Exp. Eye Res. (1992) 55, 325-335

Expression Receptor

of Interleukin-1 a, Interleukin-l/l, Antagonist in Human Retinal

and an Interleukin-I Pigment Epithelial Cells

GLENN J. JAFFEa*, LINDA VAN LE”, FIDEL VALEA”, STEPHEN HASKILL”, WENDY ROBERT!?, WILLIAM P.ARENDd, ANN STUARTb~~~ WILLIAM P. PETERSb Departments of aOphthalmology and bMedicine, Duke University Medical Center, Durham, NC, cDepartment of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, and dDivision of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, U.S.A. (Received Houston 26 August 1991 and accepted in revised form 9 December 1991) mRNA expressionand protein productionof interleukin-la, interleukin-11 and intracellular and secreted forms of an interleukin-1 receptor antagonist were measuredin visually confluent monolayersof unstimulated cultured human retinal pigment epithelial cells and after cells were stimulated with recombinantcytokines.Usingreversetranscriptionpolymerasechain reaction, transcriptsfor interleukinla and interleukin-lb were not detectedin unstimulatedcellsfrom any of six donorswhereasmRNA expressionfor both interleukin-la and interleukin-l/3 was readily inducedin all six cell lines after cells were stimulatedwith recombinant IL-1 (a or @).tumor necrosisfactor CL,or lipopolysaccharide.The combination of cycloheximide and recombinant interleukin-1 caused a 14-fold enhancement of interleukin-leeand interleukin-l/? mRNA expressionabovethat observedafter cellswere stimulatedwith interleukin-1 alone. After stimulation by interleukin-1, cells produced intracellular interleukin-1c( protein, but did not secreteit into medium.In contrast, interleukin-lp protein was not detectedin cell lysates or conditioned-mediumafter stimulation with interleukin-1. An intracellular interleukin-1 receptor antagonist was expressedconstitutively by human retinal pigment epithelial cells: mRNA transcriptswereenhancedin a doseand time dependentmannerafter cellswere exposedto recombinant interleukin-1 or tumor necrosisfactor a. In contrast, mRNA for a secretedform of the interleukin-1 receptorantagonistwasnot detectedunderbasalconditionsor after cellswerestimulatedby recombinant cytokines.Interleukin- 1 receptorantagonistprotein wasfound primarily in cell lysates; little interleukin 1 receptor antagonistprotein was secretedby the cells. The presenceof cell-associated interleukin-1 receptor antagonist was confirmed by immunocytochemistry. Levels of cell-associated IL-1 receptor antagonist protein were not significantly influenced by recombinant interleukin-1 or tumor necrosis factor a. Endogenousexpressionof interleukin-1 receptor antagonist may attenuate the effect of exogenousor endogenous interleukin-1, thus providing the RPEcell a meansof maintaininginterleukin1 homeostasis in ocular inflammatory disease. Key words: retinal pigment epithelium: interleukin-1 ; interleukin-lfi; interleukin-1 receptor antagonist; tumor necrosisfactor ; cytokines; growth factor ; mRNA ; polymerasechain reaction.

1. Introduction Interleukin-1 (IL-l) is a family of two functionally related proteins, IL-la and IL-l/3, that demonstrate a broad spectrum of effects in vitro and in many organ systems (Dinarello, 1988). Both IL-la and IL-l/3 are initially translated as intracellular precursors with a relative molecular mass(n/l,) of approximately 3 1000. Subsequently, mature products with nlr, of approximately 17 500 are releasedfrom the cell (Mosley et al., 1987; Hazuda, Lee and Young, 1988). In the eye, IL1 is found in the subretinal fluid of patients with retinal detachment (Davis et al., 1988) and is produced by retinal glial cells (Roberge, Caspi and Nusenblatt, 198 8) and limbal microvascular endothelial cells (Marceau et al., 1990). There is in vitro and in vivo evidence that IL-1 may contribute to ocular wound * For correspondence at: Duke UniversityEyeCenter,Box3802, Durham.NC 27710,U.S.A. 00144835/92/080325+11

