Overexpression of Macrophage Colony-stimulating Factor Receptor on Microglial Cells Induces an Inflammatory Response

Abstract

Microglia are important in the inflammatory response in Alzheimer's disease (AD). We showed previously that macrophage colony-stimulating factor receptor (M-CSFR), encoded by the c-fms protooncogene, is overexpressed on microglia surrounding amyloid ␤ (A␤) deposits in the APP V717F mouse model for AD. The M-CSFR is also increased on microglia after experimental brain injury and in AD. To determine the relevance of these findings, we transiently expressed M-CSFR on murine BV-2 and human SV-A3 microglial cell lines using an SV40-promoted c-fms construct. M-CSFR overexpression resulted in microglial proliferation and increased expression of inducible nitric-oxide synthase, the proinflammatory cytokines interleukin-1␣, macrophage inflammatory protein 1-␣, and interleukin-6 and of macrophage colony-stimulating factor (M-CSF) itself. Antibody neutralization of M-CSF showed that the M-C-SFR-induced proinflammatory response was dependent on M-CSF in the culture media. By using a co-culture of c-fms-transfected murine microglia and rat organotypic hippocampal slices and a species-specific real time reverse transcriptase-polymerase chain reaction assay and enzyme-linked immunosorbent assay, we showed that M-CSFR overexpression on exogenous microglia induced expression of interleukin-1␣ by the organotypic culture. These results show that increased M-CSFR expression induces microglial proliferation, cytokine expression, and a paracrine inflammatory response, suggesting that in APP V717F mice increased M-CSFR on microglia could be an important factor in A␤-induced inflammatory response.

Accelerated Phagocytosis of Amyloid-β by Mouse and Human Microglia Overexpressing the Macrophage Colony-stimulating Factor Receptor

“…Control transfections were performed in parallel with an equal volume of LipofectAMINE PLUS reagent only (transfection medium; designated by M in the figures). Transfections using the pZeoSV plasmid (Invitrogen) that contains the pTK1 backbone sequence originally used in c-fms subcloning were included as a second control, although we previously showed that pZeoSV does not induce a microglial proinflammatory response (19). Cells were transfected with the pZeoSV plasmid using the same procedure as described for pTK1.…”

“…Immunocytochemical visualization of M-CSFR was demonstrated previously (19). To quantify cells overexpressing M-CSFR, 24 h post-transfection BV-2 cells were washed with PBS buffer, reacted for 1 h with 10% normal goat serum (Zymed Laboratories Inc.), and incubated with rabbit antimouse M-CSFR antiserum (1:500) (Upstate Biotechnology Inc., Lake Placid, NY) for 16 h at 4°C.…”

“…The human SV-A3 cell line was a gift from Dr. Robert Nelson (Pfizer Central Research, Groton, CT). The SV-A3 line was an immortalized line from fetal brain microglia, and phenotypic features have been previously described (19).…”

“…We recently demonstrated that overexpression of M-CSFR by cultured microglia increases proliferation, stimulates release of pro-inflammatory and chemotactic cytokines, and induces a paracrine inflammatory response in a microglial-organotypic co-culture system (19). In the present study we sought to determine the effects of M-CSFR overexpression on A␤ phagocytosis by cultured mouse and human microglia.…”

“…The human SV-A3 cell line was a gift from Dr. Robert Nelson (Pfizer Central Research, Groton, CT). The SV-A3 line was an immortalized line from fetal brain microglia, and phenotypic features have been previously described (19).Transient transfections with the pTK1 plasmid were carried out * This work was supported by National Institutes of Health Grants MH57833, MH40041, and AG17824 and the Alzheimer's Association. The costs of publication of this article were defrayed in part by the payment of page charges.…”