Differential Chemokine Regulation by Th2 Cytokines during Human RPE–Monocyte Coculture

Abstract

purpose. To determine the effects of the potent anti-inflammatory Th2 cytokines, interleukin (IL)-4, -10, and -13, on IL-8 and monocyte chemoattractant protein (MCP) 1 production by human retinal pigment epithelial (HRPE) cells, monocytes, and HRPE cell–monocyte cocultures.

methods. Enzyme-linked immunosorbent assays were performed to determine IL-8 and MCP-1 secretion by HRPE cells, monocytes, and HRPE cell–monocyte cocultures stimulated with IL-1β or TNF-α, either alone, or in combination with IL-4, -10, or -13, at various time points.

results. IL-4 and -13, but not IL-10, enhanced constitutive and TNF-α–induced HRPE IL-8 and MCP-1 secretion. IL-4 also enhanced IL-1β–induced HRPE IL-8. IL-4 and -13 reduced monocyte IL-8 and MCP-1, whereas IL-10 reduced monocyte IL-8 but enhanced MCP-1. Overlay of monocytes onto HRPE cell cultures resulted in increased IL-8 and MCP-1 secretion. IL-8 secretion by HRPE cell–monocyte cocultures was inhibited by IL-4, -10, and -13, whereas MCP-1 was inhibited only by IL-10. These cytokines also inhibited IL-1β potentiation of IL-8, but not MCP-1 secretion by cocultures. IL-4 enhanced TNF-α potentiation of chemokine secretion by cocultures, whereas IL-10 had no effects. IL-13 potentiated TNF-α–induced MCP-1, but not IL-8 secretion by cocultures.

conclusions. IL-4, -10 and -13 have complex effects on chemokine secretion by HRPE cells, monocytes, and HRPE cell–monocyte cocultures. IL-10 appears to be the most consistently suppressive cytokine, suggesting potential therapeutic usefulness of IL-10 in the treatment of ocular inflammatory and proliferative diseases.

Monocyte infiltration is a prominent feature of proliferative vitreoretinopathy (PVR), age-related macular degeneration (ARMD), and overtly inflammatory ocular diseases including uveitis. Infiltrating monocytes are intimately associated with human retinal pigment epithelial (HRPE) cells in the epiretinal membranes of PVR, in the subretinal neovascular membranes of ARMD, and in the retina of patients with uveitis and experimental uveitis. ^{1 2 3 4 5 6} There is increasing evidence that both monocytes and HRPE cells have critical regulatory functions in development of these diseases and that monocytes and monocyte-derived cytokines could modulate HRPE functions with respect to cell proliferation and inflammatory response. ^{7 8 9 10} Therefore, it is likely that interactions between HRPE cells and monocytes are involved in the evolution of these diseases. 

We previously demonstrated that HRPE cells produce chemokines that induce leukocyte chemotaxis. ¹¹ Interleukin (IL) 8 and monocyte chemoattractant protein (MCP) 1 are the two chemokines responsible for the majority of pro-inflammatory cytokine–induced, HRPE-derived leukocyte chemotactic activity. ^{7 8 11} IL-8 is primarily chemotactic for neutrophils ¹² and eosinophils ¹³ , whereas MCP-1 attracts and stimulates monocytes ¹⁴ and lymphocytes. ¹⁵ We and others reported that these chemokines are increased in eyes from patients with PVR, uveitis, and diabetic retinopathy. ^{16 17 18} IL-8 and MCP-1, thus, may be implicated in these diseases by inducing leukocyte accumulation and activation leading to the establishment and progression of retinal diseases. We previously reported that direct interactions between monocytes and vascular endothelial cells resulted in the increased expression of IL-8 and MCP-1. ¹⁹ However, the consequences of interactions between RPE cells and monocytes are poorly understood. A better understanding of how such interactions effect chemokine production may help to improve methods for treatment of the inflammatory component of ocular diseases. 