$08.00/O

healing/inflammatory disease.For example, IL-l stimulates RPE chemotaxis (Kirchof and Sorgente, 1989) and causes intraocular inflammation (Rosenbaum et al., 1987) and retinal detachment (Carlisle et al., 1990) when injected into the vitreous cavity of rabbits. These activities are relevant to proliferative vitreoretinopathy (PVR), an inflammatory eye disease characterized by abnormal intraocular wound healing (Glaser and Lemor, 1988). A secretedIL-1 receptor antagonist (sIL-lra) (Arend et al., 1989; Hannum et al., 1990) and an intraceullar version (icIL- lra) (Haskill et al.. 199 1) have been recently described. The sIL-lra was originally identified in supernatents from IgG-stimulated monocytes (Hannum et al., 1990). It is a single peptide chain comprised of 152 amino acids and contains a leader sequence for secretion. Intracellular IL-lra is similar to sIL-lra but has an additional seven amino acids at the amino terminal, lacks a leader sequence for secretion, and is found preferentially in epithelial cells. 0 1992 AcademicPress’Limited

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Both forms are considered pure antagonists and are thought to inhibit IL-1 dependent activity at the level of the IL-l receptor (Arend et al., 1991; Haskill et al., 1991). The role of these receptor antagonists under physiologic and pathophysiologic conditions has not yet been fully characterized. The factors responsible for IL-l production and activity in ocular cells are unknown. Because IL-1 has been implicated in so many aspects of wound healing and the inflammatory response, it will be important to determine which ocular cells produce IL-l and to clarify the factors that regulate its activity. In this study we determined whether cultured human RPE cells express IL-lp and IL-l& mRNA and protein. We also determined whether RPE cells express IL-l receptor antagonist(s). We hypothesized that production of IL-1 could be influenced by exogenous cytokines such as tumor necrosis c1 (TNFa) and IL-1 produced in association with inflammation, and that concurrent expression of an IL-l receptor antagonist (IL-lra) could serve to maintain cellular homeostasis in response to these inflammatory mediators. 2. Materials

and Methods

Cell Culture Human cadaveric donor eyes were obtained from the North Carolina Organ Donor and Eye Bank within 24 hr of death. Cells were harvested from these eyes as previously described (Jaffe et al., 1990). Cells were grown in MEM with 10% fetal bovine serum (FBS) at 3 7°C in a humidified environment containing 5 % CO,. For experiments, second to fifth passage cells were seeded at a density of 3040000 cells ml-’ in 24-well culture plates or in 60-mm culture dishes and were grown to visual confluence. Purity of RPE cell cultures was confirmed by cytokeratin staining as previously described (Leschey et al., 1990). All cells used in these experiments formed a contact inhibited monolayer, exhibited polygonal morphology characteristic of RPE cells, and were stained uniformly for cytokeratins. RNA Extraction Cells were grown to visual confluence in 60-mm tissue culture dishes in MEM with 10% FBS. For experiments, cells were exposed to MEM + 10% FBS, alone or in combination with varying dosages of recombinant human IL- 1 a (Collaborative Research, Bedford, MA), IL-1B (Collaborative Research), TNFa (Amgen Biologicals, Thousand Oaks, CA), or lipopolysaccharide (LPS, 1 ,ug ml-l; Sigma, St Louis, MO) or cycloheximide ( 1 ,ug ml-‘, Sigma) at 3 7°C. 5 y0 CO, for varying time intervals. At the end of the incubation period, cells were rinsed twice with MEM, and total RNA was extracted as previously described (Chirgwin et al., 1979). RNA purity was determined by measand RNA quantity was estimated uring O%,/OD,,, from OD,,,.

ET AL.

