Rabbit Pigmented Ciliary Epithelium Produces Interleukin-6 in Response to Inflammatory Cytokines

Exp. Eye Res. (2000) 70, 271–279 doi : 10.1006\exer.1999.0787, available online at http :\\www.idealibrary.com on

Rabbit Pigmented Ciliary Epithelium Produces Interleukin-6 in Response to Inflammatory Cytokines L L O Y D N. F L E I S H ER*, M. C H R I S T I N E M  G A H A N    J E N N Y B. F E R R E L L Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, U.S.A. (Received Columbia 12 May 1999 and accepted in revised form 4 October 1999) Interleukin-6 is a multifunctional cytokine that is found in high concentrations in intraocular fluids during the uveitic response. Although monocytic cells are a major source of interleukin-6, resident intraocular cells may also contribute to its accumulation in intraocular fluids during uveitis. The purpose of this study was to determine whether interleukin-6 is produced by pigmented ciliary epithelial cells and whether agents known to stimulate interleukin-6 production, such as interleukin-1β, tumor necrosis factor-α, bacterial endotoxin, and stimulators of the adenylyl cyclase\adenosine 3h,5h-cyclic monophosphate system, increase interleukin-6 production by these cells. Primary and first-passage cultures of nontransformed rabbit pigmented ciliary epithelial cells were incubated with the test agents for varying periods of time in serum-free medium and interleukin-6 levels in the cell-conditioned medium were measured by bioassay. Little, if any interleukin-6 was released from pigmented ciliary epithelial cells incubated for up to 18 hr in serum-free medium. Interleukin-1β stimulated interleukin-6 release in a time- and concentrationdependent manner. Tumor necrosis factor-α, although ineffective alone, increased interleukin-1βinduced interleukin-6 release in a concentration-dependent manner when co-incubated with interleukin1β for 18 hr. However, tumor necrosis factor-α did not enhance interleukin-1β-induced interleukin-6 release if co-incubated with interleukin-1β for a shorter time (6 hr). A 6 hr exposure to bacterial endotoxin did not stimulate interleukin-6 release from pigmented ciliary epithelial cells. Co-incubation of pigmented ciliary epithelial cells with interleukin-1β and agents that stimulate the adenyl cyclase\ adenosine 3h,5h-cyclic monophosphate system through cell surface G-protein transduced receptors, i.e. isoproterenol, vasoactive intestinal peptide or prostaglandin E , significantly enhanced the ability of # interleukin-1β to stimulate interleukin-6 release. However, neither the adenyl cyclase activator, forskolin or the adenosine 3h,5h-cyclic monophosphate-mimetic, dibutyryl 3h,5h-cyclic monophosphate enhanced interleukin-1β-induced release of interleukin-6. These results indicate that the pigmented ciliary epithelium is one potential source of interleukin-6 and may contribute to the elevation in intraocular fluid interleukin-6 levels observed during various intraocular inflammatory episodes. Although agents that activate the adenyl cyclase\adenosine 3h,5hcyclic monophosphate system through cell surface G-protein transduced receptors increased interleukin1β-induced release of interleukin-6, the ineffectiveness of forskolin and dibutryl 3h,5h-cyclic monophosphate suggest that simply increasing intracellular 3h,5h-cyclic monophosphate is not sufficient to augment interleukin-1β-induced release of interleukin-6. The significance of interleukin-6 in the intraocular inflammatory response is discussed in terms of its proposed role in an endogenous antiinflammatory system acting through induction of interleukin-1 receptor antagonist, soluble tumor necrosis factor receptor, acute-phase proteins and corticosteroids. # 2000 Academic Press Key words : interleukin-6 ; pigmented ciliary epithelium ; interleukin-1 ; tumor necrosis factor-α ; adenosine 3h,5h-cyclic monophosphate.

1. Introduction Interleukin-6 (IL-6) is a multifunctional cytokine produced mainly by monocytes (Akira et al., 1990), but also by other cell types such as fibroblasts (Elias and Lentz, 1990), endothelial cells (May et al., 1989) and epithelial cells (Krueger et al., 1991 ; Zhou, Munster and Winchurch, 1991). IL-6 modulates proliferation of lymphoid and nonlymphoid cells and is an important component of the immune response (Akira et al., 1990 ; Krueger et al., 1991). Il-6 is also thought to participate in the inflammatory response, but the precise nature of its role is unclear. For * Author for correspondence.

