Regulation of Fcgamma receptors and immunoglobulin G-mediated phagocytosis in mouse microglia

Abstract

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F(c) receptors. In mice, both FcgammaRI and FcgammaRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-gamma, IL-4, IL-13 and GM-CSF on expression of FcgammaRI and FcgammaRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-gamma induced a 4-fold increase in the mRNA level of FcgammaRI but did not induce changes in FcgammaRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcgammaRIIb mRNA, but had no effect on FcgammaRI expression. GM-CSF increased both FcgammaRI and FcgammaRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-gamma and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcgamma receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.

Figure 1

Regulation of FcγRI mRNA expression in mouse microglia. Serum starved N9 (A) and pMG (B) were treated for 24 h with 10 U/mL IFN-γ or 10 ng/mL of IL-4, IL-13 or GM-CSF and FcγRI expression was assessed by qRT-PCR. Data are mean ±SEM of n=4 from each group presented as fold increase in expression. **: P<0.01 vs. unstimulated.

Figure 2

Regulation of FcγRIIb mRNA expression in mouse microglia. Serum starved N9 (A) and pMG (B) were treated for 24 h with 10 U/mL IFN-γ or 10 ng/mL of IL-4, IL-13 or GM-CSF and FcγRIIb expression was assessed by qRT-PCR. Data are mean ±SEM of n=4 from each group presented as fold increase in expression. *: P<0.05; **: P<0.01 vs. unstimulated.

Figure 3

Regulation of IgG-induced phagocytosis by cytokines in mouse microglia. Serum-starved pMG were treated for 24 h with 10 U/mL IFN-γ or 10 ng/mL IL-4, IL-13 or GM-CSF. Cells were incubated 30 min with iso IgG or anti-S. aureus IgG opsonized S. aureus. Data are mean ±SEM of n=4 from each group presented as fold increase in phagocytosis. *: P<0.05 vs iso IgG in unstimulated group. #: P<0.05, ##: P<0.01, ###: P<0.001 vs. anti-S. aureus IgG in unstimulated group.