Th2 cytokines such as IL-4, -10, and -13 have been demonstrated to be capable of downregulating the pro-inflammatory cytokine release of activated monocytes in various diseases. ^{20 21 22 23} In contrast, we previously reported that IL-4 induced HRPE IL-8 and MCP-1 secretion ²⁴ and that IL-10 had no significant effects on IL1-β and tumor-necrosis factor (TNF) α–induced IL-8 and MCP-1 secretion. ²⁵ It has been demonstrated that IL-10 and -13 are present in subretinal fluid from eyes with retinal detachment and that IL-4 and -10 are present in experimental autoimmune uveitis. ^{26 27} These cytokines, thus, may have an immunoregulatory role in clinical and experimental ocular inflammation. Because they have been considered to be potential anti-inflammatory therapeutic agents for in vivo use, it is prudent to evaluate their effects on the cell types that are involved in ocular inflammatory processes. However, there is little information about the effects of these cytokines on ocular cells. 

In this study, we examined the effects of the interaction between HRPE cells and monocytes on IL-8 and MCP-1 production as well as the regulatory effects of IL-4, -10, and -13 on IL-8 and MCP-1 production by HRPE cells and HRPE cell–monocyte cocultures. In addition, we tested how these immunosuppressive cytokines would modulate chemokine production in the presence of ambient pro-inflammatory cytokines, IL-1β and TNF-α, which are known to be present in retinal diseases. ^{28 29 30}  

Methods

HRPE Cell Culture

HRPE cells were isolated from donor eyes within 24 hours of death as previously described, in accordance with the Helsinki agreement. ⁷ In brief, the sensory retina was separated from the HRPE monolayer, and the HRPE cells were removed from Bruch’s membrane. Isolated HRPE cells were incubated in Dulbecco’s modified essential medium (DMEM) containing 15% fetal bovine serum, penicillin G (100 U/ml), streptomycin sulfate (100 μg/ml), and amphotericin B (0.25 μg/ml; Sigma, St. Louis, MO). The HRPE monolayers exhibited uniform immunohistochemical staining for fibronectin, laminin, and type IV collagen in a chicken wire distribution, characteristic for these epithelial cells. Cells, grown in culture up to six passages, were subcultured into 12-well plates, grown confluency, and used for experiments. 

Monocyte Culture

Human monocytes were isolated as previously described with modification, ^{19 31} in accordance with the Helsinki agreement. Peripheral blood was drawn into a heparinized syringe from healthy volunteers, diluted 1:1 in normal saline and mononuclear cells separated by density gradient centrifugation. The cells were washed and then layered onto density gradient (1.068 g/ml) for the enrichment of monocytes (Fico-Lite Monocytes; Atlanta Biologics, Atlanta, GA). The isolated cells were then washed, cytospun onto a glass slide, stained with Diff-Quick (Baxter, McGaw, IL), and differentially counted. The purity of the monocytes from the gradient was consistently 95%. ³¹  

Cell Cytokine Stimulation

Before experiments, HRPE cells in 12-well plates were incubated in serum-free medium for 12 hours. HRPE cells and monocytes were incubated in control medium, DMEM, or in the same medium also containing recombinant human IL-1β (rhIL-1β, 0.2 and 0.02 ng/ml; R&D Systems, Minneapolis, MN) or rhTNF-α (2 ng/ml; R&D Systems), either alone or in combination with rhIL-4 (100 ng/ml; R&D Systems), rhIL-10 (100 U/ml; R&D Systems), or rhIL-13 (100 ng/ml; R&D Systems) for 24 hours. We used these doses of Th2 cytokines because these were most effective in our previous studies ^{24 25} and preliminary experiments. To show that the observed effects were due to the Th2 cytokines used, monoclonal anti-human IL-4 (100 μg/ml), IL-10 (100 ng/ml), or IL-13 (30 μg/ml) antibody (anti-IL-4, -10, or -13 mAb; R&D Systems) was added to the cultures. HRPE cell cultures receiving IL-1β or TNF-α together with IL-4, -10, or -13 were either (1) preincubated for 24 hours with IL-4, -10, or -13 before the introduction of IL-1β or TNF-α in the presence of IL-4, -10, or -13; (2) preincubated with IL-4, -10, or -13 and then incubated with IL-1β or TNF-α only; or (3) simultaneously coincubated with IL-4, -10, or -13 and either IL-1β or TNF-α. For RPE cell–monocyte cocultures, enriched monocyte populations (4 × 10⁵/well) were layered onto RPE monolayers (2 × 10⁵/well) at the time of the application of IL-1β or TNF-α. To detect whether cell contact was obligatory for chemokine production, HRPE cells and monocytes were coincubated in the same cultures but separated by porous polycarbonate filters. To detect the source of the chemokines, monocytes were separated from HRPE cells after 4 hours of coincubation using cold Ca²⁺, Mg²⁺-free PBS as previously described. ¹⁹ After experimental incubations, culture media were collected, centrifuged to remove particulates, and stored at −70°C until enzyme-linked immunosorbent assay (ELISA) was performed. Cytokines were negative for endotoxin contamination as determined by the limulus amoebocyte lysate assay method (<0.05 EU/ml, BioWhittaker, Walkersville, MD). 

