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(Investigative Ophthalmology and Visual Science. 2008;49:2030-2038.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0273
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Dexamethasone Inhibits High Glucose–, TNF-{alpha}–, and IL-1β–Induced Secretion of Inflammatory and Angiogenic Mediators from Retinal Microvascular Pericytes

Alissar Nehmé and Jeffrey Edelman

From the Department of Biological Sciences, Allergan Inc., Irvine, California.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To characterize the effects of dexamethasone in human retinal pericytes (HRMPs), monocytes (THP-1), and retinal endothelial cells (HRECs) treated with high glucose, TNF-{alpha}, or IL-1β.

METHODS. HRMP and HREC phenotypes were verified by growth factor stimulation of intracellular calcium–ion mobilization. Glucocorticoid receptor phosphorylation was assessed with an anti-phospho-Ser211 glucocorticoid receptor antibody. Secretion of 89 inflammatory and angiogenic proteins were compared in cells incubated with (1) normal (5 mM) or high (25 mM) D-glucose and (2) control medium, TNF-{alpha} (10 ng/mL), or IL-1β (10 ng/mL), with or without dexamethasone (1 nM to 1 µM). The proteins were compared by using multianalyte profile testing.

RESULTS. HRMPs and HRECs expressed functional PDGFB-R and VEGFR-2, respectively. Dexamethasone induction of glucocorticoid receptor phosphorylation was dose-dependent in all cell types. High glucose increased secretion of inflammatory mediators in HRMPs, but not in HRECs. Dexamethasone dose dependently inhibited secretion of these mediators in HRMPs. For all cells, TNF-{alpha} and IL-1β induced a fivefold or more increase in inflammatory and angiogenic mediators; HRMPs secreted the greatest number and level of mediators. Dexamethasone dose dependently inhibited the secretion of multiple proteins from HRMPs and THP-1 cells, but not from HRECs (IC50 2 nM to 1 µM).

CONCLUSIONS. High glucose, TNF-{alpha}, and IL-1β induced an inflammatory phenotype in HRMPs, characterized by hypersecretion of inflammatory and angiogenic mediators. Dexamethasone at various potencies blocked hypersecretion of several proteins. Pericytes may be a key therapeutic target in retinal inflammatory diseases, including diabetic retinopathy. Inhibition of pathologic mediators may depend on delivering high levels (~1 µM) of glucocorticoid to the retina.

Diabetic retinopathy, characterized by diabetic macular edema and retinal neovascularization, is a common microvascular complication of diabetes and a leading cause of adult blindness.1 The inflammatory changes associated with this disease include leukostasis,2 increased vaso-occlusion and vascular permeability,3 and upregulation of cytokines and intracellular adhesion molecules.4 5 In fact, accelerated death of retinal capillary endothelial cells and pericytes has been described and is likely to contribute to the development of acellular, nonperfused capillaries in diabetic retinopathy, and, if extensive, to retinal ischemia, diabetic macular edema, and neovascularization.6 7 Whether subclinical inflammation contributes to this accelerated cell death remains to be determined. More important, multiple cytokines and chemokines associated with the pathogenesis of diabetic retinopathy are induced by high levels of glucose in primary human monocytes,8 the THP-1 monocytic cell line,9 10 and bovine retinal endothelial cells.11

Although several inflammatory cytokines are reported to be associated with diabetic retinopathy, particular attention has been focused on TNF-{alpha} and IL-1β. Increased levels of TNF-{alpha} and IL-1β have been found in the serum12 13 14 and vitreous13 of patients with diabetic retinopathy. Relevant to the vascular changes associated with diabetic retinopathy, these cytokines have been shown to induce inflammatory responses in human retinal endothelial cells,15 but there are no reports of their effects on retinal microvascular pericytes.

As inflammation has been implicated in the pathogenesis of diabetic retinopathy, researchers are investigating the potential use of anti-inflammatory agents, such as glucocorticoids, to treat this disease.16 17 The biological actions of glucocorticoids are mediated through the cytoplasmic glucocorticoid receptor (GR), which belongs to the nuclear receptor subfamily that includes receptors for mineralocorticoids, estrogen and thyroid hormones, retinoic acid, and vitamin D.18 On hormone binding, the activated ligand-bound receptor translocates into the nucleus and binds as a homodimer to glucocorticoid response elements within the promoter region of target genes. The GR can positively or negatively regulate gene expression, depending on the response element sequence and promoter context. The GR also modulates gene expression, independent of glucocorticoid response elements, by physically interacting with other transcription factors (e.g., activating protein [AP]-1 and nuclear factor [NF]-{kappa}B).19

Several clinical trials have been conducted to investigate the efficacy and safety of glucocorticoids, such as triamcinolone acetonide, fluocinolone acetonide, and dexamethasone, for treating macular edema associated with diabetic retinopathy.17 20 21 22 23 We have demonstrated in an animal model of retinal edema that dexamethasone can completely block retinal vascular leakage, retinal vasodilation, and vessel tortuosity.24 In vitro studies are needed, however, to gain a better understanding of the cell types, the profile of inflammatory mediators, and the cellular mechanisms underlying dexamethasone inhibition.

