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Chang Luo, Mei Chen, Heping Xu; Complement Activation in RPE Cells by Classically Activated Macrophages. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2306.
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We have shown previously that a low level of complement activation exists at the retinal-choroidal interface, and the activation increases with age. In addition, we have also shown that, with age, activated microglia/macrophages accumulate in subretinal space. Herein, we investigate the effect of differentially activated macrophages on complement gene expression by RPE.
Bone marrow-derived macrophages (BMDM, defined as M0) were cultured from C57BL/6 mice and their phenotype was determined by flow cytometry. Classically activated macrophages (M1) were induced by IFN-γ (100ng/ml) and LPS (1µg/ml) and alternatively activated macrophages (M2) by IL-4 (20ng/ml) stimulations in BMDMs. M0, M1 and M2 macrophages were washed and then incubated with normal DMEM with serum for 24 hours before supernatants were collected. Primarily cultured mouse retinal pigment epithelial (RPE) cells were treated with the supernatants from different types of macrophage for 48hrs. Complement gene expression levels were evaluated by real-time PCR.
Activation of BMDM with INF-γ and LPS induced high levels of TNF-α and iNOS expression by M1 macrophages, and Ym1, Fizz1 expression by M2 macrophages, respectively. M1 macrophages expressed high levels of complement C2, C3, C4 and CFB and low levels of MASP1 and CFH when compared to M0 macrophages. Complement gene expression was not significantly altered in M2 macrophages. The supernatants of M1 macrophages significantly increased the expression of C1r, C1s, C2, C3, C4, CFB and CFH in RPE cells. The supernatants of M0 and M2 macrophages did not affect complement gene expression in RPE cells.
Classically activated macrophages may affect complement activation by 1) directly expressing complement components; and 2) indirectly inducing complement expression by RPE cells. These cells may play an important role in complement activation at the retinal-choroidal interface in patho-physiological conditions.
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