Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist

Reagents

(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 (mapracorat; molecular weight 462.48) was provided by Bausch & Lomb (Rochester, NY), dexamethasone 21-phosphate disodium salt (dexamethasone) was obtained from Sigma-Aldrich (Steinheim, Germany), and mifepristone was purchased from Tocris Bioscience (Bristol, UK). For in vitro studies, mapracorat, dexamethasone, and mifepristone were dissolved in ethanol (10⁻² M) and further diluted as necessary in cell culture medium. The vehicle was cell culture medium containing 10 μl/ml of ethanol. For in vivo studies, mapracorat eye drops were provided by Bausch & Lomb and further diluted in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ x 2 H₂O, 1.76 mM KH₂PO₄, pH 7.4); dexamethasone was dissolved in PBS.

Roswell Park Memorial Institute-1640 (RPMI-1640) plus L-glutamine, penicillin, streptomycin, Alexa Fluor^® 488 and 568 conjugated secondary antibody, Hank’s balanced salt solution (HBSS), and MagicMark^TM XP Western Standard were purchased from Invitrogen (Carlsbad, CA). PBS, Iscove’s modified Dulbecco’s medium, and heat inactivated fetal bovine serum (FBS) were purchased from Lonza Group Ltd. (Basel, Switzerland). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained from R&D Systems (Minneapolis, MN). Interleukin-5 (IL-5), mouse monoclonal anti-chemokine (C-X-C motif) receptor 4 (CXCR4) antibody, mouse IgG₁ (isotype control), ionomycin from Streptomyces conglobatus, anti-β-actin antibody, BSA (BSA), ovalbumin (OVA) grade V, aluminum hydroxide gel, o-phenylenediamine, 30% hydrogen peroxide, Triton X-100, and peroxidase acidic isoenzyme from horseradish were obtained from Sigma-Aldrich. Nuclear and Cytoplasmatic Extraction Reagents kit, bicinchoninic acid (BCA) protein assay, and SuperSignal West Pico chemiluminescent substrate were bought from Pierce (Rockford, IL). Protran^TM was obtained from Whatman^® (Kent, UK). Anti-caspase-3 antibody was purchased from Cell Signaling (Danvers, MA). Mouse monoclonal anti-annexin I and peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Annexin V-Fluos was obtained from Roche Applied Science (Monza, Italy). Platelet-activating factor (PAF; 1-O-Octadecyl-2–0-acetyl-sn-glycero-e-phosphoryl choline) was purchased from Sigma-Aldrich and dissolved in ethanol (2 mM) and further diluted in RPMI 1640 containing 0.1% BSA. Polyacrylamide gel, N,N,N',N'-tetramethylethylenediamine, ammonium persulfate hematoxylin-Bierbrich scarlet solution, lithium carbonate, and sodium dodecyl sulfate were purchased from Sigma-Aldrich. All other reagents were of analytical grade or the highest purity available, purchased from Sigma-Aldrich. All plastic disposables were from Sarstedt (Verona, Italy).

Cell culture

Human eosinophils, isolated from whole blood by density centrifugation followed by negative selection using immunomagnetic anti-CD16 beads (purity and viability were >95%), were purchased from 3H Biomedical AB (Uppsala, Sweden) and routinely cultured in RPMI 1640 supplemented with 10% FBS, antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin), GM-CSF (70 pM), and IL-5 (30 pM). Before each experiment, cells were maintained in RPMI 1640 medium containing 0.1% FBS and in the absence of GM-CSF and IL-5.

A human mast cell line (HMC-1) [20], obtained from Pio Conti (University of Chieti, Chieti, Italy), was grown in Iscove’s modified Dulbecco’s medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO₂ in air at 37 °C.

Animals

Male Dunkin-Hartley guinea pigs (250–300 g) were purchased from Charles River (Calco, Italy). Animal procedures were performed in accordance with the Declaration of Helsinki, followed the guidelines of the University of Bologna Animal Care and Use Committee, and were comparable to those published by the Institute for Laboratory Animal Research.

