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.

In vitro modeling of human alveolar macrophage smoke exposure: enhanced inflammation and impaired function.

Pulmonary macrophages (MØs) are essential for clearance of inhaled particles, innate immunity, and lung tissue maintenance. However, the products of activated MØs have also been implicated in inflammation and tissue destruction, including in chronic obstructive pulmonary disease (COPD). Primary human alveolar macrophages (AMs) are available in limited numbers via bronchoalveolar lavage (BAL) or sputum induction, and BAL macrophages are not commonly available to all researchers. A readily available, plentiful, but representative surrogate for AMs would advance understanding of the contribution of macrophages to lung pathophysiology. Herein the authors describe a method for the in vitro derivation of AM-like cells using primary human peripheral blood monocytes differentiated in suspension with granulocyte-macrophage colony-stimulating factor (GM-CSF). The method produces a cell population with a consistent and stable phenotype. Flow cytometry reveals that GM-CSF-derived macrophages (GM-MØs) express lineage markers, immunoglobulin gamma (Fc gamma) receptors, adhesion molecules, antigen presentation coreceptors, and scavenger receptors akin to AMs. Functionally, cigarette smoke activates extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, enhances interleukin 8 (IL8) production from GM-MØs and inhibits phagocytosis, phenotypes previously described for smokers' AMs. Global transcriptional profiling revealed significant overlap in regulated genes between smokers' AMs and GM-MØs treated with cigarette smoke preparations in vitro.

Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides.

The macrophage Class A scavenger receptor MARCO (macrophage receptor with a collagenous structure) functions as a pattern-recognition receptor for bacterial components, but its role in responses to CpG oligonucleotide sequences (CpG-ODN) in microbial DNA has not been characterized. Phosphorothioate (PS)-linked CpG-ODN stimulated IL-12 and NO production in wild-type but not in MARCO-deficient, thioglycollate-elicited peritoneal macrophages. MARCO and the related class A receptor SR-A belong to a redundant system of receptors for PS ODNs. The ability of MARCO to bind CpG-ODNs and conversely, to costimulate IL-12 and NO production upon specific ligation with immobilized mAb is consistent with MARCO being a signaling receptor for CpG-ODNs, costimulating TLR9-mediated NO and IL-12 production in macrophages. In contrast to MARCO, SR-A is likely to mediate negative regulation of macrophage responses to CpG-ODNs. In particular, increased affinity toward SR-A may contribute to decreased potency of oligo G-modified CpG-ODNs in stimulating IL-12 production. The results suggest that differential involvement of activating and inhibitory membrane receptors, such as SR-A and MARCO, may underlie profound differences observed in biological activities of different ODN sequences.

Cross-linking of FcgammaR triggers shedding of the hemoglobin-haptoglobin scavenger receptor CD163.

CD163, the hemoglobin (Hb)-haptoglobin scavenger receptor, is a monocyte/macrophage-restricted member of the scavenger receptor, cysteine-rich family of proteins. In addition to being expressed on the cell surface, a soluble form of CD163 has also been reported. Like tumor necrosis factor alpha (TNF-alpha), surface CD163 is proteolytically cleaved from the plasma membrane in response to lipopolysaccharide (LPS) stimulation. As cross-linking of the Fcgamma receptor (FcgammaR) is similarly known to induce TNF-alpha shedding, the effect of FcgammaR stimulation on CD163 shedding was investigated. We found that FcgammaR stimulation resulted in a rapid release of surface CD163 into the supernatant that was blocked by inhibitors of protein kinase C and tyrosine kinases. Although LPS and FcgammaR stimulation in short-term cultures suppressed CD163 mRNA expression, long-term cultures of monocytes treated with LPS-but not with a FcgammaR cross-linking reagent-resulted in an interleukin-10-dependent recovery of surface CD163 expression. These studies suggest that the presence of immune complexes in infection or autoimmunity may radically alter the nature of CD163-dependent monocyte/macrophage processes. This may be particularly important in disease states in which immune complexes and high levels of free Hb are present, such as in autoimmune hemolytic anemia, transfusion reactions, or infections by hemolytic bacteria.

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.

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.

High-level expression of immunoreactive recombinant cat allergen (Fel d 1): Targeting to antigen-presenting cells.

BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule. OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells. METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry. RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein. CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.

CpG oligodeoxynucleotides enhance FcgammaRI-mediated cross presentation by dendritic cells.

Dendritic cells (DC) can trigger naive CD8(+) T cell responses by their capacity to cross-present exogenous antigens via the major histocompatibility complex class I pathway. The myeloid class I IgG receptor, FcgammaRI (CD64), is expressed on DC, and in vivo targeting of antigens to FcgammaRI induces strong humoral and cellular immune responses. We studied the capacity of human FcgammaRI (hFcgammaRI) to facilitate DC-mediated cross presentation and T cell activation, and assessed the effect of CpG oligodeoxynucleotides on this process. We generated hFcgammaRI expressing immature DC from hFcgammaRI transgenic and immature DC from non-transgenic mice. Antigens were targeted to Fcgamma receptors as ovalbumin immune complexes, or selectively to hFcgammaRI via ovalbumin-CD64 mAb fusion proteins. Co-incubation of immature DC with CpG ODN led to markedly increased MHC class I presentation of FcgammaR-targeted antigens. When OVA was selectively targeted to hFcgammaRI, few differences were observed between Tg and NTg DC. However, upon co-incubation with CpG ODN, hFcgammaRI-triggered cross presentation was enhanced. These results document the capacity of hFcgammaRI on DC to trigger cross presentation via MHC class I upon co-culture with CpG ODN.