Enzyme-Linked Immunosorbent Assay

ELISA was performed on serial dilutions of HRPE, monocyte, and HRPE–monocyte coculture supernatants. Antigenic IL-8 and MCP-1 were quantitated using a double-ligand ELISA method as described previously. ³² Standards included 0.5 log dilutions of rIL-8 (R&D Systems) or rMCP-1 (R&D Systems) from 5 pg to 100 ng/well. 

Semiquantitative RT-PCR

Synthetic oligonucleotide primers based on the cDNA sequences of human IL-8, MCP-1, and β-actin were prepared: IL-8, 5′-AAGCTGGCCGTGGCTCTCTTG-3′ and 5′-AGCCCTCTTCAAAAACTTCTC-3′; MCP-1, 5′-GCTCATAGCAGCCACCTTCATTC-3′ and 5′-GTCTTCGGAGTTTGGGTTTGC-3′; and β-actin, 5′-GTGGGGCGCCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTC-3′. RT-PCR was carried out in a semiquantitative manner, essentially as previously described. ¹⁸ Linearity range of the reaction was determined running 15 to 35 cycles. DNA was denatured for 5 minutes at 94°C, followed by 28, 26, and 20 PCR cycles for IL-8, MCP-1, andβ -actin, respectively. Each cycle included a 1-minute denaturation at 94°C, a 1-minute primer annealing at 65°C, and a 2-minute polymerization at 72°C. Each RT-PCR reaction mixture was analyzed by electrophoresis on a 2% agarose gel and stained with ethidium bromide. The intensity of the ethidium bromide luminescence was measured by an image sensor with a computer-controlled display. 

Statistical Analysis

Individual experiments were performed in triplicate three times on three different HRPE cell lines and monocytes isolated from the blood of three different donors on separate days. Each cell line displayed similar fold-increases or decreases compared with control levels. The representative data in figures are from one of three independent experiments. Data are expressed as means ± SEM. Various assay conditions were evaluated using ANOVA test with a post hoc analysis (Scheff é multiple comparison test); P < 0.05 was considered to be statistically significant. 

Results

IL-8 and MCP-1 Secretion by HRPE Cells

Unstimulated HRPE cell cultures consistently demonstrated basal MCP-1 production, whereas no detectable IL-8 levels were observed (Fig. 1) . IL-13 as well as IL-4 induced IL-8 and MCP-1 production, whereas IL-10 did not enhance IL-8 or MCP-1 secretion (Fig. 1 , Table 1 ). Anti–IL-4 and -13 mAb inhibited chemokine induction by IL-4 and -13, respectively. 

Incubation with IL-1β (0.2 ng/ml) produced significant increases in HRPE cell IL-8 and MCP-1 secretion as previously described (Figs. 2A 2B) . ^{7 8} Because we previously found that the effect of IL-10 on HRPE HLA-DR expression varied depending on the timing of IL-10 exposure with respect to pro-inflammatory cytokine stimulation, ²⁵ we exposed HRPE cells to IL-4, -10, and -13 before and/or during HRPE cell stimulation. When added with IL-1β simultaneously, IL-4 potentiated IL-1β–induced IL-8 secretion but had no significant effects on IL-1β–induced MCP-1 secretion. When maintained with IL-1β or removed after preincubation, IL-4 had no effects on IL-1β–induced IL-8 and MCP-1. IL-10 and -13 did not modulate IL-8 and MCP-1 secretion induced by IL-1β under any of the experimental conditions (Figs. 2A 2B) . The effects of Th2 cytokines on low-dose (0.02 ng/ml) IL-1β–induced chemokine secretion were similar to those on 0.2 ng/ml IL-1β–induced chemokine secretion (data not shown). However, when added with IL-1β simultaneously, IL-13 significantly potentiated IL-1β–induced IL-8 secretion by twofold. When maintained with IL-1β after preincubation, IL-4 potentiated IL-1β–induced MCP-1 significantly by 1.5-fold (data not shown). 

