Cytokine Production by Retinal Pigmented Epithelial Cells

Cytokine Production by Retinal Pigmented Epithelial Cells Peter A. Campochiaro The Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The retinal pigmented epithelium (RPE) has many functions through which it assists the photoreceptors in visual transduction. It regulates the transport of retinoids and nutrients to the photoreceptors, phagocytizes and digests effete rod outer segments, absorbs stray light, and contributes to retinal adhesion. As the site of the outer blood-retinal barrier, it limits the access of fluid and plasma components to the outer retina. In addition to these well-recognized day-to-day maintenance functions, the RPE also plays an active role in retinal wound healing. This is demonstrated by the fact that pigmented chorioretinal scars occur after several types of insults. Histopathologic studies have confirmed the participation of RPE in such scars occurring after trauma (Cleary and Ryan, 1980), laser photocoagulation (Wallow et al., 1973), cryopexy (Lincoff et al., 1981), and subretinal neovascularization (Miller et al., 1986). After retinal detachment, the RPE proliferates and migrates into the subretinal space (Machemer and Laqua, 1975). This attempt at wound healing is usually aborted after successful retinal reattachment surgery, but in 10% of cases it is not, resulting in a condition called proliferative vitreoretinopathy (Machemer et al., 1978; Cowley et al., 1989). In this disease process, RPE and other cells migrate onto the surface of the retina and proliferate, forming epiretinal membranes that exert traction on the retina often leading to redetachment. This is the major cause of failure after retinal reattachment surgery (Cowley et al., 1989). Thus, the RPE plays a very active role in normal and pathologic retinal wound healing. A better understanding of the details of the RPE’s participation would provide important information that could have clinical implications. An interesting feature of the RPE is that it is normally mitotically inactive and undergoes cell division only when it is participating in wound healing. Therefore, it was somewhat surprising to find that adult human RPE do not readily achieve density-dependent growth arrest when culInternarional Review of Cytology, Vol. 146

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tured on plastic and that even when serum is withdrawn, they will continue to grow (Bryan and Campochiaro, 1986). These observations suggest that under certain conditions, cultured human RPE many simulate the woundhealing phenotype of RPE in situ and provide a useful tool with which to study this aspect of RPE function. These observations also suggest that RPE cells may be able to stimulate their own growth. To investigate this hypothesis, medium conditioned on RPE cells was examined for its ability to stimulate cell proliferation. These studies demonstrated that RPEconditioned media are capable of stimulating the growth of RPE and several other cell types, indicating that cultured RPE may produce one or more cytokines and secrete them into their media (Bryan and Campochiaro, 1986). The growth-promoting activity in RPE-conditioned media was found to be trypsin-sensitive and a portion of it was heat-stable (Bryan and Campochiaro, 1986). Since platelet-derived growth factor (PDGF) is a heatstable polypeptide that plays an important role in wound healing, studies were initiated to determine if PDGF was present in RPE-conditioned media. Conditioned media were exhaustively dialyzed against 0.5 N acetic acid, lyophilized, and subjected to Western blot analysis, using as primary antibody an IgG fraction prepared from antiserum directed against human PDGF. Native PDGF was resolved as a band with M , 30 kDa under nonreducing conditions, while multiple bands at 36-39 kDa and a single band at 18.5 kDa were resolved under reducing conditions from RPEconditioned media. These bands were denser in media conditioned for 48 hr than that conditioned for 24 hr, and when media were conditioned on [ 35Slmethionine-labeled RPE cells, loaded on an anti-PDGF IgG affinity column, eluted, and resolved by SDS-PAGE, radioautography showed the same bands at 36-39 kDa and 18.5 kDa. Similar bands were resolved from acid extracts of RPE cells, but no bands were detected in RPEconditioned media or cell extracts when nonimmune goat IgG fractions were substituted for the primary antibody. When conditioned media were prepared from human fibroblast cells lines and probed with the anti-PDGF IgG, no bands were detected. These data suggest that human RPE cells constitutively produce and secrete several polypeptides that are immunologically similar to platelet PDGF, but are of relative molecular masses different than the 30 kDa of platelet PDGF or A chain or B chain homodimers. These polypeptides have therefore been referred to as PDGF-like proteins (Campochiaro et al., 1989). The mitogenic and chemotactic activities in RPE-conditioned medium are not attenuated by adding anti-PDGF IgG to the medium, but they are significantly decreased when the conditioned medium is absorbed on an anti-PDGF affinity column (Campochiaro et al., 1989). When the column is eluted with acetic acid, the bound activity is recovered and can be effec-