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example, IL-6 is generally considered to be an inflammatory cytokine (Akira et al., 1990), yet it can also inhibit local acute inflammatory responses (Ulich et al., 1991). It has been suggested that IL-6 is part of an endogenous anti-inflammatory system acting through induction of IL-1 receptor antagonist, soluble tumor necrosis factor (TNF) receptor, acute-phase proteins and corticosteroids (Barton and Jackson, 1993 ; Dinarello, 1992 ; Tilg et al., 1994). The importance of IL-6 in the intraocular inflammatory response has also been the subject of controversy. For example, although intravitreally injected IL6 induced a uveitic response in Lewis rats similar to that produced by endotoxin (Hoekzema et al., 1991), Bamforth, Lightman and Greenwood (1996) found # 2000 Academic Press

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that intravitreally injected IL-6 had no effect on the integrity of the blood–retinal barrier of Lewis rats. Increased levels of IL-6 in intraocular fluids have also been associated with intraocular inflammation. Elevated levels of IL-6 have been found in aqueous humor (Murray et al., 1990 ; Norose et al., 1994) and vitreous (de Boer et al., 1992) of patients with uveitis and in vitreous of patients with retinal detachments complicated by proliferative vitreoretinopathy (Limb et al., 1991). Furthermore, the increase in IL-6 levels in aqueous humor correlated with the initiation and severity of the uveitic response following injection of endotoxin into the footpad of Lewis rats (De Vos et al., 1994 ; Hoekzema et al., 1991). At the height of this uveitic response IL-6 levels in aqueous humor actually exceeded those in serum and this was suggested to be due to intraocular production of IL-6 (De Vos et al., 1994). In contrast, Rosenbaum et al. (1998) found that intravitreal injection of IL-6 did not produce uveitis in mice and endotoxin was an effective uveitogenic agent when injected into the vitreous of mice lacking the IL-6 gene. Although the role(s) played by IL-6 in the intraocular inflammatory response is unclear, IL-6 levels increase markedly in intraocular fluids during uveitis, and resident intraocular cells are likely contributors to this increase. However, little is known concerning which intraocular cells are responsible for this IL-6 production. One potential intraocular site for IL-6 production is the ciliary epithelium, a cellular bilayer separating aqueous humor from the vascular stroma of the ciliary body. The inner layer of the ciliary epithelium, the pigmented ciliary epithelium (PE), occupies an especially strategic location since it is the first cellular barrier encountered by inflammatory leukocytes exiting capillaries within the stroma of the ciliary body during the initial stages of the intraocular inflammatory response. These leukocytes release cytokines, such as IL-1 and TNF-α, that could stimulate de novo synthesis of IL-6 by PE cells. IL-6 could then diffuse into the aqueous humor through inflammationassociated disruptions in the nonpigmented ciliary epithelial (NPE) monolayer. Although we reported that IL-1β increased the ability of prostaglandin (PG) E to stimulate adenosine 3h,5h-cyclic monophosphate # (cAMP) synthesis in rabbit PE, in vitro (Fleisher et al., 1996) little is known about the effects of cytokines on PE or the capacity of this epithelial tissue to produce cytokines. In the present investigation we utilized cultures of nontransformed rabbit PE to determine whether IL-1β, TNF-α and bacterial endotoxin, which works largely by inducing production of IL-1 and TNFα (Beutler and Cerami, 1987 ; Dinarello, 1991), can induce IL-6 synthesis. Furthermore, since cAMP has been implicated in the regulation of IL-6 synthesis (Cinque et al., 1992 ; Dendorfer, Oettgen and Libermann, 1994 ; Tatsuno et al., 1991), we examined whether agents that stimulate cAMP production or activate the adenylyl cyclase system can either induce

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IL-6 production by PE or modify the effects of IL-1β and TNF-α on its production.