Eosinophil cell apoptosis

To assess glucocorticoid-induced apoptosis, cells were double stained with annexin V-Fluos and propidium iodide (PI). Annexin V-Fluos was used according to the manufacturer’s instructions. Briefly, the cells were washed in PBS and suspended in annexin V-Fluos labeling solution (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl₂) with PI added (1 µg/ml). The suspension was incubated at room temperature for 10 min and analyzed in the BD FACS Canto II flow cytometry system (Becton, Dickinson and Company, Franklin Lakes, NJ). Eosinophils were gated on the basis of their forward and side light scatter, with cell debris excluded from analysis. Cells showing positive staining with annexin V (i.e., both early apoptotic annexin V⁺/PI^- cells and late apoptotic/secondary necrotic cells annexin V⁺/PI⁺) were considered to be apoptotic. A two-way dot plot was prepared to verify the percentage of apoptotic cells. Annexin V^-/PI^- cells were used as control, and annexin V^-/PI⁺ cells were considered necrotic [21].

Flow cytometry

Fluorescence-activated cell sorting (FACS) was performed to measure cell surface expression of annexin I and CXCR4 as indicators of glucocorticoid-mediated transactivation [22]. Human eosinophils were double-stained with a red dye-conjugated secondary antibody to trace changes in the expression of CXCR4 or annexin I and a green dye-conjugated annexin V to exclude apoptotic cells from the analysis. The cells were counted and transferred to a 24-well plate (10⁶ cells/well) and serum starved (0.1% FBS) for 24 h. They were then exposed to dexamethasone or mapracorat for 24 h at 37 °C in 5% CO₂ plus air.

At the end of the incubation, the cells were harvested and each sample was divided into two tubes to run parallel tests for annexin I and CXCR4 surface expression. After rinsing all samples with an HBSS solution containing 1% BSA, the cells were incubated for 45 min on a shaker with anti-annexin I or anti-CXCR4 antibodies (1:200) on ice; the negative control was incubated with an isotype-specific control antibody.

The cells were then washed twice with HBSS/BSA buffer before exposure to the Alexa Fluor® 568-conjugated secondary antibody. The excess of unbound antibody was washed away, and all samples were incubated for 15 min in the presence of annexin V-Fluos. The cells were then rinsed and resuspended in HBSS/BSA buffer and were ready for analysis in the BD FACS Canto II flow cytometry system. Electronic gates were set on annexin V-negative cells and CXCR4 or annexin I-positive cells. Data from 10,000 cells/sample were analyzed using dedicated software (Becton, Dickinson and Company). The percentage of CXCR4 or annexin I-positive cells was calculated [22].

Western blotting

Human eosinophils were centrifuged and resuspended in 100 µl of Cytoplasmatic Extraction Reagent (CER) I buffer (included in the Nuclear and Cytoplasmatic Extraction Reagents kit). After 10 min incubation on ice, 5.5 µl of CER II buffer was added and the suspension was resuspended by vortexing, incubated on ice for 1 min, and resuspended. The cytoplasmic fraction was separated by centrifugation at 16,000× g for 5 min. The protein content was quantified using a BCA protein assay (Pierce). The proteins of the cytoplasmic extract (50 µg) were denatured at 95 °C for 3 min, then loaded and separated by 15% sodium dodecyl sulfate-PAGE. MagicMark^TM XP Western Standard as a molecular weight standard was used.

Proteins were transferred to Protran^TM nitrocellulose membranes, which were blocked with 5% nonfat milk in Tris buffered saline (10 mM Tris-HCl, pH 8, containing 150 mM NaCl) plus 0.1% Tween-20 for 1 h at room temperature (25 °C). The blots were probed overnight at 4 °C in Tris buffered saline containing 0.1% Tween-20, 5% nonfat milk, and antibodies with dilutions of 1:1,000 for caspase-3 monoclonal antibody or 1:5,000 for β-actin antibody (used as a loading control for cytoplasmic cell lysates). The former detects endogenous levels of procaspase-3 (around 32 kDa; p32) and its large subunit cleavage product of approximately 17 kDa (p17) [23]. The membranes were incubated with peroxidase-conjugated secondary antibodies at a dilution of 1:8,000. Blots were finally developed with SuperSignal West Pico chemiluminescent substrate for 5 min. The substrate was prepared by mixing (1:1) the SuperSignal West Pico Stable Peroxidase Solution and the SuperSignal West Pico Luminol/Enhancer Solution. After drainage of the solutions, chemiluminescence was acquired using a luminescent image analyzer LAS-3000 (Fujifilm, Tokyo, Japan).