cDAJA Synthesis Total RNA was converted to cDNA using a modification of a previously described technique (Kawasaki, 1990). One microgram of total RNA in 10 id of DEPC treated water was added to 10 /Al of reverse transcription mixture consisting of 2 j~l of 10 x PCR buffer [SO0 mM KCl, 100 mM Tris-HCl, pH 9, 15 mM MgCl, 0.01% (w/v) gelatin], 4 jr1 of 5 mM dNTP (Pharmacia LKB Biotech. Inc. Piscataway, NJ), 1 ,ul of Maloney Murine Leukemia virus reverse transcriptase (200 U ml-‘, BRL, Gaithersberg, MD. ), 1~1 of random hexamers (100 pg ml ‘. Pharmacia LKB Biotech. Inc), 0.5 td of RNasin (40000 U mll’ ; Promega, Madison, WI), and 1.5 1’1 of DEPC-treated water. This mixture was incubated for 45 min at 37°C and then 5 min at 90°C. cDNA was stored frozen at - 70°C. PCR Arnpli$cation One microliter of cDNA mixture was added to a PCR reaction mixture consisting of 1 x PCR buffer, 0.5 pmol dNTP, 5 pmol of IL-lp, IL-la, sIL-lra, icIL-lra or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer pairs, 1.2 5 U TAO polymerase (Promega), and distilled water in a total volume of 50 /rl. The sequences of the primer pairs have been previously described [IL-la, IL-lp (Wang, Doyle and Mark. 1989), icIL-lra, sIL-lra (Haskill et al., 1991). and GAPDH (Maier et al., 1990)]. The reaction mixture was overlaid with mineral oil and amplified in a PCR thermal cycler (Perkin-EImer/Cetus. Norwalk, CT) as follows: denaturation at 94°C for 1 min. primer annealing at 5 5°C for 2 min and extension at 72°C for 3 min. cDNA from plasmid pAWlO (Wang et al., 1989) was used as positive control PCR template for IL-lx and IL-l/j. A plasmid containing full-length cDNA for icIL- 1ra was used as icIL- 1ra positive contro1 template, and cDNA from activated macrophages was used as sIL- H-a positive control template. PCR reaction mixtures without the addition of cDNA template were used as negative controls. Positive control cDNA template was serially diluted and amplified with corresponding specific primer pairs in parallel with unknown samples. To verify that equivalent quantities of mRNA in unknown samples were reverse transcribed to cDNA, samples were also incubated with GAPDH primer pairs as previously described (Maier et al., 1990). To insure that PCR products were evaluated during the exponential phase of the amplification process, 10-/d aliquots were removed from the reaction mixture at different cycles. PCR products from the unknown samples and dilution series were run on a 2% agarose gel (Utrapure, BRL) with TBE (TBE: Tris. 2 1 g l-l, boric acid 11 g 1-l. EDTA 0.002 M, pH = 8 ) running buffer. Gels were stained with ethidium bromide and photographed with Polaroid film (type 55). The intensity of the bands was quantified using

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densitometric measurements obtained from the photographic negatives as previously described (Frye, Benz and Liu, 1989; Rychlik, Spencer and Rhoads, 1990). A high resolution black and white camera (Dage/MTI, Michigan City, IN) coupled to an image processor (Imaging Technology), driven by JAVA densitometric software (Handel Scientific, Corte Madera, CA) was used to perform the densitometric measurements. Relative differences in mRNA expression among samples were determined by comparison with the standard dilution curve as previously described (Van Le et al., 1990; Jaffe et al., 1991). Measurements obtained from the dilution series were fit to a regression line using Cricket Graph software on a Macintosh computer. The relative concentration of mRNA was determined by comparison with the calculated regression line. Restriction Enzyme Analysis Restriction enzyme analysis was used to confirm the identity of PCR products as previously described (Koos and Olson, 1989). IL-la, IL-lp, and icIL-lra PCR products have unique restriction sites for HinD III, Msp I, and Hga I, respectively. Samples of IL-la, n-l/3, and icIL-lra PCR products were digested with HinD III, A&p I and I-@ I, respectively. Restriction digests were run on agarose gels and bands were visualized with ethidium bromide to verify that the actual size of the bands corresponded to the predicted size. Collection of Conditioned Medium and Cell Lysates

RPE-conditioned medium and cell lysates were collected for measurement of protein secretion and cell-associated protein, respectively. Cells were grown to visual confluence in quadruplicate wells of 24-well tissue culture plates containing MEM and 10% FBS. Cells were rinsed twice with MEM and incubated for varying time intervals in the presence of 400 ~1 of MEMf 1% FBS alone or in combination &ith recombinant human IL-la (5 Uml-‘), IL-lp (5 U ml-‘), TNFcc (10 ng ml-‘), or lipopolysaccharide (1 ,ug ml-‘) as indicated below. Conditioned medium was removed for protein immunoassay then cells were rinsed twice with MEM and lysed by triple freeze-thaw in 400 ~1 of MEM+ 1 “/o FBS. Conditioned medium and cell lysates were centrifuged to remove particulate debris and stored at - 70°C until assayed. Cells were seeded in parallel for cell counts. 2 L-l cx and IL-1 /3 Protein Immunoassay

