a modified Wright stain, subsequently examined by light microscopy. Panel , ZK1 cells were unable to ingest unopsonized SRBC after lysis. Some free SRBC were present without lysis as background. Panel , approximately 80% of ZK1 cells were positive phagocytosis of opsonized SRBC.<p><b>Copyright information:</b></p><p>Taken from "Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines"</p><p>http://www.particleandfibretoxicology.com/content/5/1/7</p><p>Particle and Fibre Toxicology 2008;5():7-7.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2427050.</p><p></p

Growth rate of the continuous murine alveolar macrophage single cell clones ZK1 (◆), ZK2 (■) and ZK6 (▲)

Cells were seeded in six-well plate at 2 × 10cells/ml and incubated at 37°C in a 5% CO-humidified atmosphere in RPMI/10% FBS complete medium. Three wells per clone were harvested with cold PBS at 14 h and 18 h post seeding, and cells from each well were counted by a hemocytometer with trypan blue exclusion of dead cells. The obtained average cell count for each clone at each time point was plotted against the time. Doubling time (mean generation time = mgt) was calculated according the formula: N = N2. N is the number of cells at any time T; N, is the number of cells at an initial point. The doubling time of ZK cell lines is approximately 14 hours.<p><b>Copyright information:</b></p><p>Taken from "Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines"</p><p>http://www.particleandfibretoxicology.com/content/5/1/7</p><p>Particle and Fibre Toxicology 2008;5():7-7.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2427050.</p><p></p

Modified Wright staining of ZK1 and ZK2 cell lines compared with primary alveolar macrophages

Primary AMs were isolated from wild type (WT) C57BL/6 mice and from MARCOand SR-AI/II(MS) mice. ZK1 and ZK2 cells were identified as macrophages by their large, dark nuclei and abundant pale, granular cytoplasm containing numerous vacuoles.<p><b>Copyright information:</b></p><p>Taken from "Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines"</p><p>http://www.particleandfibretoxicology.com/content/5/1/7</p><p>Particle and Fibre Toxicology 2008;5():7-7.</p><p>Published online 2 May 2008</p><p>PMCID:PMC2427050.</p><p></p