2. Materials and Methods Cell Culture Rabbit PE cell cultures were prepared from male Dutch Belted rabbits (Franklin Rabbitry, Wake Forest, NC, U.S.A.) based upon methods developed by Fain and Farahbakhsh (1989). Preparation of primary cultures of rabbit PE cells and validation of their purity has been previously reported (Fleisher et al., 1996). With the exception of the initial time course, the IL-1β dose-response experiments, and part of the TNF-α\IL1β experiments (in which both primary and firstpassage PE were used), all experiments were conducted with first-passage PE cells. Treatment of animals conformed to the NIH Guide for the Care and Use of Laboratory Animals, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and was approved by the University Institutional Animal Care and Use Committee. In order to prepare PE cells, rabbits were killed by exposure to an atmosphere of 100 % CO and eyes # enucleated and rinsed in Hanks’ balanced salt solution (HBSS). The posterior segment was removed 4 mm posterior to the limbus and the anterior segment pinned to a dissecting dish (with 5 mm hardened Sylgard ; Dow Corning, Midland, MI, U.S.A.) containing HBSS. The lens was gently removed leaving the capsule in place and a 1 mm strip (around the ciliary body ring) dissected from the remaining anterior segment. The strip was transferred to fresh HBSS, cut into four segments, and transferred to growth medium (GM) consisting of minimum essential medium (MEM) with Earle’s salts and -glutamine (Gibco BRL, Grand Island, NY, U.S.A.) plus gentamicin (50 µg ml−" ; Gibco BRL, Grand Island, NY, U.S.A.), kanamycin (100 µg ml−" ; Sigma Chemical Co., St. Louis, MO, U.S.A.), additional -glutamine (3 m), and fetal bovine serum (FBS, 15 % ; Hyclone, Logan, UT, U.S.A.). The strips from both eyes were rinsed with Hanks’ Ca#+- and Mg#+-free balanced salt solution (HCMF-BSS) and incubated with HCMF-BSS containing 2n4 % dispase (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and 100 m sorbitol (Sigma Chemical Co., St. Louis, MO, U.S.A.) for 37 min. Strips were then rinsed with HCMF-BSS, transferred to GM, and the epithelial bilayer peeled off and transferred to fresh GM. Strips of epithelial cells were rinsed in HCMF-BSS and incubated for 1 hr in HCMF-BSS containing 0n5 m EDTA and 0n05 % trypsin (Difco, Detroit, ME, U.S.A.). The cells plus the trypsin-EDTA were then mixed with GM, centrifuged (5 min at 300 g), the cell pellet rinsed by centrifugation (2i with GM), and the cells suspended by trituration of the pellet with 3 ml GM. In order to produce primary cultures, cells were then plated (60 000 cells well−")

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on 6 well tissue culture plates (Costar T3506, Cambridge, MA, U.S.A.) and incubated at 37mC in 95 % air\5 % CO and 95 % humidity. These conditions # support the growth of PE, but not NPE cells (Fain and Farahbakhsh, 1989). PE cells reached confluence within 7–9 days. In order to produce first-passage PE, confluent cultures of primary PE were rinsed (2i with HCMFBSS) and 1 ml of HCMF-BSS containing 0n5 m EDTA and 0n1 % trypsin added. After 15 min incubation at 37mC in 95 % air\5 % CO and 95 % humidity, cells # were gently agitated and transferred to 15 ml centrifuge tubes and washed 3i by centrifugation (5 min at 300 g) in GM. A cell suspension was then prepared by trituration with GM (3 ml). The cell suspension was counted and diluted to 1n5i10' cells ml−" in GM containing 0n59  DMSO (ATCC, Rockville, MD, U.S.A.). Aliquots were frozen at k70mC. First-passage cultures were started by quickly thawing the cells, washing 3i by centrifugation (5 min at 300 g) in GM, resuspending the cells, and plating them on 6 well tissue culture plates (Costar F3506, Cambridge, MA, U.S.A.) at 37mC in 95 % air\5 % CO and 95 % humidity # (60 000 cells well−"). All subsequent experiments (i.e. with primary or first-passage PE) were conducted in serum-free medium.

Interleukin-6 Bioassay The presence of IL-6 in the incubation medium of the PE cells (i.e. the cell-conditioned medium ; CCM) was measured by a $H-thymidine uptake assay using the IL-6-dependent murine hybridoma T1165 as previously described (Van Snick et al., 1986). The T1165 cells were maintained and supplied by Dr Susan Tonkonogy (North Carolina State University, Raleigh, NC, U.S.A.). For the IL-6 bioassay, 2-, 4-, 8and 16-fold dilutions of the PE CCM were added to 96 well flat bottom plates in triplicate (50 µl well−" ; Costar T3595, Cambridge, MA, U.S.A.). T1165 cells were washed 3i by centrifugation (6 min at 300 g) in RPMI medium containing 5 % FBS, 50 µg ml−" gentamicin, 2 m glutamine, 1 m pyruvate and 50 µ 2-mercaptoethanol (all from Gibco BRL, Grand Island, NY, U.S.A.). T1165 cells were then added to each well (1i10& cells ml−" ; 50 µl well−") for a final volume of 100 µl. After 48 hr incubation at 37mC in 95 % air\5 % CO in 95 % humidity, cells were pulsed # with 20 µCi of $H-thymidine (ICN, Costa Mesa, CA, U.S.A.) for 18 hr, harvested (PhD Cell Harvester, Cambridge, MA, U.S.A.), counted in a liquid scintillation counter (Wallac 1409, Gaithersburg, MD, U.S.A.), and the amount incorporated into DNA calculated. This measurement reflects the amount of biologically active IL-6 present in the sample. In the absence of IL-6, the T1165 cells will not proliferate and will die by 48 hr (background). Units of IL-6 bioactivity per milliliter are defined as the reciprocal of