IL- 1a and IL- l@ protein in conditioned medium and cell lysates were measured by quantitative enzyme linked immunoassay (ELISA) as previously described (Kenny et al., 198 7 ; Masada et al., 1990). Monoclonal antibodies to IL- 1 a and IL-l/Y, generously provided by Marvin Masada, were used to detect iL-la and IL-la with a sensitivity of 6 pg ml-l (Kenney et al., 1987)

327

and 15 pg ml-’ (Masada et al.. 1990). respectively. These monoclonal antibodies detect both the 31 kDa precursor form and the 17 kDa mature form of IL-la and IL-l/3, respectively (Sandborg et al., 1989). Because these assays are not influenced by the presence of fetal bovine serum, they are useful for measuring IL-la. or IL-l/3 in tissue culture medium containing FBS (Kenney et al., 198 7). Results are expressed as pg protein per lo3 cells. Mature IL-la and IL-lp bind to the identical receptor and have equivalent biological effects (Dinarello and Schindler, 1990). To determine the effect of exogenous IL- 1 on XL-101and IL- 1 p protein production we used IL-la as an inducing agent for IL-lp protein production, and IL-l/? as an inducing agent for IL-la protein production. Because the ELISA for n-la does not detect IL-l/j’ and vice versa, this strategy insures that measurement of B,-lee and IL-lp in RPE cell lysates and conditioned medium represents cellular production rather than exogenous IL-l. IL-l Receptor Antagonist Protein Immunoassay

Interleukin- 1 receptor antagonist protein was measured by quantitative sandwich ELISA as previously described (Malyak et al., 1991). The affinity purified rabbit polycolonal antibody used for these assays detects both the secreted and intracellular form of the receptor antagonist but does not cross-react with either IL-la or IL-l/?. In this assay, the interleukin 1 receptor antagonist is detected with a maximum sensitivity of 250 pg ml-‘. Results are expressed as pg protein per 1 O3cells. The recombinant IL-lra used as a standard in this assay was obtained from Dr Robert C. Thompson, Synergen Inc., Boulder, CO. lmmunocytochemistry

To examine the distribution of IL-lra in cultured RPE, cells were seeded at 20000 cells ml-’ in gelatin coated &well glass chamber slides (LabTek, Napier IL). Cells were grown for 72 hr in MEM + 10 % FBS fixed in acetone, and stored at -20°C until use. Slides were air-dried and incubated with 0.3 Y0hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. They were rinsed with phosphatebuffered saline (PBS) then incubated with 1% normal goat serum in PBS for 20 min to block non-specific binding. Slides were incubated for 30 min with primary antibody to IL-lra (identical to that used in ELISA as described above), rinsed with PBS, then incubated for 30 min with a biotinylated goat antirabbit affinity-purified secondary antibody (Vector Laboratories, Burlingame, CA). They were rinsed with PBS, incubated for 30 min with Vectastain Elite ABC reagent (Vector Laboratories), rinsed with PBS, and incubated with the DAB substrate kit for horseradish peroxidase (Vector Laboratories) for approximately

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FIG. 1. PCR-cDNA gel showing IL-la mRNA expression in cultured human RPE. Amplification for 32 cycles. A, Cells exposed to varying concentrations of IL-lb (U ml-‘) or TNFa (ng ml-‘) for 4 hr. Lanes of dilution curve correspond to serial three-fold dilutions of pAWlO plasmid positive control cDNA template as indicated (undiluted plasmid cDNA = 03 pg ml-‘). B, Cells exposed to IL-l/1 ( 5 IJ ml-‘) or TNFa ( IO ng ml-‘) for varying time periods. Lanes of dilution curve correspond to serial threefold dilutions of pAWlO plasmid positive control cDNA template as indicated (undiluted plasmid cDNA = 0.3 pg ml-l). 4 min. Finally, slides were rinsed with tap water and cell nuclei were counterstained with methyl green. For negative controls, an antigen-absorbed antisera was used in place of the primary antibody. It was created by adding IL-lra protein to affinity-purified polyclonal rabbit IL-lra antibody. The mixture was placed on ice for 30 min and centrifuged. The supernatent was collected and diluted in PBS containing O-1 yO normal rabbit serum to a concentration similar to that used for the primary antibody.