TNF-α (2 ng/ml) also enhanced both IL-8 and MCP-1 production (Figs. 2C 2D) . IL-4, but not IL-10, potentiated IL-8 and MCP-1 secretion induced by TNF-α under all conditions (Figs. 2C 2D) . When maintained or removed after preincubation, IL-13 potentiated TNF-α–induced IL-8 and MCP-1 secretion. When added with TNF-α simultaneously, IL-13 potentiated TNF-α–induced IL-8, but not MCP-1. 

IL-8 and MCP-1 Secretion by Monocytes

Unstimulated monocytes consistently demonstrated basal IL-8 and MCP-1 production (Fig. 3A 3B) . IL-1β and TNF-α increased monocyte IL-8 and MCP-1 (Fig. 3C 3D) . IL-4, -10, and -13 all reduced constitutive as well as IL-1β (0.2 ng/ml)- and TNF-α (2 ng/ml)–induced IL-8 secretion as previously described (Figs. 3A 3C) . ^{33 34} IL-4 and -13 reduced basal as well as IL-1β– and TNF-α–induced MCP-1 secretion, whereas IL-10 potentiated MCP-1 (Figs. 3B 3D) . Anti–IL-4, -10, and -13 mAb blocked the effects of IL-4, -10, and -13 on chemokine production, respectively (Figs. 3A 3B) . 

IL-8 and MCP-1 Production by HRPE Cell: Monocyte Cocultures

The direct overlay of monocytes onto HRPE cell cultures consistently resulted in increased IL-8 and MCP-1 production, whereas coincubation of HRPE cells and monocytes in the same cultures, but separated by porous polycarbonate filters, did not induce the secretion of these chemokines significantly. (Figs. 4A 4B) . When monocytes were incubated with HRPE cells for 4 hours and separated for an additional 24 hours, 98% of MCP-1 and 72% of IL-8 production was derived from HRPE cells (Table 2) . 

We studied the effects of IL-4, -10, and -13 on chemokine secretion due to HRPE cell–monocyte interaction. Because HRPE cells produce majority of IL-8 and MCP-1 production by HRPE–monocyte cocultures (Table 2) and the effect of Th2 cytokines on HRPE chemokine production is differently regulated by the timing of their exposure (Fig. 2) , we exposed HRPE cells to IL-4, -10, and -13 before and/or during the HRPE cell–monocyte coculture assays. Coincubating with IL-4, -10, or -13, with or without preincubation, reduced IL-8 secretion by HRPE cell–monocyte cocultures (Fig. 4C) . When removed after preincubation, only IL-4 reduced IL-8 secretion. IL-4 and -13 had no effects on MCP-1 by cocultures under all conditions (Fig. 4D) . Coincubation with IL-10, with or without preincubation, reduced MCP-1 secretion. When removed after preincubation, IL-10 had no significant effects on MCP-1 secretion. Anti–IL-4, -10, and -13 mAb blocked the inhibitory effects of the Th2 cytokines on chemokine secretion. 