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the dilution of supernatant that produces 50 % of the maximal counts per min in each assay. These values are multiplied by 10, because the bioassay volumes are 0n1 ml. The sensitivity of the IL-6 bioassay is 1 U ml−". Reagents Human recombinant IL-1β (specific activity : 1n5i10) U mg−" protein) and human recombinant TNF-α (specific activity : 2n86i10( U mg −" protein) were purchased from R & D Systems, Minneapolis, MN, U.S.A. Isoproterenol, vasoactive intestinal peptide, PGE , forskolin, dibutyryl cAMP and endotoxin # (lipopolysaccharide from E. coli, sera type 055 ; B5) were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Statistical Analysis Differences between experimental groups were analysed using one-way ANOVA. When analysis involved more than two means, the significance of differences between them was determined using Tukey’s HSD test. The null hypothesis was rejected at P 0n05. Data represent meanp..(.). Treatments were routinely run in triplicate. 3. Results Effect of IL-1β on IL-6 Production Since IL-1 is a potent agonist for IL-6 production (Akira et al., 1990 ; Gimble et al., 1991) and we previously found that IL-1β enhanced PGE -stimulated # cAMP production by cultured rabbit PE cells (Fleisher et al., 1996), we first examined the effects of this inflammatory cytokine on PE production of IL-6. Using the dose of human recombinant IL-1β employed in our previous investigation (150 U ml−") a time-course study was conducted. Separate groups of PE cells (both primary and first-passage cells) were exposed to IL-1β for periods up to 18 hr. Since both primary and firstpassage cells responded similarly to IL-1β, the results were combined and are illustrated in Fig. 1. IL-6 levels in the incubation medium were highest after 3 and 6 hr exposure to IL-1β (P 0n001 at both times compared to time 0). By 18 hr, IL-6 levels were still significantly greater than at time 0 (P 0n002), but lower than at the 3 and 6 hr time points. A low level of IL-6 [6n9p4n5 ; meanp..(.)] was detected at the zero time point (i.e. following addition and immediate removal of serum-free medium containing 150 U ml−" of IL-1β). Parallel groups of PE cells incubated for 0, 3, 6 and 18 hr in serum-free medium without IL-1β exhibited no detectable release of IL-6 into the CCM (data not shown). Based upon the kinetics of IL-6 accumulation, a complete IL-1β dose-response analysis was conducted

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F. 1. Time course for IL-1β-induced IL-6 release from PE cells. Primary and first-passage cultures of Dutch Belted rabbit PE cells were incubated in serum-free medium supplemented with IL-1β (150 U ml−") for periods ranging 0–18 hr. At the conclusion of each incubation period IL-6 was measured in the cell-conditioned medium by a $Hthymidine uptake assay using the IL-6-dependent murine hybridoma T1165. Data are expressed as meanp..(.) for n l 6. * l Significantly different from time 0 (P 0n001 for 3 and 6 hr ; P 0n002 for 18 hr).

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F. 3. Effect of TNF-α on IL-6 release from PE cells. Primary cultures of Dutch Belted rabbit PE cells were incubated in serum-free medium or serum-free medium supplemented with IL-1β (75 U ml-"), TNF-α (400– 2000 U ml−"), or IL-1β plus each dose of TNF-α for 6 ( ) and 18 hr (5). At the conclusion of the 6 and 18 hr incubation periods IL-6 was measured in the cell-conditioned medium by a $H-thymidine uptake assay using the IL-6dependent murine hybridoma T1165. Data are expressed as meanp..(.) for n l 3 (6 hr incubation) and n l 6 (18 hr incubation). * l Significantly different from IL-1β alone (P 0n001).

Effects of TNF-α and Endotoxin on IL-6 Production

F. 2. Dose–response relationship for IL-1β-induced IL-6 release from PE cells. Primary ( ) and first-passage (5) cultures of Dutch Belted rabbit PE cells were incubated in serum-free medium or serum-free medium supplemented with IL-1β (50–500 U ml−") for 6 hr. At the conclusion of the 6 hr incubation period IL-6 was measured in the cellconditioned medium by a $H-thymidine uptake assay using the IL-6-dependent murine hybridoma T1165. Data are expressed as meanp..(.) for n l 6. * l Significantly different from medium alone (P 0n001).

using an incubation time of 6 hr. In order to ensure that primary and first-passage cells behaved similarly, two dose-response analyses were conducted ; one with primary PE, the second with first-passage PE derived from the same cells used to make the primary cultures. As illustrated in Fig. 2, the dose–response relationships for primary and first-passage PE were quite similar. No IL-6 production was observed in PE cells incubated for 6 hr in medium without IL-1β. The threshold dose of IL-1β required to induce IL-6 production was 50 U ml−" and the ability of IL-1β to stimulate IL-6 production increased in a linear fashion up to a dose of 500 U ml−". Doses of IL-1β less than 50 U ml−" were tested. However, since they did not stimulate IL-6 production they were not included in Fig. 2.