3. Results Steady-state mRNA Expression of IL-I r and IL-7 p : Induction by /nflamrnatory Mediators IL- ICC and IL- l/j’ mRNA expression was measured in cell lines from six different donors. IL-la and IL-lp mRNA were not expressed by any of the six cell lines under basal conditions. Both IL-la: and IL- Ifi mRNA transcripts were readily induced in all six cell lines after exposure to IL-la (5 U ml-‘), IL-l/j (5 U ml-‘).

TNFcl (10 ng ml-‘), or LPS (1 pg ml-‘). The identity of the PCR products was confirmed by restriction enzyme analysis. Additional experiments were conducted to determine the dose and time dependency of IL-1 mRNA expression. Both IL-1~ and IL-l/1 mRNA were expressed in a dose and time dependent manner after exposure to IL-l/s and TNFr (Figs 1 and 2). IL-la expression was maximal 4 hr after exposure to IL-l/j and 2 hr after exposure to TNFa and declined thereafter [Fig. l(B)]. IL-11 mRNA expression was maximal 4-8 hr after exposure to IL-l/j’ and 2 hr after exposure to TNFa and declined thereafter [Fig. 2(B)]. The same samples amplified with GAPDH primer pairs produced bands of equivalent intensity. confirming that equivalent amounts of mRNA were reverse transcribed. E’ect of’ Cycloheximide on K-l

mRNA Expression

To determine whether induction of IL-la and IL-l/j mRNA expression depended on new protein synthesis,

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FIG. 2. PCR-cDNa gel showing IL-l/J mRNA expression in cultured human RPE. Amplification for 32 cycles. A, Cells exposed to varying concentrations of IL-lp (LJml-l) or TNFa (ng ml-‘) for 4 hr. Lanes from dilution curve correspond to serial threefold dilutions of pAWlO plasmid positive control cDNA template (undiluted plasmid cDNA = 0.3 pg mlkl). B, Cells exposed to IL-l/1 (5 U ml-‘) or TNFcr (10 ng ml-l) for varying time periods. Lanes from dilution curve correspond to serial three-fold dilutions of pAWI plasmid positive control cDNA template (undiluted plasmid cDNA = 0.3 pg ml-‘).

visually confluent RPE cells were stimulated for 4 hr with cycloheximide alone, or with recombinant IL-la in the presence or absence of cycloheximide (1 ,ug ml-‘). Cycloheximide alone caused induction of IL-la and IL-lb mRNA to a level that was comparable to that produced by IL-la alone. Cycloheximide significantly potentiated the inductive effect of IL-la. In cells treated with the combination of IL-la and cycloheximide, steady-state levels of both IL-la and IL-l/j’ mRNA were 14-fold higher than in cells treated with IL-k alone. IL-1 TX,IL-1 p Protein Production Levels of IL-la and I7;-1fl protein were assayed in RPE-conditioned medium and cell lysates under basal conditions and after cells were stimulated with IL-la or IL-l/?. K-la: was not detected in RPE cell lysates under basal conditions but was present by l-4 hr after exposure to IL-l/?. Maximum IL-la levels were measured 4-8 hr after exposure to IL-lb, and by 48 hr IL-la was no longer detectable (Fig. 3). Lipopoly-

FIG. 3. Levelsof IL-la protein in RPEcell lysatesfrom a representative cell line. Cells were stimulated with ILP (5 U ml-l) aloneor in combinationwith LPS(1 pugml-‘) for 1. 4, 8, 24 or 48 hr. Time 0 point obtainedby adding ILlp+LPS followedby immediateremoval. IL-la protein was undetectableat time 0, and 1 hr and 48 hr after stimulation with IL-l/3. Valuesrepresentmean+s.n of quadruplicate samples.(a) ILlP: (m) IL-lp+LPS.

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FIG. 4. PCR-cDNA gel showing icll-lra mRNA expression in cultured human RPE Amplification for 32 cycles. A, Cells exposed to varying concentrations of IL-~/J’ (U ml-l) or TNFa (ng ml-l) for 4 hr. Lanes from dilution curve correspond to serial ten-fold dilutions of icIL-lra plasmid positive control cDNA template (undiluted plasmid cDNA = 2.2 pg ml-‘). B, Cells exposed to IL-l@ (5 U ml-l) or TNFa. (10 ng ml-l) for varying time periods. Lanes from dilution curve correspond to serial ten-fold dilutions of icIL-lra plasmid positive control cDNA template (undiluted plasmid cDNA = 2.2 pg rnlkl).