When added to HRPE cell–monocyte cocultures, IL-1β (0.2 ng/ml) enhanced both IL-8 and MCP-1 production (Figs. 5A 5B) . When HRPE cell–monocyte cocultures were exposed to IL-1β and IL-4, -10, or -13 after preincubation of HRPE cells with the Th2 cytokines, IL-4 and -10, but not IL-13, reduced IL-1β–induced IL-8 secretion (Fig. 5A) . When removed after preincubation, only IL-4 reduced IL-8. When IL-1β, the Th2 cytokines, and monocytes were added to HRPE cells simultaneously, IL-10 and -13 reduced IL-8 secretion by cocultures. IL-4, -10, and -13 did not modulate MCP-1 secretion induced by IL-1β under any of the experimental conditions (Fig. 5B) . The effects of Th2 cytokines on low-dose (0.02 ng/ml) IL-1β–induced chemokine secretion resembled those on 0.2 ng/ml IL-1β–induced chemokine secretion (data not shown). However, when IL-1β and -4 were added simultaneously, IL-4 significantly reduced IL-1β–induced IL-8 secretion by HRPE cell–monocyte cocultures by 44%. Coincubating with IL-1β and -13 with preincubation of IL-13 also significantly reduced IL-1β–induced IL-8 secretion by 40% (data not shown). 

When added to HRPE cell–monocyte cocultures, TNF-α (2 ng/ml) also enhanced both MCP-1 and IL-8 production (Figs. 5C 5D) . IL-4, but not IL-10, enhanced IL-8 and MCP-1 secretion under all experimental conditions (Figs. 5C 5D) . Coincubation of TNF-α and IL-13, with or without preincubation of HRPE cells with IL-13, potentiated TNF-α–induced MCP-1, but not IL-8. When removed after preincubation, IL-13 did not modulate TNF-α–induced chemokine secretion. 

Semiquantitative RT-PCR showed that IL-4 and -10, but not IL-13, reduced IL-8 mRNA expression after 5 hour HRPE cell–monocyte coculture (data not shown). All of them had no effects on MCP-1 mRNA expression. The mRNA levels confirmed the ELISA results of the effects of Th2 cytokines on IL-1β- and TNF-α–induced chemokine secretion by HRPE cell–monocyte cocultures (data not shown). 

Discussion

Homeostatic mechanisms exist in the eye by which the immune system attempts to limit the inflammatory process and its destructive effects on ocular tissues. Examples of this include the enhanced expression of IL-1 receptor antagonist (IL-1ra) in the eyes with uveitis and TGF-β expression in the eyes with PVR. ^{35 36} Recent studies have shown that treatment with exogenous Th2 cytokines such as IL-4, -10, and -13 may alter the cytokine balance mitigating inflammation in several experimental diseases. ^{37 38 39} Because HRPE cells and monocytes are important sources of chemokines, ^{7 8 34} and are closely associated in the histopathologic lesions of eyes with uveitis, ARMD, and PVR, ^{1 2 3 5 6} the effects of these Th2 cytokines on HRPE cells and monocyte chemokine secretion may be important in regulating ocular inflammatory responses. 

In addition to confirming our previous results about the effects of IL-4 and -10 on HRPE IL-8 and MCP-1 secretion, ^{24 25} we found that IL-13 enhances constitutive as well as TNF-α– and 0.02 ng/ml IL-1β–induced HRPE chemokine secretion. We also found that direct overlay of monocytes onto HRPE cells consistently results in increased secretion of both IL-8 and MCP-1. This appears to be dependent on cell-to-cell contact, because coincubation of HRPE cells and monocytes in the same cultures, but separated by porous polycarbonate filters, does not induce the secretion of the high levels of these chemokines measured after direct overlay of human monocytes onto HRPE cells. Therefore, HRPE cell–monocyte interactions are likely to induce additional leukocyte accumulation and activation, leading to the progression of ocular inflammatory diseases by enhancing expression of IL-8 and MCP-1. Recent studies have shown that the direct adhesion of cells resulting in gene expression may be dependent on various adhesion molecule such as ICAM-1, VCAM-1, and integrin family receptors on the surfaces of interacting cells. ^{40 41 42} This suggests that cell surface molecules might also control the chemokine induction that is caused by direct contact of HRPE cells and monocytes. In addition, we found that addition of IL-4, -10, and -13 resulted in inhibition of the chemokine production by HRPE cell–monocyte cocultures. The effects of the Th2 cytokines were modified in the presence of known pro-inflammatory cytokines, IL-1β and TNF-α, and by the timing of the Th2 cytokine exposure. Semiquantitative RT-PCR generally confirmed the ELISA results of the effects of Th2 cytokines on chemokine secretion by HRPE cell–monocyte cocultures. 