TNF-α is another inflammatory cytokine that can stimulate IL-6 production (Akira et al., 1990). Furthermore, TNF-α and IL-1β interact synergistically to produce uveitis when injected into the vitreal chamber of the rabbit eye (Fleisher, Ferrell and McGahan, 1992). Therefore, in separate 6 and 18 hr incubation experiments, the effects of TNF-α, IL-1β and combinations of IL-1β and TNF-α on IL-6 production by primary rabbit PE were assessed. As illustrated in Fig. 3, TNF-α (400–2000 U ml−") did not induce IL-6 production by PE during either a 6 or 18 hr incubation. Furthermore, IL-6 levels in CCM of PE exposed to combinations of IL-1β and TNF-α for 6 hr were not different from those of PE exposed to IL-1β (75 U ml−") alone for the same time period. In contrast, in the 18 hr incubation experiment, TNF-α significantly increased the effects of IL-1β on IL-6 accumulation in a dose-dependent manner at 1000 and 2000 U ml−" (P 0n001). First-passage PE cells were also exposed to bacterial endotoxin (0, 1, 10, 100 and 1000 ng ml−") for 6 hr. No IL-6 production was observed at any concentration of endotoxin (data not shown). Effects of Agents that Increase Intracellular cAMP Levels on IL-1β-induced IL-6 Production Several signal transduction systems, including the adenylyl cyclase system, are involved in IL-1stimulated IL-6 gene expression (Cinque et al., 1992 ; Dendorfer et al., 1994 ; Tatsuno et al., 1991). Therefore, we examined the effects of agents that stimulate or mimic the effects of the adenylyl cyclase\cAMP system on IL-1β-induced IL-6 production by rabbit PE. The cAMP inducers used were

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F. 4. Effects of isoproterenol, VIP and PGE on IL-1β# induced IL-6 release from PE cells. First passage cultures of Dutch Belted rabbit PE cells were incubated in serum-free medium supplemented with IL-1β (200 U ml−" ) or serumfree medium supplemented with IL-1β plus isoproterenol (10 µ ; 5), VIP (50 n ; 5), or PGE (10 µ ; 5) for 6 hr. # At the conclusion of the 6 hr incubation period IL-6 was measured in the cell-conditioned medium by a $H-thymidine uptake assay using the IL-6-dependent murine hybridoma T1165. Data are expressed as meanp..(.) for n l 3–6. * l Significantly different from IL-1β alone compared to IL-1β plus isoproterenol (P 0n02), IL-1β plus VIP (P 0n004), and IL-1β plus PGE (P 0n01). #

F. 5. Effects of forskolin and dibutyryl cAMP on IL-1βinduced IL-6 release from PE cells. First passage cultures of Dutch Belted rabbit PE cells were incubated in serum-free medium supplemented with IL-1β (200 U ml−" ; ) or serum-free medium supplemented with IL-1β plus forskolin (1 µ ; 5) or dibutyryl cAMP (10 µ ; 5) for 6 hr. At the conclusion of the 6 hr incubation period IL-6 was measured in the cell-conditioned medium by a $H-thymidine uptake assay using the IL-6-dependent murine hybridoma T1165. Data are expressed as meanp..(.) for n l 3.

isoproterenol, vasoactive intestinal peptide (VIP), PGE , forskolin and dibutyryl cAMP. Isoproterenol, # VIP and PGE activate the adenylyl cyclase\cAMP # system through cell surface receptor-G-protein transduced mechanisms (Asano et al., 1984 ; Bhattacherjee, Rhodes and Paterson, 1993 ; Finch, Sreedharan and Goetzl, 1989), forskolin directly activates the catalytic subunit of adenylyl cyclase (Seamon and Daly, 1981), and dibutyryl cAMP is a cell-permeant cAMP agonist (Posterak and Weiman, 1974). We have previously reported that isoproterenol, VIP, PGE and forskolin # stimulate cAMP production in rabbit PE (Fleisher et al., 1996). The three cAMP inducing agents that act through