Eosinophil migration assay

The migration of human eosinophils was assayed using Transwell^TM inserts (pore size 5 μm) and 24-well culture plates (Corning Costar, Cambridge, MA). Briefly, the cells (2×10⁵) were suspended in 0.2 ml RPMI-1640 medium containing 0.1% BSA, treated for 2 h with mapracorat or dexamethasone (0.001–10 μM), and transferred to the upper compartment of the transwell insert. PAF (200 μl of a 10⁻⁶ M solution) was added in the lower compartment of the transwell insert. Vehicle-treated (control) cells contained the same concentration of ethanol used to dissolve PAF and were further diluted in RPMI 1640 containing 0.1% BSA. After 2 h incubation in an atmosphere of 95% air and 5% CO₂, the number of cells that had migrated from the upper to the lower compartment was counted using a hemocytometer. The calculated half maximal inhibitory concentration (IC₅₀) indicates the concentration of mapracorat or dexamethasone causing 50% inhibition of the maximal number of cells that had migrated in comparison to control cells.

Cytokine and chemokine assays

Human eosinophils or HMC-1 cells (5×10⁵/ml) were suspended in RPMI 1640 containing 0.1% FBS, plated onto 24-well tissue culture plates, and pre-incubated in duplicate with dexamethasone or mapracorat for 45 min before adding ionomycin (2 μM). After 18 h stimulation at 37 °C in a 5% CO₂ atmosphere, IL-8 was measured in supernatants obtained from eosinophils with a commercial enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems. The threshold sensitivity was 5 pg/ml and the inter- and intra-assay variations were less than 5%. The supernatants obtained from HMC-1 cells were aliquoted in duplicates for IL-6, IL-8, chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation, normal T-cell expressed and secreted (RANTES), and tumor necrosis factor-α (TNF-α) analysis using high-throughput multiplex Luminex technology (Luminex 200 System; Luminex, Austin, TX) [9] and Beadview software version 1.0 (Upstate Cell Signaling Solutions, Temecula, CA). Standard curves for known concentrations of recombinant human cytokines were used to convert median fluorescence intensities 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.

The calculated IC₅₀ indicates the concentration of mapracorat or dexamethasone causing 50% inhibition of the maximal cytokine or chemokine release detected in control cells.

Active anaphylaxis in the guinea pig

Male Dunkin-Hartley guinea pigs were actively immunized by intraperitoneal (i.p.) injection of 200 μg OVA in 2 ml saline with 40 mg aluminum hydroxide (positive control) or saline alone (negative control) [24]. This immunization procedure was repeated after one week. Three weeks after the first immunization, mapracorat and dexamethasone (0.4%, weight/volume) eye drops were instilled into the conjunctival sac (30 µl/eye) of the treated guinea pigs, and 45 min later the animals were challenged with 30 ml/eye of saline solution, containing 100 mg/ml OVA, instilled into the conjunctival sac. Negative controls received saline alone. Conjunctival clinical symptoms were rated blind on both eyes using the following scale: 0, no symptoms; 1, slight conjunctival redness with or without tears; 2, mild conjunctival redness with tears and mild chemosis; 3, mild conjunctival redness with tears and moderate chemosis; 4, severe conjunctival redness with tears and partial lid eversion; 5, severe conjunctival redness with tears and lids more than half closed. The animals were euthanized 24 h after OVA challenge by i.p. injection of Tanax^® (3 ml/kg; Hoechst AG, Frankfurt-am-Main, Germany), and the conjunctivas were carefully excised and each divided into two samples for subsequent investigations. One sample was fixed in 10% buffered paraformaldehyde solution and paraffin embedded; slides, 6 μm thick, were stained with Luna’s eosinophil stain to determine eosinophil accumulation and distribution. To perform Luna’s eosinophil stain, slides were desiccated in xylene, stained with hematoxylin-Biebrich scarlet solution, differentiated in 1% acid alcohol, and subsequently stained with lithium carbonate. Eosinophil granules stain red-orange [25]. The number of eosinophils in each field was counted under light microscopy (500× magnification). In the other sample eosinophil peroxidase activity was measured.

