A Role for Connexin43 in Macrophage Phagocytosis and Host Survival After Bacterial Peritoneal Infection

Our findings reveal a novel role for Connexin43 (Cx43) in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and implicate Cx43 in the regulation of the host response to microbial infection. We demonstrate that  Cx43, a gap junction protein, is expressed by macrophages, and localizes to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation.
As shown, expression of adenovirus GFP-Cx43 reverses the impairment in phagocytosis observed in Cx43- deficient macrophages. A, Macrophages were harvested from the livers of embryonic Cx43_/_, Cx43_/_, andCx43_/_ mice at gestational age embryonic days 16–18, immunostained with Abs against the macrophage marker CD45, and examined by confocal microscopy. Representative CD45 staining (iiii) and the corresponding DIC images (iv-vi) are shown. B, Embryonic macrophages were harvested from livers of Cx43_/_, Cx43_/_, and Cx43_/_ mice and allowed to internalize opsonized RBCs. The phagocytosis index is shown. Representative of five separate experiments. p _ 0.05 vs wild-type macrophages. C, Macrophages were harvested from Cx43_/_, Cx43_/_, and Cx43_/_ mice and infected with adenoviruses expressing GFP (yellow bars), GFP-wtCx43 (green bars), or GFPdnCx43 (blue bars). Cells were then allowed to undergo phagocytosis of opsonized RBCs as described in Materials and Methods. The phagocytosis index (number of macrophages with at least one internalized particle per 100 cells relative to GFP-infected macrophages from each strain) is shown. †, p _ 0.05 vs GFP-infected cells from wild-type mice. Note that in each case the rate of phagocytosis was decreased in GFP-infected cells from _/_ and _/_ mice vs _/_ mice. _, p _ 0.05 vs GFP-infected macrophage for each strain. Note that in each case infection with GFPwtCx43leads to a significant increase in phagocytosis; __, p _ 0.05 vs GFP-wtCx43. Note that for each mouse strain, infection with GFP-dnCx43 leads to a significant decrease in the rate of phagocytosis compared with infection with wild-type Cx43. Representative of at least five separate experiments with more than five mice and 100 cells per group.
Anand RJ, Dai S, Gribar SC, Richardson W, Kohler JW, Hoffman RA, Branca MF, Li J, Shi XH, Sodhi CP, Hackam DJ
2008 Dec 15
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January 22, 2011
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Last Update
January 22, 2011