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cell-surface receptors had similar effects on the ability of IL-1β to stimulate IL-6 production by first-passage PE cells. When co-incubated with IL-1β (200 U ml−") for the 6 hr incubation period, isoproterenol (10 µ), VIP (50 n) and PGE (10 µ) significantly increased # the ability of IL-1β to stimulate IL-6 production (P 0n02, P 0n004 and P 0n01, respectively ; see Fig. 4). PE incubated with medium or mediumj isoproterenol, VIP or PGE for 6 hr produced no # detectable IL-6 (data not shown). In contrast to the results with isoproterenol, VIP and PGE , neither a # direct stimulator of the catalytic subunit of adenylyl cycase, forskolin (1 µ) or a cAMP-mimetic, dibutyryl cAMP (10 µ), increased IL-1β-induced IL-6 production in first-passage PE cells (Fig. 5). PE incubated with medium, mediumjforskolin or mediumj dibutryryl cAMP for 6 hr produced no detectable IL-6 (data not shown). 4. Discussion Situated between the NPE and the vascular stroma of the ciliary body, the PE monolayer occupies a strategic location in the anterior portion of the eye. Its intimate proximity to the vascular stroma of the ciliary body allows for rapid ion uptake and transport to the NPE ; a process integral to aqueous humor secretion (Butler et al., 1994 ; Edelman, Sachs and Adorante, 1994). Furthermore, this proximity increases the likelihood that inflammatory cells exiting capillaries within the vascular stroma of the ciliary body during the intraocular inflammatory response will immediately interact with the PE monolayer. These inflammatory cells release soluble mediators, such as the cytokines IL-1 and TNF-α, which can bind to and dramatically alter the activity of the PE. Surprizingly, despite the strategic location of the PE and the potential for significant cytokine-mediated interactions, little information exists concerning the effects of these cytokines on the activity of the PE. In this investigation we have shown that cultured rabbit PE cells can be induced to release IL-6 in a time- and concentrationdependent manner when stimulated with IL-1β (Figs. 1 and 2). This suggests that the PE is one potential source of intraocular IL-6 and would help to explain a previous report that aqueous humor levels of IL-6 exceeded those in plasma during endotoxin-induced uveitis in rats (De Vos et al., 1994). Although IL-6 is generally thought of as a product of monocytic cells, epithelial cells such as rat alveolar type II epithelial cells (Crestani et al., 1994), the A549 human pulmonary epithelial cell line (Crestani et al., 1994), and retinal pigment epithelial (RPE) (Elner et al., 1992 ; McKillop-Smith and Forrester, 1995) cells have also been shown to produce IL-6 in response to IL-1 stimulation. The ability of RPE cells to produce IL-6 is especially interesting since both PE and RPE have the same neuroectodermal origin and PE is the anterior extension of RPE.

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Rabbit PE cells exhibited little, if any basal release of IL-6. In all time-course and dose-response experiments no detectable accumulation of IL-6 was measurable if PE cells were incubated in serum-free medium without cytokines. The zero time point of the IL-1β time course (Fig. 1), which was generated by adding and immediately removing serum-free medium containing IL-1β, exhibited a low level of IL-6 [6n9p4n5 U ml−", meanp..(.) ; n l 6]. Since it is unlikely that this brief exposure to IL-1β induced IL-6 production, IL-1β could have stimulated release of a small pool of preexisting IL-6 into the CCM. Indeed, a membranebound form of IL-6 has been described (Decandia et al., 1995 ; Oxholm et al., 1991). Apparently the presence of IL-1β is required for release of IL-6 to occur since IL6 was never present in zero time point controls to which serum-free medium without IL-1β was added and immediately removed (data not shown). It should be noted that less IL-6 accumulated in the 18 hr medium compared to the 3 or 6 hr time points (Fig. 1). This suggests that IL-1β may be inducing a protease that degrades IL-6 and\or a substance that inhibits the IL-6 bioassay. Additional studies analysing IL-6 mRNA levels and looking at smaller windows of IL-6 accumulation could help to clarify this issue. In contrast to the effects of IL-1β, the inflammatory cytokine TNF-α did not stimulate accumulation of IL6 by rabbit PE cells. However, TNF-α did enhance the ability of submaximal doses of IL-1β (75 U ml−") to stimulate IL-6 accumulation by these cells. This synergistic effect of TNF-α and IL-1β was only observed after 18 hr incubation with both cytokines (Fig. 3). These results are comparable to those reported for IL-1β and TNF-α on IL-6 production in rat alveolar type II epithelial cells where the effects of the two cytokines were synergistic after 24 hr, but not after 4 hr incubation (Crestani et al., 1994). In contrast to the effects of IL-1β and IL-1β plus TNF-α, bacterial endotoxin, which works largely by inducing production of IL-1 and TNF-α (Beutler and Cerami, 1987 ; Dinarello, 1991), did not stimulate release of IL-6 from PE cells during a 6 hr exposure. However, it is possible that longer exposure to endotoxin might have induced IL-6 release from PE cells. Interestingly, in human RPE cells, although TNF-α did induce some IL-6 production, IL-1β was a significantly more powerful stimulus. Furthermore, endotoxin failed to induce either IL-6 mRNA expression or IL-6 production in these human RPE cells (Elner et al., 1992). The effect of TNF-α on IL-1β induced IL-6 accumulation was also interesting in terms of its kinetics. Examination of Fig. 1 reveals that IL-1β increased accumulation of IL-6 up to the 6 hr time point. However, by 18 hr the ability of IL-1β to stimulate IL6 accumulation had declined considerably to less than half that seen at 6 hr. In contrast, TNF-α induced enhancement of IL-1β-induced IL-6 accumulation was not observed until the 18 hr time point (Fig. 3). These results suggest that IL-1β and TNF-α may act in