IL-10 has been shown to ameliorate some experimental inflammatory disorders including uveitis. ^{38 43 44} In this study, IL-10 could inhibit IL-8 production by HRPE cell–monocyte cocultures when monocytes were added in the presence or absence of IL-1β. IL-10 also inhibited MCP-1 production by HRPE cell–monocyte cocultures and monocyte IL-8 secretion. In contrast, IL-10 had no significant effects on IL-1β–induced MCP-1 or TNF-α–induced IL-8 and MCP-1 secretion by HRPE–monocyte cocultures or IL-1β– and TNF-α–induced HRPE chemokine secretion. The continued presence of IL-10 appears to be necessary, because its effects did not persist after its removal from cultures that were subsequently exposed to pro-inflammatory cytokines or monocytes. The effects of IL-10 did not depend on preincubation, whereas preincubation was needed to inhibit HRPE cell HLA-DR expression in our previous study. ²⁵ Although IL-10 enhanced monocyte MCP-1 secretion as described previously, ³³ the amount of secretion by monocytes is much smaller than that by HRPE–monocyte cocultures. Taken together, these findings support the use of IL-10 as a potentially therapeutic suppressor of ocular inflammatory processes in the clinical setting. In addition, Ongkosuwito et al. ⁴⁵ demonstrated that IL-10 detected in ocular fluids with acute retinal necrosis ranged from 29 to 3927 pg/ml. Because we used 100 U/ml of IL-10 corresponding to 3400 pg/ml of IL-10, endogenous IL-10 might also have suppressive effects on chemokine expression under clinically relevant conditions. 

Previous reports demonstrated that IL-4 and -13 share a common subunit that is important in signal transduction and that they share many actions. ^{46 47} They are produced by T cells, mast cells, basophils, and macrophages. ^{24 48 49 50} In this study, IL-4 and -13 inhibited IL-8 production by HRPE cell–monocyte cocultures in the presence or absence of IL-1β as well as constitutive and IL-1β– and TNF-α–induced monocyte IL-8 and MCP-1 production. In contrast, IL-4 and -13 enhanced TNF-α–induced HRPE cell–monocyte coculture MCP-1 secretion and constitutive and TNF-α–induced HRPE chemokine secretion. The actions of IL-4 in this study mostly paralleled those of IL-13. However, IL-4, but not IL-13, had additional enhancing effects on chemokine production. It enhanced 0.2 ng/ml IL-1β–induced HRPE IL-8 secretion and TNF-α–induced IL-8 secretion by HRPE cell–monocyte cocultures. This may explain, in part, reports of IL-13, but not IL-4, inhibiting experimental uveitis. ^{39 51 52} IL-4 and -13 have been thought to modulate inflammation, promote host repair, and restrict tissue damage. ⁵³ Our findings suggest that these cytokines may have biphasic effects, inducing HRPE chemokine secretion at the beginning of ocular inflammation leading to the recruitment of leukocytes and inhibiting chemokine production in the presence of infiltrating monocytes, thereby restricting damage to tissue. 

Inflammatory cytokines like IL-1β and TNF-α are considered to be important in the pathophysiology of the inflammatory component of numerous ocular disorders. They have been identified on epiretinal membranes removed surgically from patients with PVR and on epiretinal membranes produced experimentally. ^{28 29} In addition, intravitreal injection of IL-1β and TNF-α induces strong inflammatory responses. ⁵⁴ Th2 cytokines have been demonstrated to inhibit monocyte IL-1β and TNF-α production. ^{55 56} However, in this study, in contrast to the inhibitory effects of Th2 cytokines on IL-1β–induced IL-8 production by HRPE cell–monocyte cocultures, IL-4 and -13 enhanced TNF-α–induced chemokine secretion by these cocultures, whereas IL-10 had no significant effects. These data suggest that the Th2 cytokines, particularly IL-10, in combination with TNF-α blockade might assist to inhibit ocular inflammation most effectively. The overall effects of Th2 cytokines appear to depend on the types of ambient cytokines present, resulting in subtle alterations of homeostatic balance during inflammation. This may explain why Th2 cytokine effects were not always prominent in our study. Further elucidation of Th2 cytokine effects in the presence of both IL-1β and TNF-α may lead to better understanding of the complex mechanisms of inflammation. 