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tively neutralized with anti-PDGF IgG. This suggests that the PDGF-like proteins demonstrated by Western blot are responsible for a significant portion of the chemotactic and mitogenic activity in RPE-conditioned media. However, it is likely that sources of activity other than PDGF-like proteins are present, because even the antibody affinity column did not remove all activity (Campochiaro et al., 1989). Several cell types have been demonstrated to produce PDGF-like proteins, including macrophages (Shimokado et al., 1985; Martinet et al., 1986; Mornex et al., 1986), smooth muscle cells (Nilsson et al., 1985; Sejersen et al., 1986), vascular endothelial cells (Di Corleto and BowenPope, 1983), and several types of tumor cells (Graves et al., 1984; Betsholtz et al., 1983). Each of these cell types express the gene for PDGF A and/or B chain, and RPE cells do also, although in very low abundance (our unpublished observations). Bradham et al. (1991) have demonstrated that the major PDGF-related molecule secreted by umbilical vein endothelial cells is a monomer of 36-38 kDa and that PDGF A and B chains account for only a small amount of biological activity in human umbilical vein endothelial cell-conditioned media. This monomer is a cystine-rich 349 amino acid peptide that is antigenically related to PDGF, but is not a product of the PDGF A or B chain genes. It is a product of a separate gene whose cDNA has a40% sequence homology with the cDNAs of both the A and B chains of PDGF. This novel peptide has been named connective tissue growth factor (CTGF), and when it is subjected to Western blot analysis using anti-PDGF IgG, it shows multiple bands between 36 and 39 kDa, in a pattern that is strikingly similar to the Western blots of the PDGF-like proteins in RPE-conditioned media. Preliminary studies indicate that the mRNA for CTGF is constituitively expressed by cultured human RPE cells (Gary Grotendorst, Ph.D., personal communication). Additional studies are needed to determine how much of the activity in RPE-conditioned media can be attributed to CTGF compared to PDGF A and B chain dimers. In addition to PDGF-like proteins, RPE cells have been demonstrated to produce other cytokines. Schweigerer et al. (1987a) demonstrated basic fibroblast growth factor (bFGF), but not acidic FGF transcripts in cultured bovine RPE and mitogenic activity in cell extracts indistinguishable from bFGF based upon its immunologic and biologic characteristics. Sternfeld et al. (1989) had similar results in cultured human RPE and also demonstrated saturable bFGF binding to RPE cells. Western blots showed an 18-kDa protein in cell lysates that cross-reacts with anti-bFGF antibody, but no cross-reactive protein could be identified in RPE-conditioned medium, even when it was run over a heparin-agarose affinity column. Since bFGF lacks a signal sequence for secretion (Abraham et al., 1986), it may not be present in conditioned media in substantial amounts; but recent

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evidence in other cell types known to produce bFGF suggests that it may get into conditioned media in very small amounts, possibly by cell injury or other mechanisms and may be involved in autocrine stimulation of growth (Schewigerer er al., 1987b; D’Amore et al., 1990). This along with in uiuo studies demonstrating that antibodies against bFGF inhibit normal (Broadley et al., 1989) and abnormal (Lindner and Reidy, 1991) wound healing, provide indirect evidence that bFGF production by RPE could play a role in normal and abnormal retinal wound healing. Another member of the FGF gene family, FGF-5, has been demonstrated to be expressed in RPE cells (Bost et al., 1992). Since FGF-5 contains a signal sequence, it is likely to be secreted and therefore may be more likely than bFGF to account for some of the mitogenic activity in RPE-conditioned media and may also be a more likely candidate for an RPE-derived paracrine stimulator of cell growth. Transforming growth factor-/3 (TGF-p) is another cytokine that has been implicated in wound repair. It has been suggested that RPE cells express TFG-p type 2 and that levels of TFG-/3 type 2 in the vitreous cavity are elevated in patients with proliferative vitreoretinopathy (Connor et al., 1989). In addition to producing factors that are likely to directly stimulate cell proliferation and extracellular matrix production in retinal wound healing, under certain circumstances, cultured RPE are also capable of secreting cytokines that recruit leukocytes and macrophages. When stimulated with the inflammatory cytokines interleukin l p or tumor necrosis factor a, RPE cells produce interleukin 8 (Elner et al., 1990), a neutrophil chemotactic factor, and monocyte chemotactic protein (Elner et al., 1991). Macrophage colony stimulating factor is constitutively produced by cultured RPE and its secretion is also markedly stimulated by interleukin 1/3 and tumor necrosis factor a (Jaffe et al., 1992). These findings provide additional evidence that the RPE may be a major participant in retinal wound healing, capable of stimulating repair somewhat independently or acting in concert with inflammatory cells to greatly magnify the wound healing response. In addition to a potential role in retinal wound healing, cytokines produced by the RPE may have a trophic influence in development and maintenance of the neural retina. This is particularly true for bFGF, which is a known neurotrophic agent in other systems (Togari et al., 1985; Walicke et al., 1986) and for which there is mounting evidence that it could play a similar role in the retina (Park and Hollenberg, 1989; Li and Turner, 1988; Factorovich et al., 1990). Surgical removal of the retina in the stage 22-24 chick embryo followed by implantation of slow-release, plastic implants containing bFGF results in retinal regeneration (Park and Hollenberg, 1989). The effect is dependent upon the dose of bFGF, and although