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a coordinated manner to maintain IL-6 release by PE. IL-1β would serve as the primary stimulus for IL-6 production. TNF-α, although ineffective by itself, would prolong the effectiveness of IL-1β as an IL-6 inducer by maintaining the PE in an IL-1β-responsive state. As alluded to earlier, this could be the consequence of TNF-α-induced suppression of IL-1βinduced production of an IL-6 protease and\or inhibitor of the IL-6 bioassay. Several signal transduction systems are involved in the regulation of IL-6 gene expression including the cAMP-protein kinase A (PKA) system (Norris et al., 1994 ; Zhang et al., 1988). Each cAMP-PKA system activator tested in the present investigation, i.e. isoproterenol, VIP, PGE , forskolin and dibutyryl # cAMP, has been shown to increase IL-6 release from a variety of cell types including rat thymic epithelial cells (von Patay et al., 1998), a human bronchial epithelial cell line (Mullol et al., 1997), murine peritoneal macrophages (Martinez et al., 1998), rat astrocytes (Grimaldi et al., 1994 ; Maimone et al., 1993) and rat pleural resident monocytic cells (Oh-ishi et al., 1996). However, none of these cAMP-PKA system activators stimulated IL-6 release when incubated with PE cells for 6 hr (data not shown). On the other hand, isoproterenol, VIP and PGE did increase IL-1β# induced IL-6 release when co-incubated with IL-1β for 6 hr (Fig. 4). However, neither forskolin nor dibutyryl cAMP had this additive effect (Fig. 5). The ability of isoproterenol, VIP and PGE , agents that increase # intracellular cAMP levels through receptor-mediated G-protein transduced mechanisms, to enhance IL-1βinduced IL-6 production is consistent with a facilitative role for cAMP in IL-1-induced IL-6 production. However, the inability of forskolin, a directly-acting adenylyl cyclase activator, and dibutyryl cAMP, a cAMP-mimetic to enhance IL-1β-induced IL-6 production is inconsistent with this hypothesis. If IL1β-induced IL-6 release was mediated though the cAMP-PKA system, as has been reported in other cell systems (Cinque et al., 1992 ; Maimone et al., 1993 ; Zhang et al., 1988), then all the cAMP-PKA activators tested would be expected to increase IL-1β-induced IL6 from PE cells. However, the results of the present investigation suggest that the mechanism(s) regulating IL-6 release in rabbit PE cells is more complex. The inability of forskolin and dibutyryl cAMP to enhance IL-1β-induced IL-6 release raises the possibility that G-proteins play an essential role in this process. It should also be noted that IL-1 can stimulate IL-6 release by mechanisms independent of the cAMPPKA system, such as the phospholipase C-PKC cell signalling system (Grimaldi et al., 1995 ; Lieb et al., 1998 ; Norris, 1990). Furthermore, extensive crosstalk occurs between cAMP-PKA and phospholipase CPKC systems (Garrel et al., 1997 ; Perez-Martı! nez et al., 1998). Indeed the complex nature of the intracellular signalling systems controlling IL-6 production is exemplified in neonatal rat astrocytes where

