BOL-303242-X, a novel selective glucocorticoid receptor agonist, with full anti-inflammatory properties in human ocular cells

Materials

EpiLife medium and human corneal growth supplement were purchased from Cascade Biologics (Portland, OR). Fibroblast Medium and fibroblast growth supplement were purchased from ScienCell (San Diego, CA). CS-C medium and acidic fibroblast growth factor (aFGF) were purchased from Cell Systems (Kirkland, WA). RPMI-1640 medium and DMEM medium were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was purchased from Hyclone (Logan, UT). Charcoal/Dextran-treated fetal bovine serum (CD-FBS) and Micro BCA protein assay kit were purchased from Fisher Thermo Scientific (Waltham, MA). Dexamethasone (DEX), triamcinolone acetonide (TA), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), and proteinase inhibitor cocktail were from Sigma (St. Louis, MO). BOL-303242-X (ZK 245186; R-1,1,1-trifluoro-4-(5-fluoro-2,3-dihydrobenzofuran-7-yl)-4-methyl-2-{[(2-methyl-5-quinolyl)amino]methyl}pentan-2-ol) was provided by Bayer Schering Pharma (Berlin, Germany). IL-1ß was from R&D Systems (Minneapolis, MN). Alamar Blue solution was from Biosource (Camarillo, CA). Human multiplex-cytokine and sICAM-1 Luminex kits were obtained from Millipore (Billerica, MA). Antibodies for the western blotting were purchased from Cell Signaling Technology (Danvers, MA), Santa Cruz Biotech (Santa Cruz, CA), Zymed (San Francisco, CA), and Millipore. All other reagents were purchased from standard commercial sources and were of the highest available quality and purity.

Cells and cell treatments

Primary HCEpiCs were purchased from Cascade Biologics and maintained in EpiLife medium containing human corneal growth supplement (bovine pituitary extract 0.2% [v/v], bovine insulin [5 µg/ml], hydrocortisone [0.18 µg/ml], bovine transferrin [5 µg/ml], and mouse epidermal growth factor [1 ng/ml]). Primary HConFs were purchased from ScienCell and maintained in fibroblast medium (FM) containing fibroblast growth supplement (undisclosed formulation) and 2% FBS. Primary HRECs were purchased from Cell Systems and maintained in CS-C medium containing acidic fibroblast growth factor (aFGF) and 10% FBS. THP-1 cells were purchased from ATCC (Manassas, VA) and maintained in RPMI-1640 medium containing 10% FBS. HONAs were isolated according to Yang & Hernandez [23], characterized by immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) antibody with 90–95% positive cells, and maintained in astrocyte defined serum-free medium (ADSF) containing 3% CD-FBS and G5 astrocyte supplement (insulin [5 ng/ml], human transferrin [50 ng/ml], selenite [5.2 pg/ml], biotin [10 pg/ml], hydrocortisone [3.6 pg/ml], FGF [5 pg/ml], and EGF [10 pg/ml]). These cells were all cultured in the above media, containing 100 U/ml of penicillin and 100 µg/ml of streptomycin, at 37 °C in a humidified incubator with 5% CO₂. Prior to treatments, cells were seeded in 24-well plates and cultured until confluence. Cells were pretreated with test agents (BOL-303242-X, DEX, or TA) for 2 h, and then further treated with either vehicle (0.1% DMSO), LPS, IL-1ß, BOL-303242-X, DEX, TA, or combinations of them in basal medium (HCEpiCs and HONAs) or in basal medium plus CD-FBS (HConF, HREC, and THP-1) for 18 h. Each treatment was performed in triplicate. After conditioned media were collected for the cytokine assay by Luminex, cells were used for the Alamar Blue assay. For the ICAM-1 and IL-1ß assays of HRECs, cells were lysed in NP-40 lysis buffer (PBS containing 0.1% NP-40 and 10 µl/ml proteinase inhibitor cocktail), transferred to Eppendorf tubes, and sonicated. After centrifugation at 10,000× g for 5 min, the supernatant from each triplicate sample was transferred to a fresh Eppendorf tube for IL-1ß and ICAM-1 Luminex and protein measurements.