Eosinophil peroxidase assay

Eosinophil peroxidase was assayed in conjunctival samples obtained as described above. The tissues were washed twice with ice-cold PBS, weighed, and homogenized with 50 mM Tris-HCl buffer (pH 8.0) using a Potter–Elvejehm glass/teflon homogenizer (Wheaton, Millville, NJ) on ice. After addition of 350 μl of 50 mM Tris-HCl buffer and 150 μl of 0.1% Triton X-100, the homogenates were placed in an ice bath for 1 h. The substrate solution (400 μl of 50 mM Tris-HCl buffer containing 0.1% Triton X-100, 1 mM o-phenylenediamine, and 0.5 mM hydrogen peroxide) was added to 200 μl of the sample and incubated at 37 °C for 10 min. The reaction was stopped with 200 μl of 4 M H₂SO₄. Absorbance was measured using a spectrophotometer (JASCO V-530, Jasco, Great Dunmow, Essex, UK) at 490 nm. A standard curve was plotted with different concentrations of peroxidase diluted in 50 mM Tris-HCl buffer (pH 6.0) containing 1 mM o-phenylenediamine and 0.5 mM hydrogen peroxide. Eosinophil peroxidase activity was measured adopting a method based on the oxidation of o-phenylenediamine by eosinophil peroxidase in the presence of hydrogen peroxide. One unit corresponds to 1 mmole of hydrogen peroxide decomposed for 10 min, and the results were expressed as eosinophil peroxidase levels (mU of enzyme/mg wet tissue) [24].

Data analysis

All data were expressed as mean±standard error of the mean (SEM) for the number of experiments indicated. Statistical comparisons were made, as required, by the Student t test, ANOVA, and post-hoc Newman–Keuls test or two-way ANOVA with Bonferroni post test; differences of p<0.05 were considered significant. For the clinical score, each group comprised five animals. Nonparametric analysis of the scores assigned to the conjunctival symptoms was done using the Friedman test followed by Dunn's post-hoc comparison. IC₅₀ or half maximal effective concentration (EC₅₀) values and associated 95% confidence limits (CL) correspond to the molar drug concentration producing 50% of its own maximal effect and were generated by sigmoidal nonlinear curve fitting of the concentration-response data performed by Prism through a non-weighted iterative process (Prism version 4.0, GraphPad Software, Inc., San Diego, CA).

Effect of mapracorat on spontaneous eosinophil apoptosis

Peripheral human blood eosinophils cultured for up to 48 h with 0.1% FBS and in the absence of prosurvival cytokines showed time-dependent spontaneous apoptosis, determined by flow cytometry to evaluate their ability to bind annexin V and exclude PI (Figure 1). Exposure of eosinophils cultured in 0.1% FBS and in the absence of prosurvival cytokines to mapracorat or to the reference glucocorticoid dexamethasone (0.001–10 μM) for 48 h enhanced constitutive eosinophil apoptosis in a concentration-dependent manner. Mapracorat was equally potent as dexamethasone but displayed a higher efficacy. IC₅₀ values were mapracorat 0.142 μM (95% CL 0.021–0.941 μM) and dexamethasone 0.142 μM (95% CL 0.023–0.884 μM). Mapracorat did not cause any significant change of the concentration-response curve in comparison to dexamethasone (two-way ANOVA with Bonferroni post test); interestingly it showed higher efficacy than dexametasone (mean±SEM E_max=91±2.62 versus 78±2.23; n=6; p<0.01; Student t test).

In eosinophils cultured with the vehicle, apoptosis was similar to cells cultured in 0.1% FBS for 48 h (Figure 1A). In eosinophils cultured in 0.1% FBS and exposed for 24 h to mapracorat and dexamethasone (0.1, 1.0, and 10 μM), a lower but significant apoptosis was observed in comparison to eosinophils cultured with the vehicle (data not shown).

Apoptosis induced by mapracorat was confirmed by the characteristic morphologic features on light microscopy reported for glucocorticoids [23], such as cell shrinkage and intense chromatin condensation (data not shown).

To confirm whether eosinophil apoptosis is induced by mapracorat through the glucocorticoid receptor, we investigated the effect of mifepristone (10 μM) [26]. This glucocorticoid receptor antagonist prevented apoptosis induced by 5 and 10 μM mapracorat or dexamethasone in human eosinophils (Figure 1).