In this study, Th2 cytokines did not inhibit constitutive chemokine secretion by HRPE cells, suggesting that the effects of these Th2 cytokines on HRPE chemokine expression may depend on presence of closely associated inflammatory cells (i.e., monocytes). Thus, Th2 cytokine inhibitory effects on chemokine expression during ocular inflammation maybe most prominent at the onset of leukocyte infiltration, acting as a negative feedback mechanism. As a result, Th2 cytokines could be candidates as therapeutic agents in diseases, such as PVR, in which leukocytes persist in the lesions or in conventionally treated uveitis where subclinical inflammation is still present and may be exacerbated with corticosteroid taper. 

In conclusion, the results of the present study, together with our previous observations, demonstrated that IL-4, -10, and -13 have complex effects on HRPE chemokine secretion that likely depend on the types of ambient cytokines and presence of inflammatory cells. Our data suggest the potential therapeutic usefulness of these cytokines, particularly IL-10, in the treatment of ocular proliferative and inflammatory diseases. 

 Supported by National Institutes of Health Grants EY09441 and EY007003 and Research to Prevent Blindness–Olga Keith Weiss Award (VME).

 Submitted for publication September 27, 2000; revised January 29, 2001; accepted February 7, 2001.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effects of IL-4, -10, and -13 on IL-8 (A) and MCP-1 (B) secretion by HRPE cells. HRPE cells were incubated with IL-4, -10, or -13 in the presence or absence of anti-IL-4, -10, or -13 mAb for 24 hours, respectively. The conditioned media were assayed for MCP-1 and IL-8 using ELISA. Values represent means ± SEM (n = 3). *P < 0.05;** P < 0.01, compared with control. No St, no stimulant.

Table 1.

 

Summary of Effects of IL-4, -10, and -13 on IL-8 and MCP-1 Production

	IL-8					MCP-1
		IL-4	IL-10	IL-13	IL-4	IL-10	IL-13
HRPE	↑	→	↑	↑	→	↑
HRPE+ IL-1β (0.2 ng/ml)	↑*	→	→	→	→	→
HRPE+ TNF-α (2.0 ng/ml)	↑	→	↑	↑	→	↑, †
Monocytes	↓	↓	↓	↓	↑	↓
Monocytes+ IL-1β (0.2 ng/ml)	↓	↓	↓	↓	↑	↓
Monocytes+TNF-α (2.0 ng/ml)	↓	↓	↓	↓	↑	↓
HRPE–Monocytes	↓	↓, ‡	↓, ‡	→	↓, ‡	→
HRPE–Monocytes+ IL-1β (0.2 ng/ml)	↓, §	↓, ∥	↓, ¶	→	→	→
HRPE–Monocytes+ TNF-α (2.0 ng/ml)	↑	→	→	↑	→	↑, ∥

 Significant differences were observed

*  when HRPE cells were coincubated with IL-4 and IL-1β simultaneously;

†  with pre+co-incubation or only preincubation of IL-13 with TNF-α in HRPE cultures;

‡  with pre+co-incubation or simultaneous coincubation of IL-10 or IL-13 with monocytes in HRPE cultures;

§  with IL-4 pre+coincubation or only preincubation with IL-1β + monocytes in HRPE cultures;

∥  with pre+coincubation or simultaneous coincubation of IL-10 or IL-13 with IL-1β or TNF-α + monocytes in HRPE cultures; and

¶  when HRPE cells were co-incubated with IL-13, IL-1β, and monocytes simultaneously.

Figure 2.

