The Phagocytosis of Rod Outer Segments is Inhibited by Selected Drugs in Retinal Pigment Epithelial Cell Cultures

Abstract

The effects of tamoxifen, toremifene and chloroquine on the phagocytosis of rod outer segments by retinal pigment epithelium were evaluated in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cell culture. Retinal pigment epithelial cells were exposed to different concentrations of tamoxifen (1-20 mM), toremifene (1-20 mM) and chloroquine (1-1000 mM), and challenged with FITC-labeled rod outer segments for 24 hr. The phagocytized (bound and ingested) rod outer segments were measured fluorometrically, and the effect of the drugs on the phagocytosis was determined. The cytotoxicity of the drugs was evaluated by measuring their effects on mitochondrial enzyme activities (WST-1-test). The results showed that the test compounds inhibited the phagocytosis of rod outer segments in both D407 and pig retinal pigment epithelial cells. The phagocytic activity was more sensitive to tamoxifen (EC 50 7.2 mM for D407 cells and 3.6 mM for pig retinal pigment epithelial cells) and toremifene (EC 50 6.2 mM and 3.1 mM respectively) than to chloroquine (EC 50 77.2 mM for D407 cells). The inhibition of rod outer segment phagocytosis in both cell cultures started at lower dose levels of test compounds than the cytotoxicity indicated by the WST-1-test. The experiments were carried out both in serum-free medium and serum-containing medium. Serum seemed to be a critical factor in the medium and caused difficulties in the interpretation of the results.The phagocytosis of rod outer segments by the retinal pigment epithelium is one of the most important functions of these cells and essential for retinal homeostasis (Young 1969). The mechanisms of phagocytosis are complex and incompletely understood. The stages involve the recognition and binding of rod outer segments to retinal pigment epithelial cells, ingestion of rod outer segments, and digestion by lysosomal enzymes. The phagocytosis of rod outer segments is a highly specific receptor-mediated process (Hall & Abrams 1984;Mayerson & Hall 1986). However, cultured retinal pigment epithelial cells of one species are able to phagocytize rod outer segments isolated from another species (Reid et al. 1992). Disturbances in the phagocytosis can result in the accumulation of rod outer segment debris in the subretinal space, which can cause retinal degeneration and eventual blindness (Bok & Hall 1971; Edwards & Szamier 1977). Several agents, such as lysosomal enzyme inhibitors (Kennedy et al. 1994), protein glycosylation inhibitors (Hall et al. 1988;Boyle & McLaughlin 1990) and compounds that increase intracellular cAMP, reduce the rod outer segment phagocytosis by retinal pigment epithelial cells (Edwards & Bakshian 1980;Hall et al. 1993;Kuriyama et al. 1995).Chloroquine has been shown to be oculotoxic. It accumulates in the retina and causes degeneration in photoreceptors and retinal pigment epithelium in vivo (Rosenthal et al. 1978). In vitro chloroquine reduces the viability of retinal Author for correspondence: Hanna Tähti, Medical School, Universi...

Retinal Pigment Epithelial Phagocytosis and Metabolism Differ from Those of Macrophages

“…In our study we investigated the phagocytotic and defense mechanisms of RPE in comparison to monocytes. In our RPE phagocytosis model, we experienced only a weak enhancement effect of phagocytosis by serum, but data published most recently with experiments using serum-free and serum-containing medium revealed serum to be a critical factor for the uptake of rod outer segments [11]. It is not clear which serum component may be responsible for opsonization, as we did not find the classical complement receptors CR1 (CD35), CR2 (CD21) or CR3/MAC-1 (CD11b).…”

“…The studies in most cases employed primary RPE of animal origin. These data showed that several drugs including tamoxifen, toremifene, cloroquine [11] as well as corticosteroids [12] or N(G)-nitro-L-arginine [13] provoke a reduced phagocytosis of RPE cells. The latter data suggesting a nitric oxide and cGMP second-messenger system as the main effector pathway but also activation of cyclooxygenase 2 by phagocytosis of photoreceptor outer segments seem to play a role in regulating RPE function.…”

Kinetics of Inhibition of Glutamate Uptake by Antioestrogens

“…Long exposures (24 hr) to 7.5 μM tamoxifen and toremifene have been found to hamper the viability of the cells and induce cytotoxicity (Mannerström et al 2002). Only slight differences between the effects of these two amphiphilic, structurally very similar drugs have been discernible in retinal pigment epithelial cells (Toimela et al 1998; Mannerström et al 2001). A major different feature is the hepatocarcinogenity of tamoxifen in the rat (Hard et al 1993).…”

“…It is considered to be a component of the blood‐retina barrier with tight junctions between epithelial cells (Törnquist et al 1990) and it has a role in maintaining retinal glutamate homeostasis. In previous studies, retinal pigment epithelium has been found to be a sensitive target of antioestrogenic effects (Toimela et al 1998; Pappas et al 1999; Mannerström et al 2001; Engelke et al 2002; Mäenpää et al 2002). We have recently shown that antioestrogenic drugs inhibit glutamate uptake in cultured retinal pigment epithelal cells (Mäenpää et al 2002).…”

Ocular side effects of biological agents in oncology: what should the clinician be aware of?

“…So far, it has been proven that tamoxifen crosses the blood-aqueous barrier and can be detected both in the aqueous humor and in the vitreous 64. In vitro, tamoxifen may have toxic effects on cells of the retinal pigment epithelium65 and inhibit its phagocytic activity 66. In cell culture, retinal pigment epithelial cells and photoreceptor cells undergo cell death within 18 hours after exposure to tamoxifen 67…”