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CM under any of the conditions tested. Although IL-18 mRNA was readily induced by IL-la or LPS, IL-18 protein was not detected in cell lysates or in RPE-CM under basal conditions or after stimulation by IL-1~. LPS or IL-la combined with LPS.

Steady-state rnRNA Expression oJ K-1 Receptor Antagonists

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FIG. 5. Levels of IL-1 receptor antagonist protein in RPE cell lysates from a representative cell line. Cells in duplicate wells were stimulated with IL-Q? (5 U ml-‘) or (10 ng ml-l) for 1. 4. 8, 24 or 48 hr. Time 0 point obtained by adding IL1B or TNFa followed by immediate removal. Values represent mean ( +s.D.). (H) IL-lb stimulated: (m) TNF stimulated.

saccharide alone did not induce IL-la, but potentiated the stimulatory effect of IL-lb at all time points tested (Fig. 3). In contrast, IL-la was not secreted into RPE-

Intracellular IL-l receptor antagonist mRNA expression was measured under basal conditions in three of three cell lines tested. The identity of the icIL-lra PCR product was verified by restriction enzyme analysis. mRNA expression was enhanced in a dose and time dependant manner after exposure to.recombinant IL-1p and TNFa (Fig. 4). Expression was maximal 4 hr after exposure to IL-1p and 4-8 hr after exposure to TNFcw. and declined thereafter. In contrast, in the same three cell lines, sIL-lra mRNA was not expressed significantly under conditions sufficient to induce expression of IL-la. IL-l/J, and icIL-lra (not shown). The same samples amplified with GAPDH primers yielded bands of equal intensity, verifying that

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FIG. 6. A. Phase contrast microscopy showing immunohistochemical staining of cultured human RPE using affinity pu.rified antibody to IL-lra. Arrow corresponds to cell nucleus stained with methyl green. B, Negative control: staining conditions were the same as (A) except that affinity purified IL-lra antibody pre-incubated with recombinant IL-lra protein was substitute ed for affinity purified IL-1 ra antibody. Arrow corresponds to cell nucleus stained with methyl green, Original magnification x 625.

equivalent amounts of mRNA were reverse transcribed among samples. 1.b1 Receptor Antagonist Protein Production

IL-lra protein production was assayed in three different cell lines by ELISA. IL-lra was detected in cell lysates from unstimulated cells in all three cell lines.

The amount of protein in Iysates averaged 1.9 pg 1o-” cells (range: 0.6-3.8 pg lo-” cells). In contrast, nnuch smaller quantities of IL-lra were detected in conditioned medium. In two cell lines. IL-lra was: not detected in conditioned medium at anv time teste d. In one cell line, low levels (0.18-0.8 pg 1 Oe3cells) (If ILlra were measured by 24 hr. Although IL-lp and TNFa caused increased IL-lra mRNA expression, they

332 did not alt.er IL-lra protein in a statistically significant manner in any cell line tested. Figure 5 shows results from a representative experiment. The presence of IL-lra in RPE cells was confirmed immunocytochemically in three of three cell lines tested. Virtually all unstimulated cells stained positively for IL-lra and for the nuclear counter stain methyl green [Fig. 6(A)]. Although the intensity of staining varied among cells, immunoperoxidase positive granules were distributed evenly without a given cell. It was not possible to distinguish significant qualitative differences in staining intensity among unstimulated cells and cells stimulated up to 24 hr with IL-l/3. In contrast, in negative controls, cell nuclei were positively stained with methyl green, but cells did not stain positively for IL-lra [Fig. 6(B)].