Multiplex Luminex technology

Cytokine content in the culture medium or cell lysate was analyzed using multiplex Luminex technology [24-26]. Up to 26 cytokines (Eotaxin; Fractalkine; G-CSF; granulocyte macrophage colony-stimulating factor [GM-CSF]; IL-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12(p40); IL-12(p70); IL-13; IL-15; IFN-γ; interferon-inducible protein-10 [IP-10]; MCP-1; macrophage inflammatory protein-1β [MIP-1α]; transforming growth factor-α [TGF-α]; TNF-α, regulated upon activation, normal T-cell expressed, and secreted [RANTES]; and vascular endothelial growth factor [VEGF]) were measured according to the manufacturer's instructions. Data were analyzed using a Luminex 200 (Luminex, Austin, TX) and Beadview software v1.0 (Upstate Cell Signaling Solutions, Temecula, CA). Standard curves for known concentrations of recombinant human cytokines were used to convert median fluorescence intensities (MFI) to cytokine concentrations in pg/ml. Only the linear portions of the standard curves were used to quantify cytokine concentrations, and in instances where the fluorescence reading exceeded the linear range of the standard curve, an appropriate dilution was performed to ensure that the concentration was in the linear portion of the curve. IL-1ß and ICAM-1 concentrations in the cell lysate were normalized based on the protein content of the sample and expressed as pg/mg protein.

Western blot analysis for IκB-α, NFκB, and MAPKs

Cells were pretreated with vehicle, BOL-303242-X or DEX for 2 h, and then further treated with vehicle, IL-1ß, or either IL-1ß plus BOL-303242-X or DEX in basal EpiLife medium for 10 min or 30 min. Cells were washed with ice-cold PBS and lysed in cell lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol, and 1:1,000 proteinase inhibitor cocktail). Cells were sonicated, and centrifuged at 12,000 rpm. Protein concentration was determined using the Micro BCA protein assay kit. Proteins in aliquots of cell lysate (approximately 30 µg protein) were separated by SDS-polyacrylamide electrophoresis (SDS-PAGE) on 10% gels and transferred to nitrocellulose membranes. Membranes were blocked with 1% BSA and exposed to rabbit anti-phospho-p38 (Thr180/Tyr182) antibody (Cell Signaling), rabbit anti-phospho-p65 NFκB (Ser536) antibody (Cell Signaling), rabbit anti-IκB-α antibody (Santa Cruz Biotech), or rabbit anti-phospho-JNK (Thr183/Tyr185) antibody (Santa Cruz Biotech). The blots were washed, and exposed to horseradish peroxidase-conjugated anti-rabbit secondary antibody. After washing, blots were incubated in enhanced chemiluminescence solutions and chemiluminescent bands were visualized using the FluorChem imaging system (Alpha Innotech, San Leandro, CA) [25]. Blots were then stripped and re-probed as appropriate with rabbit anti-p38 antibody (Cell Signaling), rabbit anti-JNK antibody (Santa Cruz), rabbit anti-p65 NFκB antibody (Cell Signaling), or mouse anti-keratin K3/K76 antibody (Millipore; a specific marker of corneal epithelial cells), as loading controls. The experiment was repeated three to four times. Analysis of western blot band density for phosphorylated and non-phosphorylated p65 NFκB, JNKs, and p38, as well as keratin K3/K76 in captured digital images, was done using the Alpha-Innotech Chemi-Imager software (Alpha Innotech). Levels of phosphorylated p65 NF-κB, JNKs, and p38 were normalized to non-phosphorylated p65 NF-κB, JNKs, and p38 from the same sample on the same membrane. Levels of IκB-α protein were normalized to keratin K3/K76. For quantitation of JNK phosphorylation, the densities of two JNK isoforms, JNK1 (46 kDa) and JNK2/3 (54 kDa), were determined separately and then summed together. The experiment was repeated three to four times.

