Biochemical and Biophysical Research Communications
MafB enhances the phagocytic activity of RAW264.7 macrophages by promoting Fcgr3 expression
Introduction
Tissue macrophages play critical roles in defense against infectious pathogens. Indeed, a lack or loss of function of these macrophages resulted in systemic fetal infections in vivo [1]. Macrophages survey for invading microbes using pathogen recognition receptors, such as toll-like receptors and mannose receptors, and subsequently phagocytose and kill these organisms via the intracellular production of nitrogen oxide and superoxide.
Two primary macrophage ontogeny scenarios have been described [2]. In the first scenario, hematopoietic progenitor cells differentiate into blood monocytes and subsequently develop into tissue macrophages upon recruitment. Alternatively, resident macrophages can proliferate within local tissues. In the first scenario, the process of the cell maturation is of particular importance. Macrophages acquire specific functions through terminal differentiation, and several transcription factors, including PU.1, MafB, and Irf8, are essential for this process [3], [4], [5].
MafB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a member of the Maf protein family, and plays a key role in terminal monocyte/macrophage differentiation [3]. Specifically, MafB homodimers bind to MafB recognition element (MARE), resulting in transcriptional transactivation of MARE-regulated genes [6]. As reported previously, alveolar macrophages from MafB gene-targeted mice exhibited significantly decreased phagocytosis of opsonized beads [7]. Meanwhile, Tillmanns and colleagues [8] demonstrated that ectopic expression of MafB yielded enhanced phagocytic activity [8]. While these data demonstrate that MafB influences the phagocytic capacity of macrophages, the regulatory mechanism underlying this process remains unclear.
Opsonization is essential for the phagocytosis of immunoglobulin-associated pathogens. Because macrophages recognize the Fc portion of immunoglobulin through cell surface-expressed Fc receptors (FcRs), immunoglobulin-associated pathogens are easily recognized and phagocytosed by macrophages [9], [10]. In particular, FcRs for IgG (FcγRs) play an important role in the phagocytosis of opsonized pathogens by macrophages. Among this family of receptors, Fcgr3 (CD16) was reported to be expressed only in certain macrophage subtypes, and to play an important role in host defense [11], [12], [13].
To date, the effect of MafB on Fcgr3 expression has yet to be investigated. In this study, we evaluated whether MafB influences the phagocytic activity of macrophages by modulating the expression of Fcgr3 and/or other FcγRs using a macrophage cell-line that constitutively expresses a MafB-specific short hairpin (sh)-RNA (RAW264.7-MafB-shRNA).
Section snippets
Cultivation of shRNA-expressing macrophage cell lines
As described previously, the RAW264.7-MafB-shRNA cell line [14] was generated by transfection of RAW264.7 cells with a plasmid vector containing the shRNA MafB sequence using a GenePORTER2 system (Gene Therapy Systems Inc., San Diego, CA, USA) [15]. As a control, RAW264.7 cells were transfected with a vector encoding a scrambled-sequence shRNA, generating the RAW264.7-control-shRNA cell line. Cells were maintained in Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY, USA) supplemented
RAW264.7-MafB-shRNA cells exhibited significantly reduced Fc receptor mediated-phagocytosis
The phagocytic index for IgG-coated opsonized PE-labeled beads was significantly lower in RAW264.7-MafB-shRNA cells than in RAW264.7-control-shRNA cells (Fig. 1A and B). Moreover, compared to the control cell population, RAW264.7-MafB-shRNA cells exhibited significantly decreased mean numbers of phagocytosed beads per cell (Fig. 1C and D).
Analysis of Fcgr2b and Fcgr3 mRNA expression in RAW264.7-MafB-shRNA cells
While RAW264.7-MafB-shRNA cells exhibited markedly reduced mRNA expression of Fcgr3, compared with RAW264.7-control-shRNA cells (Fig. 2A), there was no
Discussion
In this study, we observed significantly reduced levels of Fc receptor-mediated phagocytosis in RAW264.7-MafB-shRNA, compared with RAW264.7-control-shRNA cells (Fig. 1). In addition, mRNA and cell surface Fcgr3 expression levels were significantly lower in RAW264.7-MafB-shRNA cells than in control cells (Fig. 2, Fig. 3). Meanwhile, the observed defect in phagocytosis could be rescued by transient expression of Fcgr3 in RAW264.7-MafB-shRNA cells (Fig. 4). While a previous study demonstrated that
Disclosure statement
ICME Form for Disclosure of Potential Conflicts of Interest are attached.
Acknowledgments
This work was supported by Grants from the Japan Society for the Promotion of Science (23390220, 26461498, 26461177, and 26461153). The authors thank Emiko Nishidate, Hiroko Sasaki, Yuki Miyano, Junko Higuchi, and Eiji Tsuchida for their invaluable technical assistance.
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