4. Discussion In the current study we have shown that human RPE cells do not produce IL-l a or IL-Ip mRNA under basal conditions, but IL-la and IL-l/? transcripts are rapidly induced after cells are exposed to inflammatory mediators. Similarly, fibroblasts do not express IL-lp mRNA basally, but do so when stimulated by TNFa. Unlike RPE, however, fibroblasts do not express IL-la transcripts under conditions which induce accumulation of IL-l/j mRNA (Yamamoto, El-Hajjaoui, and Koeffler, 1989). In other cells adherence alone is sufficient to induce IL-1 mRNA. For example, peripheral human blood monocytes adhered to plastic express IL-lp transcripts without additional inflammatory stimuli (Haskill et al., 1988). Other cell-types including keratinocytes (Kupper et al., 1986) and astrocytoma cells (Lee, Simon and Young, 1989) express both IL-la and IL-l/3 mRNA constitutively, while melanoma cells express only IL-la transcripts constitutively (Kock et al., 1989). Thus, control of mRNA expression is cell-type specific and may reflect the different roles of these cells normally and pathologically. The kinetics of IL-l SIand IL-l/j mRNA accumulation in human RPE are similar to that found in other cell types. For example. in human monocytes, IL-1 mRNA is detected by 1 hr after stimulation with LPS, and was maximal by 6 hr (Matsushima et al., 1986). Similarly, in fibroblasts, IL-l mRNA is expressed within an hour of exposure to inflammatory mediators (Yamamoto et al., 1989). Rapid induction of IL-1 mRNA would provide the cell with a means to regulate IL-l production in response to brief exposures to exogenous inflammatory stimuli. New protein synthesis was not necessary for induction of increased IL-l& and IL-lg mRNA. The addition of cycloheximide to IL-la treated cells markedly enhanced, rather than inhibited levels of ILICC and IL-l/J mRNA expression. It is possible that cycloheximide could block synthesis of IL- 1 ra protein,

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thereby increasing the effective concentration of exogenous IL- 1 added to cells to stimulate IL- 1 mRNA expression. It is unlikely that the ‘superinductive ’ effect observed with cycloheximide was mediated through the IL-lra, however, because cycloheximide alone, without exogenous IL-1 was sufficient to induce expression of IL-la and IL-lg mRNA. The superinductive effect of cycloheximide in RPE cells is of greater magnitude, but otherwise similar to that observed in human fibroblasts (Yamamoto et al., 1989). Cycloheximide mediated superinduction of macrophage colony stimulating mRNA has also been reported in human monocytes (Horiguchi, Sariban and Kufe, 1988). It has been hypothesized that cycloheximide inhibits a protein that destabilizes mRNA (Horiguchi et al., 1988). This explanation could account for the ‘ superinductive effect ’ observed in the current study. We had originally hypothesized that IL-1 secreted by RPE cells might influence cellular behaviour in an autocrine or paracrine manner. However, we found that neither IL-la nor IL-l/3 protein were secreted by RPE cells despite the presence of IL-1% and IL- 111 mRNA. Rather, IL-la was sequestered within the cells, while IL-l/j was not detected at all. Similarly, keratinocytes preferentially produce IL- 1 x and sequester most of it intracellularily (Kupper et al., 1987 ; Blanton et al., 1989). Melanoma cells (Kock et al., 1989) and astrocytoma cells (Lee, Simon and Young, 1989) also preferentially produce IL- 1x. but unlike RPE. these cells release significant quantities into their media. It is possible that RPE lack the cellular machinery necessary for IL- 1z secretion. Alternatively, secretion may require a ‘second signal ‘, In peripheral blood monocytes, while adherance is sufficient to induce mRNA transcription. secretion requires a second signal such as LPS (Haskill et al., 1988). In RPE, the nature of such a second signal is unclear, but presumably it would differ from signals like LPS since LPS alone or in combination with IL-l/? did not induce RPE cells to secrete IL-la. Because IL-la was sequestered and not secreted by RPE cells. our data raise the possibility that IL-l;1 influences cells in an ‘intracrine ’ manner. A similar role for IL-l as an intracrine modulator in keratinocytes has previously been proposed (Blanton et al.. 1989). Recently, it was shown that antisense oligonucleotides to IL-la inhibited in vitro senescence of human endothelial cells (Maier et al.. 1990). This report provided the first evidence that IL- 1 z could modulate cell activities at an intracellular level. The role of intracellular IL-lx in human RPE remains to be determined. The ELISA used in the current study detects both the precursor and processed forms of ILICC. and thus is a measure of the total amount of biologically active IL-1 a, IL-1s. unlike 11,-l/1. is biologically active in its intracellular precursor form (Mosley et al., 1987; Young, Hazuda and Simon, 1988). Because intracellular IL-1 a was induced by