Cellular metabolic activity

Cellular metabolic activity was determined by the Alamar Blue assay [27]. Briefly, after removal of medium, cells were incubated with 1:10 diluted Alamar Blue solution for 3 h at 37 °C in a humidified incubator with 5% CO₂. The plate was read fluorometrically by excitation at 530–560 nm and emission at 590 nm. Relative fluorescence units (RFU) were used to determine cell metabolic activity.

Data analysis and statistics

Data were expressed as means±SEM unless otherwise indicated. Statistical analysis was conducted after evaluating the data for compliance with normality and variance homogeneity criteria. If these criteria were not met, data were Box-Cox-transformed and then the analysis was completed on the transformed data. Comparison of treatment effects across groups was performed using two-way analysis of variance (ANOVA) followed by the Tukey-Kramer test (JMP 7 software, SAS Institute, Cary, NC) on either raw or Box-Cox-transformed data [28,29] using either LPS or IL-1ß treatment as a reference. For all assays, a p≤0.05 was predetermined as the criterion of statistical significance. A Student’s t-test on either raw or Box-Cox-transformed data [28,29] was used to determine whether LPS or IL-1ß was effective at increasing cytokine release compared to the vehicle control. Dose-response curve data were fitted to a re-parameterized four-parameter logic equation [30], and this equation, below, permitted estimation of the IC₅₀ values for each drug treatment.

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BOL-303242-X demonstrates activity similar to classical glucocorticoids in inhibiting LPS- or IL-1ß-induced cytokine release in four different primary human ocular cells

HConFs, HCEpiCs, HONAs, and HRECs were pretreated with the test agents, BOL-303242-X, dexamethasone (DEX), or triamcinolone acetonide (TA), for 2 h, and then further treated with vehicle, LPS, IL-1ß, test agents, LPS plus test agents, or IL-1ß plus test agents for 18 h. No statistically significant effects of BOL-303242-X or dexamethasone on cellular metabolic activity, an index of cell viability, were observed in the four cell types studied. Under basal conditions (without LPS or IL-1ß stimulation), cytokine levels in the conditioned medium were very low, with almost all the cytokines studied below the limits of detection by Luminex technology. Furthermore, no statistically significant effect on cytokine release from either BOL-303242-X or the two glucocorticoids was observed.

BOL-303242-X significantly inhibited G-CSF, IL-6, IL-8, and MCP-1 release from HConFs at doses of 1–100 nM in a dose-dependent manner (Figure 1). The effects of BOL-303242-X were similar to those of DEX, with estimated IC₅₀ values of 1 nM for DEX versus 7 nM for BOL-303242-X with G-CSF, 5 nM for DEX versus 2 nM for BOL-303242-X with IL-6, 10 nM for DEX versus 4 nM for BOL-303242-X with IL-8, and 18 nM for DEX versus 7 nM for BOL-303242-X with MCP-1. The differences observed in IC₅₀ between BOL-303242-X and DEX were not considered statistically significant because confidence limits on these values overlapped for all cytokines tested. Furthermore, a significantly higher activity of BOL-303242-X was observed when compared to DEX at the 1 nM, 10 nM, 1 µM, and 10 µM doses with IL-6 (Figure 1). In addition, there was a greater inhibition of MCP-1 release by BOL-303242-X as compared to DEX at 10 µM (Figure 1). Together, these data demonstrate the full anti-inflammatory properties of BOL-303242-X in HConFs.

In HCEpiCs, BOL-303242-X significantly inhibited IL-1β-induced IL-6 release at 10 nM and TNF-α release at 100 nM, in a dose-dependent manner (Figure 2). Whereas at lower doses, DEX induced significantly greater effects than BOL-303242-X for TNF-α, both compounds showed similar activity at reducing IL-6 and TNF-α levels at the highest doses tested (Figure 2), indicating the full agonist activity of the compound as an anti-inflammatory agent.