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the polarization of the regenerated retina is reversed, it is otherwise intact. In RCS rats with inherited retinal dystrophy, the mutant gene is in the RPE and leads to loss of photoreceptor cells. Degeneration of photoreceptors is prevented by transplantation of normal RPE into the subretinal space (Li and Turner, 1988). The effect extends beyond the boundaries of the RPE grafts, suggesting the possibility that a diffusible factor might be involved. Injection of bFGF into the subretinal space or vitreous cavity of RCS rats results in photoreceptor rescue, mimicking the effect of RPE transplantation (Faktorovich et al., 1990).This provides indirect evidence that basic FGF and/or a related molecule (e.g., FGF-5) produced by the RPE might be involved in photoreceptor maintenance. The RPE may produce other trophic agents, in addition to the FGFs, that play a role in photoreceptor development and/or maintenance. Human fetal RPE-conditioned media (RPE-CM) potentiates the neuronal differentiation of Y79 retinoblastoma cells (Tombran-Tink and Johnson, 1989). A 50-kDa protein responsible for the neuronal differentiating activity has been purified from fetal RPE-CM and has been termed pigment epithelial-derived factor (Tombran-Tink et al., 1991). The purified protein stimulates neurite outgrowth in Y79 retinoblastoma cultures in concentrates as low as 50 ng/ml, suggesting that it may play a role in retinal development. Thus, cultured RPE cells are capable of producing a number of cytokines. Information concerning cytokine production by RPE in uiuo is much less available. This is due in part to the technical difficulty of identifying and localizing cytokine production in uiuo, due to their small size, low concentrations, and homologies among various cytokines leading to potential problems with cross-reactivity. More so than most antigens, the results of immunohistochemical studies may vary depending upon methods of tissue preparation and antibodies employed. This is typified by studies investigating the immunolocalization of bFGF in the retina. In bovine and human eyes, Hanneken et al. (1989, 1991) emphasized the staining of basement membranes (primarily vascular basement membranes, but also Bruch’s membrane). In rats, Connolly et al. (1991) found bFGF staining in some neural cells, along blood vessels, and in the RPE of developing and adult retinas. Using different methods of tissue preparation and a battery of bFGF antibodies in primate retina, Hageman et al. (1991) found fixative-independent staining of the interphotoreceptor matrix and fixative-dependent staining of basement membranes, the RPE, and possibly Muller cells. Thus, it appears likely that bFGF is produced by RPE in v i m , and it could be a source (and/or target) for the bFGF that is apparently sequestered in the interphotoreceptor matrix. This provides indirect evidence for a potential trophic role for bFGF in the retina. Recently, expression of acidic FGF has been demonstrated in RPE in

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uitro (Kitaoka et al., 1992) and in situ (Baudouin et al., 1990). As noted above, vitreous aspirates from patients with PVR, a disease in which there is RPE proliferation, show elevated levels of TGF-/3 type 2 (Connor et al., 1989),but it has not been directly demonstrated that RPE produce TGF-p in uiuo. Using an antibody that cross-reacts with TGF-P types 1 but not TGF-P type 2, Lutty et al. (1991) found staining in photoreceptor outer segments and no staining in RPE in human retina. Using a panel of antibodies raised against synthetic peptides specific to TGF-PI, TGF-P2, or TGF-P3, Anderson et al. (1991) found labeling of Miiller cells and photoreceptors with all three antibodies and labeling of vascular endothelial cells only with the antibody directed against vascular endothelial cells. There was no staining for any of the three TGF-P isoforms in the RPE. More studies are needed for each of the cytokines expressed by RPE in culture to determine the circumstances in which they are produced in uiuo and the effects of inhibiting them with antibodies or other agents. This could provide new insight into photoreceptor-RPE interactions, as well as normal and abnormal retinal wound healing, and could have clinical implications for such diverse disease processes as retinal dystrophies, retinal degenerations, subretinal neovascularization, and proliferative retinopathies. Acknowledgment This study was supported by PHS Grant 05951.

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