Collagen phagocytosis by human retinal Müller cells in culture

Abstract

Abstract: : Purpose: Recently, packages of vitreous collagen (type II) and interruptions in the internal limiting lamina (ILL) were found in the basal and equatorial areas of human retinas; in most cases Müller cells were closely related. These findings are suggestive of a process of interactive remodeling, in which both phagocytosis and synthesis of vitreous and ILL collagens may occur. Here, we study the possible role of Müller cells in phagocytosis of collagen type II (vitreous) and collagen type IV (ILL). Methods: Spontaneously immortalized human Müller cells in culture (MIO–M1; GA Limb, UK) were exposed to 2.0 µm fluorescent beads coated with BSA and human collagen types I, II, and IV and to non–coated beads for 2, 12, 24, and 48 hours. Cells were harvested after the incubation periods and evaluated by flow cytometry. To verify phagocytosis, cells exposed to beads were also embedded in EPON and evaluated by TEM. We checked main characteristics of Müller cells with several antibodies. Statistical analysis was done by ANOVA and Student’s t–test. Results: The results (n=3) showed that Müller cells gave preference to phagocytosis of beads coated with collagen type II compared with collagen type I, which in its turn was better phagocytized than collagen type IV, BSA and non–coated beads. Immunohistochemical analysis revealed that Müller cells were positive, under all tested circumstances, for vimentin, CRALBP, and only for about 5% GFAP. TEM evaluation confirmed internalized beads. Conclusions: Our observations demonstrate that Müller cells in vitro prefer to phagocytize fibrillar collagen types I and II. Moreover, collagen type II seems to be specifically phagocytized compared with type I. In contrast, the phagocytosis of collagen type IV is comparable to the control coatings. The first results may support our hypothesis that a process of vitreoretinal remodeling occurs in adult human eyes.

Keywords: Muller cells • extracellular matrix