 

Effects of IL-4, -10, and -13 on IL-1β (0.2 ng/ml; A and B)– or TNF-α (2.0 ng/ml; C and D)–induced IL-8 (A and C) and MCP-1 (B and D) production by HRPE cells. □, HRPE cells were preincubated with IL-4, -10, or -13 and then coincubated with IL-1β or TNF-α together with IL-4, -10, or -13; ▪, HRPE cells were preincubated with IL-4, -10, or -13 and incubated with IL-1β or TNF-α only; , IL-1β or TNF-α and IL-4, -10, or -13 were added to HRPE cells simultaneously and coincubated. IL-8 and MCP-1 protein levels in supernatants were detected by ELISA. Values represent means ± SEM (n = 3). *P < 0.05;** P < 0.01, compared with protein secretion from HRPE cells with IL-1β or TNF-α only. No St, no stimulant.

Figure 3.

 

(A and B) Effects of IL-4, -10, and -13 on constitutive monocyte IL-8 (A) and MCP-1 (B) secretion. Monocytes were incubated with IL-4, -10, or -13 in the presence or absence of anti–IL-4, -10, or -13 mAb, respectively. Values represent means ± SEM (n = 3).* P < 0.05; **P < 0.01, compared with control. No St, no stimulant. (C and D) Effects of IL-4, -10, and -13 on IL-1β (0.2 ng/ml)– and TNF-α (2.0 ng/ml)–induced monocyte IL-8 (C) and MCP-1 (D) secretion. Monocytes were coincubated with IL-1β or TNF-α together with IL-4, -10, or -13. The conditioned media were assayed for MCP-1 and IL-8 using ELISA. Values represent means ± SEM (n = 3). *P < 0.05;** P < 0.01, compared with protein secretion from monocytes with IL-1β or TNF-α alone.

Figure 4.

 

(A and B) Production of IL-8 (A) and MCP-1 (B) on cocultures of HRPE cells with monocyte (Mo). HRPE cells were cocultured with monocytes for 24 hours. The conditioned media were assayed for MCP-1 and IL-8 by ELISA. HRPE+Mo, monocytes were overlayed onto HRPE cells. HRPE/Mo, HRPE cells and monocytes were coincubated in the same cultures, but separated by porous polycarbonate filters. Values represent means ± SEM (n = 3). (C and D) Effects of IL-4, -10, and -13 on IL-8 (C) and MCP-1 (D) by HRPE cell–monocyte cocultures. □, HRPE cells were preincubated with IL-4, -10, or IL-13 and coincubated with monocytes and IL-4, -10, or -13; ▪, after preincubation, HRPE cells were coincubated with monocytes only; , monocytes and IL-4, -10, or -13 were added to HRPE cells simultaneously and coincubated in the presence or absence of anti-IL-4, -10, or -13 mAb, respectively. IL-8 and MCP-1 protein levels in supernatants were detected. Values represent means ± SEM (n = 3). *P < 0.05;** P < 0.01, compared with control. No St, no stimulant.

Table 2.

 

Chemokine Secretion by HRPE and Monocyte Separated after Cocultures

	IL-8 (ng/ml)	MCP-1 (ng/ml)
HRPE/monocyte removed	5.75 ± 0.11 (72%)	24.05 ± 0.28 (98%)
Monocyte/detached from HRPE	2.26 ± 0.14 (28%)*	0.374 ± 0.12 (2%), †

*   P < 0.01, compared with HRPE/monocyte removed (IL-8).

†   P < 0.01, compared with HRPE/monocyte removed (MCP-1).

Figure 5.

 

Effects of IL-4, -10, and -13 on IL-1β (0.2 ng/ml; A and B)– or TNF-α (2.0 ng/ml; C and D)–induced IL-8 (A and C) and MCP-1 (B and D) production by HRPE cell–monocyte cocultures. □, after preincubation with IL-4, -10, or -13, HRPE cells were coincubated with monocytes, IL-1β or TNF-α, and IL-4, -10, or -13; ▪, after preincubation, HRPE cells were coincubated with monocytes and IL-1β or TNF-α. , monocytes, IL-1β or TNF-α and IL-4, -10, or -13 were added to HRPE cells simultaneously and coincubated. IL-8 and MCP-1 protein levels in supernatants were detected by ELISA. Values represent means ± SEM (n = 3). *P < 0.05;** P < 0.01, compared with protein secretion from HRPE cell–monocyte cocultures with IL-1β or TNF-α alone. No St, no stimulant.