To determine whether caspases were activated during these processes, caspase-3 activation during mapracorat- or dexamethasone-induced human eosinophil cell apoptosis was investigated by western blotting. In agreement with data reported in Figure 1A, human eosinophils cultured for 24 h in 0.1% FBS and in the absence of prosurvival GM-CSF and IL-5 constitutively expressed the inactive form of procaspase-3 (p32) and lower levels of its active subunit p17 (Figure 2). There was a marked concentration-dependent increase of the p17 subunit in cells exposed for 24 h to mapracorat or dexamethasone (0.001–10 μM; Figure 2). Increase of the p17subunit in cells exposed to mapracorat was higher than that observed in cells exposed to the same concentration of dexamethasone (Figure 2). We detected no changes in the amount of p32 and p17 in cells cultured for 24 h in the presence of the vehicle in comparison to control cells cultured for 24 h in 0.1% FBS and in the absence of prosurvival cytokines (control cells; Figure 2).

Effect of mapracorat on cytokine-sustained eosinophil survival

Prosurvival cytokines, particularly GM-CSF and IL-5, have been implicated in inhibiting eosinophil apoptosis, while glucocorticoids have been reported to reverse cytokine-sustained cell survival [22,27]. As previously described, human eosinophils cultured for 48 h in 0.1% FBS undergo significant apoptosis determined by flow cytometry to evaluate their ability to bind annexin V and exclude PI in comparison to control cells routinely maintained in medium containing 10% FBS (Figure 3). As expected, GM-CSF (70 pM) or IL-5 (30 pM) prevented eosinophil apoptosis. This effect was reversed in a concentration-dependent manner by dexamethasone or mapracorat (Figure 3A,B). Mapracorat was equally as potent as dexamethasone; inhibition of GM-CSF-induced eosinophil survival results were mapracorat IC₅₀ 0.154 μM (95% CL 0.079–0.301 μM) and dexamethasone IC₅₀ 0.160 μM (95% CL 0.82–0.311 μM); mapracorat did not cause any significant change in the concentration-response curve in comparison to dexamethasone (two-way ANOVA with Bonferroni post test); interestingly it showed higher efficacy than dexametasone (E_max=67±2.2 versus 47±2.1; n=6; p<0.01). Inhibition of IL-5-induced eosinophil survival was mapracorat IC₅₀ 0.156 μM (95% CL 0.073–0.335 μM) and dexamethasone IC₅₀ 0.173 μM (95% CL 0.010–0.280 μM). Mapracorat did not cause any significant change in the concentration-response curve in comparison to dexamethasone (two-way ANOVA with Bonferroni post test), whereas it showed higher efficacy than dexametasone (E_max=68±1.6 versus 51±2.3; n=6; p<0.01). However, this effect is abolished when GM-CSF or IL-5 are used at higher concentrations [27]. Dexamethasone and mapracorat were not, in fact, able to reverse cytokine-sustained survival in the presence of GM-CSF 200 pM or IL-5 100 pM (data not shown).

Effect of mapracorat on eosinophil migration induced by platelet-activating factor

PAF (10⁻⁶ M) added in the lower compartment of transwell inserts induced a significant increase of eosinophil migration (mean±SEM 1,830±38 cells; n=6) over controls (172±34 cells; n=6, p<0.01). Mapracorat and dexamethasone (0.001–10 μM) caused a concentration-dependent reduction of eosinophil migration from the upper to the lower compartment of the transwell insert. Mapracorat was equally as potent as dexamethasone; inhibition of PAF-induced eosinophil migration results were mapracorat IC₅₀ 0.114 μM (95% CL 0.025–0.313 μM) and dexamethasone IC₅₀ 0.148 μM (95% CL 0.035–0.402 μM); mapracorat did not cause any significant change of the concentration-response curve in comparison to dexamethasone (two-way ANOVA with Bonferroni post test); interestingly it showed higher efficacy than dexamethasone (E_max=93±4.0 versus 74±6.5; n=6; p<0.01). Mifepristone (10 μM) co-incubated with mapracorat or dexamethasone (5 or 10 μM) prevented eosinophil migration (data not shown).