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exogenous inflammatory cytokines, we hypothesize that it may serve to influence RPE activities in ocular inflammatory diseases such as PVR and uveitis. In RPE cells, induction of IL-1 mRNA expression and synthesis of IL-1 protein requires the presence of inflammatory mediators such as IL-l and TNFa. If IL1 produced by RPE cells is to be physiologically or pathophysiologically relevant, an ocular source of ‘inducing agent ’ is required. In inflammatory diseases such as PVR, vitreous levels of TNFcl and other cytokines are increased above normal (Little et al., 199 1). Retinal glial cells produce IL- 1 (Roberge et al,, 1988) and subretinal fluid may be a further source of IL-1 (Davis et al., 1988). Recently, it was shown that GM-CSF transgenic mice have severe ocular inflammation characterized by influx of macrophages. These macrophages express IL-la and TNFcr mRNA. PVR membranes contain macrophages and retinal glial cells in close proximity to RPE. Thus, in ocular inflammatory diseases such as PVR. increased levels of IL-1 and TNFa produced by glial cells and macrophages could induce IL-1 in RPE in a paracrine manner. Whether other conditions or mediators are sufficient to induce IL-1 production in RPE remains to be determined. Inhibition of IL-1 activity may occur at various locations. IL-l signalling is initiated when mature IL1 binds to a cell membrane receptor (Dinarello, 198 8 ). It is then internalized and accumulates in the cytosol, eventually localizing to the nuclear membrane or nucleus (Mizel et al., 1987; Curtis et al., 1990). sILlra blocks IL-l function by binding to IL-l receptors and probably inhibits binding at the cell membrane level (Eisenberg et al., 1990). It has been postulated that icll-lra inhibits intracellular IL-1 activity (Haskill et al., 1991). In preliminary studies. we found that RPE cells expressed mRNA for the intracellular but not secreted form of the IL-l receptor antagonist (Haskill et al., 1991). In the current study, we have expanded these observations to show that mRNA expression of the intracellular receptor antagonist is enhanced by exposure to IL-la and IL-l/J. However, IL-lra protein is produced constitutively and is not significantly influenced by exogenous cytokines. It is possible that we were unable to measure small increases in IL-lra production in response to IL-l and TNFa. Alternatively, increased translation and/or secretion of IL-lra may require additional factors that have yet to be identified. IL-l receptor antagonist was found primarily in RPE cell lysates. Although the ELISA and immunocytochemical techniques used in these studies do not discriminate between icIL- 1 ra and sIL-lra, the mRNA data together with the protein data suggest that the cells produce mostly icIL-lra. In RPE cells, icIL-lra may thus function to inhibit the activity of endogenous IL-la produced in response to inflammatory mediators, thereby providing the RPE cell a means of maintaining IL-1 homeostasis in ocular inflammatory disease.

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The levels of IL-l receptor antagonist in RPE cells were lower than those measured in keratinocytes (Haskill et al., 1991). Because keratinocytes constitutively produce high concentrations of cell associatedIL-1 (Kupper et al., 1987; Blanton et al., 1989), higher concentrations of IL-1 receptor antagonist may be required in these cells to regulate intracellular IL-1 mediated events. The quantity of IL-1 receptor antagonist in RPE cell lysates were similar to the levels of IL-la. In contrast, when exogenous IL-l-receptor antagonist is administered in vivo or in vitro, a tenfold or greater excess is required to block IL- 1 mediated activities (Arend et al., 1991). High extracellular IL-1 receptor antagonist concentrations may be required because cells express excess n-1 receptors on the cell surface (Dower and Urdal, 1987). It is likely that lower intracellular levels of IL-1 receptor antagonist may be required to block IL-1 activity, because only receptors internalized in response to activation by IL- 1 would be subject to inhibition by intracellular IL-l receptor antagonist. Future studies are needed to clarify the functional role of intracellular IL- 1 receptor antagonist in RPE, and the quantity necessary to block IL-1 mediated events. Acknowledgements This investigation was supported in part by NIH grant EY0916-1 (D-Jaffe), Research to Prevent Blindness Inc., and the Adler Foundation. Dr Jaffe is a Robert McCormack Scholar. Alice Wang generously provided plasmid AW108.

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