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PKC activators increased IL-1-induced IL-6 release ; PKC inhibitors had the opposite effect. Activators of the cAMP-PKA system also increased IL-1-induced IL6 release. However, inhibitors of this system had no effect on IL-1-induced IL-6 production (Norris et al., 1994). The controversy regarding the role of IL-6 in the intraocular inflammatory response cannot be resolved at this time. A predominantly inflammatory role for IL6 is a logical conclusion based upon several relationships observed between endotoxin and cytokines ; i.e. endotoxin induces uveitis (Bhattacherjee, 1975 ; Rosenbaum et al., 1980), endotoxin works largely through induction of IL-1 and TNF-α (Beutler and Cerami, 1987 ; Dinarello, 1991), and IL-1 and TNF-α are potent agonists for IL-6 production (Akira et al., 1990 ; Gimble et al., 1991 ; Littlewood et al., 1991 ; Sironi et al., 1989). Furthermore, IL-6 levels increase in intraocular fluids during episodes of intraocular inflammation (Limb et al., 1991 ; Murray et al., 1990 ; Norose et al., 1994) and intravitreally injected IL-6 induces uveitis in Lewis rats (Hoekzema et al., 1991, 1992). During uveitis induced by footpad injection of endotoxin in Lewis rats, IL-6 levels significantly increase in aqueous humor and these changes correlate with the initiation and severity of the uveitic response (De Vos et al., 1994 ; Hoekzema et al., 1991). Although these findings are consistent with a predominantly inflammatory role for IL-6 in the intraocular inflammatory response, other reports strongly suggest that IL-6 is not a significant mediator of the uveitic response and that its function may be primarily anti-inflammatory in nature. Rosenbaum et al. (1998) have pointed out that elevated levels of a putative inflammatory mediator at an inflamed site do not prove that the substance is enhancing the local inflammatory response. They suggest that IL-6 could reduce the severity of uveitis or simply function as a bystander rather than an active participant. In the Lewis rat, Hoekzema et al. (1992) reported that intravitreally injected human recombinant IL-6 produced both an anterior and posterior uveitis, yet Bamforth, Lightman and Greenwood (1996) found that intravitreally injected murine recombinant IL-6 had no effect on the integrity of the blood–retinal barrier of these rats, although a small infiltration of inflammatory cells was observed. Although different forms of recombinant IL-6 were used in these two studies, it is unlikely that human recombinant IL-6 was more active than mouse recombinant IL-6 as a uveitogenic agent in the Lewis rat. It should be noted that the IL-6-induced uveitis reported by Hoekzema et al. (1992) was based upon PMN infiltration of the retina ; blood–retinal barrier integrity was not assessed. Perhaps the most convincing evidence against an inflammatory role for IL-6 in uveitis is the recent report by Rosenbaum et al. (1998) that intravitreally injected mouse recombinant IL-6 was not uveitogenic in normal mice, but endotoxin was

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uveitogenic when injected into the vitreal chamber of IL-6 knockout mice. Although these results do no rule out an inflammatory role for IL-6 in idiopathic uveitis, it is unlikely that IL-6 actively promotes endotoxininduced uveitis. It should be pointed out that redundancy in function is a prominent characteristic of inflammatory mediators. Therefore, the contributions of IL-6 to the inflammatory response to endotoxin may be compensated for by other mediators. Alternatively, the effects of IL-6 may be primarily anti-inflammatory. Indeed, Rosenbaum et al. (1998) suggested that IL-6 might actually have a protective role in endotoxininduced uveitis since IL-6-gene-deficient mice exhibited significantly more infiltrating inflammatory cells in the aqueous and vitreous humors than congenic control mice. The concept of IL-6 as an anti-inflammatory cytokine is supported by studies in several nonocular systems. IL-6 protected mice against death in an endotoxin-galactosamine model of septic shock (Barton and Jackson, 1993) and IL-6 inhibited endotoxin-induced TNF-α production in cultured human monocytes (Aderka, Le and Vilcek, 1989). In human blood mononuclear cells, IL-6 suppressed endotoxin- or phytohemaglutinin-induced production of IL-1β and TNF-α, apparently at the transcriptional level (Schindler et al., 1990). Furthermore, in mice infected with Yersinia enterocolitica, neutralization of IL-6 with anti-IL-6 antiserum suppressed expression of IL-1 receptor antagonist mRNA in Peyer’s patches and synthesis of IL-1 receptor antagonist in circulating neutrophils (Jordan et al., 1995). They suggested that IL-6 stimulated induction of IL-1 receptor antagonist and that the subsequent inhibition of IL-1 activity was part of a negative feedback loop facilitating resolution of the inflammatory response. This hypothesis was further strengthened by a subsequent report that intravenous infusion of IL-6 in humans significantly increased plasma levels of IL-1 receptor antagonist and soluble TNF receptor (TNFsRp55) (Tilg et al., 1994). Consequently, several groups have suggested that IL-6 is not primarily an inflammatory cytokine, but is a part of an endogenous anti-inflammatory system acting through induction of IL-1 receptor antagonist, soluble TNF receptor, acute-phase proteins and corticosteroids (Barton and Jackson, 1993; Dinarello, 1992; Tilg et al., 1994). Although there is no clear consensus regarding the physiological significance of IL-6 in the inflammatory response, we have demonstrated that the PE is a source of this pluripotent cytokine. Furthermore, cytokines such as IL-1β, which is generally considered to be an inflammatory modulator, significantly increased its production and release. The inflammatory cytokine TNF-α, although ineffective by itself, significantly increased the ability of IL-1β to simulate IL-6 release from these ocular epithelial cells. Considering the significant amounts of IL-6 produced during

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inflammation and its potential anti-inflammatory properties, further investigation into its role in the uveitic response is clearly warranted. Acknowledgements The authors would like to thank Dr Susan Tonkonogy for her generous supply of T1165 cells. Supported by funds from the state of North Carolina.

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