Mapracorat has less transactivation activity than dexamethasone

Activated glucocorticoid receptors bind recognition sites in the promoters of certain genes to activate their transcription; this is known as transactivation. The CXCR4 receptor and annexin I can be considered markers of glucocorticoid-induced transactivation [22]. To determine whether mapracorat maintains transactivation on binding to the glucocorticoid receptor, we used flow cytometry to determine the induction of CXCR4 receptor and annexin I in eosinophil cells exposed for 24 h to mapracorat (0.01–10 μM) in comparison to the positive effect elicited by dexamethasone (0.01–10 μM). As reported in Figure 4A, dexamethasone (1, 5, and 10 μM) induced a significant concentration-dependent increase of the CXCR4 receptor expression; conversely, mapracorat up to 5 μM did not change CXCR4 receptor expression in comparison to vehicle-treated or control eosinophils. However, 10 μM mapracorat partially increased this receptor on the cell surface; this elevation was significantly lower than that induced by 1, 5, and 10 μM dexamethasone. Results were similar for annexin I, which is the other marker of glucocorticoid-induced transactivation investigated here (Figure 4B). These data indicate that mapracorat is less potent than dexamethasone in activating transactivation mechanisms regulated by glucocorticoid agents.

The upregulating effect of 1, 5, and 10 μM dexamethasone or 10 μM mapracorat on CXCR4 or annexin I expression cannot be explained by its apoptosis-inducing activity. Treated eosinophil cells were double stained with annexin V and anti-CXCR4 or anti-annexin I and their expression was detected in cells stained negatively with annexin V.

Effect of mapracorat on cytokine secretion

Glucocorticoids inhibit cytokine production and secretion in immune cells [11]. This has been called transrepression and contributes to their anti-inflammatory activity [7,15]. In view of the substantial apoptosis caused by mapracorat in peripheral blood eosinophils, we investigated its effect on IL-8 release from ionomycin-treated eosinophils. IL-8 is produced by eosinophils [28] and is involved in eosinophil migration and survival, which are two relevant aspects in chronic allergic diseases [29]. We also investigated the compound’s action on cytokine and chemokine secretion in the human mast cell line HMC-1 as these can greatly influence eosinophil activity in inflamed ocular tissues [2].

Mapracorat and dexamethasone (0.001–10 μM) both reduced IL-8 release induced by ionomycin in eosinophils cells in a concentration-related manner (Figure 5). Mapracorat displayed potency similar to dexamethasone (mapracorat IC₅₀ 0.020 μM, 95% CL 0.013– 0.132 μM; dexamethasone IC₅₀ 0.064 μM, 95% CL 0.0037–0.109 μM) and did not cause any significant change of the concentration-response curve in comparison to dexamethasone (two-way ANOVA with Bonferroni post test). Similarly, both antagonized the release induced by ionomycin of the following cytokines from HMC-1 cells: IL-6, IL-8, CCL5/RANTES, and TNF-α (IC₅₀ results are reported in Table 1). Mapracorat was equally as potent as dexamethasone in inhibiting ionomycin-induced secretion of IL-6, IL-8, CCL5/RANTES, and TNF-α.

Effect of mapracorat on conjunctival symptoms and conjunctival eosinophil recruitment in ovalbumin-sensitized guinea pigs

Guinea pigs were actively immunized by i.p. injection of OVA and 2 weeks later were challenged with OVA instilled into the conjunctival sac. One hour after challenge, during the early phase ocular reaction, swelling of the eyelids and chemosis were more marked in treated animals than controls, but the difference was significantly reduced by 0.4% mapracorat or dexamethasone eye drops given before treatment (30 µl/eye 45 min before OVA; Figure 6). During the late phase of allergic conjunctivitis, 6 h after challenge, there was still a significant reduction in the severity of conjunctival symptoms in treated guinea pigs with both compounds.

The guinea pigs were euthanized by intraperitoneal injection of 3 ml/kg of Tanax® 24 h later and histological analysis showed numerous eosinophils infiltrating the conjunctiva. The infiltration was much less marked in mapracorat- or dexamethasone-treated guinea pigs than in OVA-treated animals (Figure 7A,B). Similarly, eosinophil peroxidase activity, taken as an indicator of eosinophil infiltration, increased 24 h after antigen challenge in OVA-treated guinea pigs, whereas there was a noteworthy reduction in mapracorat or dexamethasone-treated animals (Figure 7C).