In HONAs, both BOL-303242-X and TA inhibited LPS-induced IL-6 and IL-8 release, and a statistically significant inhibitory effect on IL-6 and IL-8 was observed at doses as low as 10 nM (Figure 3). At this dose, the activity of both compounds was very close to maximal, making it difficult to estimate IC₅₀ values (<10 nM). In HONAs, BOL-303242-X behaved as a full anti-inflammatory agent, with efficacies comparable to those observed for TA in these cytokines (Figure 3), as seen in HConFs and HCEpiCs.

BOL-303242-X and TA both inhibited LPS-induced increases in cellular levels of IL-1ß and ICAM-1 in HRECs, and a statistically significant reduction of LPS-induced increases in the cellular levels of IL-1ß and ICAM-1 was observed at doses ranging from 0.1 to 10 µM (Figure 4).

BOL-303242-X reduces LPS-induced cytokine release in THP-1 cells

Monocytes play a critical role in the inflammatory process; therefore, the effect of BOL-303242-X on cytokine release in human THP-1 monocytes was also evaluated. Both BOL-303242-X and DEX inhibited LPS-induced IL-6, IL-12p40, and MCP-1 release in a dose-dependent manner, and a statistically significant inhibitory effect on IL-6, IL-12p40, and MCP-1 release was observed at 1 nM (Figure 5). DEX was slightly more efficacious than BOL-303242-X (86% compared to DEX for IL-6, 75% compared to DEX for IL-12p40, and 93% compared to DEX for MCP-1). However, the estimated IC₅₀s for both drugs with these cytokines were comparable (2 nM for DEX versus 2 nM for BOL-303242-X with IL-6, 2 nM for DEX versus 2 nM for BOL-303242-X with IL-12p40, and 1 nM for DEX versus 1 nM for BOL-303242-X with MCP-1). Again, the differences observed in IC₅₀ between BOL-303242-X and DEX were not considered statistically significant because confidence limits on these values overlapped for all cytokines tested.

BOL-303242-X reduces MAPK phosphorylation/activation in HCEpiCs

To investigate the potential mechanisms underlying the anti-inflammatory activity of BOL-303242-X, its effects on the MAPKs p38 and JNK were determined in HCEpiCs. Immunoblotting was performed using phospho-specific antibodies against JNKs at Thr183/Tyr185, and against p38 at Thr180/Tyr182. These sites have been demonstrated to be associated with the activation state of these proteins [31]. Treatment of HCEpiCs with IL-1β for 30 min significantly increased the phosphorylation state of JNK and p38 MAP kinases (Figure 6). IL-β-induced phosphorylation of p38 and JNKs was significantly reduced by BOL-303242-X or DEX at both 1 µM and 10 µM doses (Figure 6). The activity of both compounds was comparable (107% compared to DEX at 1 µM [100%] for p38, 128% compared to DEX at 10 µM [100%] for p38, 121% compared to DEX at 1 µM [100%] for JNKs, and 126% compared to DEX at 10 µM [100%] for JNKs).

BOL-303242-X reduces NFκB phosphorylation and increases levels of IkB in HCEpiCs

The effect of BOL-303242-X on the NFκB signal transduction cascade was investigated by measuring NFκB phosphorylation and IκB-α degradation. Treatment of HCEpiCs with IL-1β for 10 min significantly increased the phosphorylation state of p65 NFκB at Ser536, a site that has been demonstrated to be associated with NFκB’s activation state [32,33]. Under basal conditions, NFκB is prevented from entering the nucleus by its interaction with IκB; therefore, a key step in the activation of NFκB is the degradation of IkB [33]. After IL-1β treatment, there was a significant decrease in cellular IκB-α levels, consistent with IκB-α degradation. IL-β-induced p65 NFκB phosphorylation at Ser536 was significantly reduced by 10 µM BOL-303242-X and both 1 µM and 10 µM DEX (Figure 7). Moreover, 10 µM BOL-303242-X and DEX significantly increased the levels of IκB-α in the cell lysate (Figure 7). These effects would be expected to result in an effective reduction of the ability of NFkB to induce inflammation by combining these two mechanisms; i.e., there would be reduction of transcription factor phosphorylation and elevation of levels of the inhibitor IκB.