The purpose of our in vitro study was to investigate whether dexamethasone inhibits TNF-{alpha}– or IL-1β–induced secretion of inflammatory mediators from primary human retinal microvascular pericytes (HRMPs), human monocytes (THP-1 cells), and primary human retinal microvascular endothelial cells (HRECs).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Culture Conditions
Primary HRMPs and HRECs (Cell Systems, Kirkland, WA) were maintained in MCDB-131 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) antibiotics/antimycotics, 10 ng/mL epidermal growth factor, 1 µg/mL hydrocortisone, and 0.2 mg/mL complete medium (Endo Gro, MCDB-131 complete medium; VEC Technologies, Rensselaer, NY). Characteristics of HRECs (Fig. 1A) and HRMPs (Fig. 1B) were confirmed by immunocytochemical examination and morphologic observation. Briefly, more than 95% of HRECs tested positive for von Willebrand factor and uptake of diacetylated low-density lipoprotein. For HRMPs, more than 80% of cells tested positive for cytoplasmic {alpha}-actinin and desmin intermediate filaments, and less than 2% of HRMPs tested positive for von Willebrand factor and uptake of diacetylated low-density lipoprotein. Cells from the human THP-1 acute monocytic leukemia cell line (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated FBS, 1% (vol/vol) antibiotics-antimycotics, and 0.05 mM 2-mercaptoethanol. All cells were used within passages 2 to 10.


Figure 1
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  		FIGURE 1. Digital phase-contrast images of (A) HRECs and (B) HRMPs. Cells (HRECs at passage 7 and HRMPs at passage 6) were cultured in MCDB-131 complete medium on fibronectin-coated and plastic tissue culture flasks, respectively. Magnification: (A) x4; (B) x10.

 
Calcium-Ion (Ca2+) Mobilization in Response to Growth Factor Stimulation
Human monocytes express the gene for VEGFR-1 but not that for VEGFR-2.25 As Ca2+ mobilization depends on activation of VEGFR-2, the Ca2+ assay could not be performed in THP-1 monocytes. To confirm that HRMPs or HRECs express functional PDGF-BR or VEGFR-2, respectively, the cells were plated in 96-well black, clear-bottomed plates (Biomedtech Laboratories, Tampa, FL) that were precoated with h-fibronectin at 12,000 cells/well in 150 µL of MCDB-131 complete medium. The cells were loaded with the fluorescent Ca2+ indicator dye fluo-4 (2 µM; Invitrogen-Molecular Probes, Eugene, OR) for 45 minutes at 37°C and then stimulated for 30 minutes with VEGF165, VEGF-121, PlGF, PDGF-BB (R&D Systems, Minneapolis, MN), or VEGF-E (Axxora, San Diego, CA) at 1, 10, 50, and 100 ng/mL. A fluorometric imaging plate reader (FLIPR; Molecular Devices Corp., Sunnyvale, CA) was used to measure changes in intracellular Ca2+-dependent fluo-4 fluorescence. All determinations were performed in triplicate.

Dexamethasone-Dependent Phosphorylation of Glucocorticoid Receptors
To determine whether dexamethasone can induce phosphorylation of GRs in HRMPs, THP-1, and HRECs, we used an anti-phospho-Ser211 antibody capable of detecting phosphorylation of receptors by Western blot analysis. Dexamethasone-induced phosphorylation of GRs on Ser211 and subsequent proteasome-mediated receptor degradation have been shown to act as biomarkers for activated GRs in vivo.26 The day before the experiment, confluent HRMPs, THP-1, or HRECs, growing in 100-mm tissue culture plates (BD Labware, Bedford, MA), were washed once with phosphate-buffered saline (PBS) and stepped to 1% FBS-containing medium with antibiotics-antimycotics. The day of the experiment, the cells were incubated with cell culture–tested dexamethasone (Sigma-Aldrich, St. Louis, MO) at 10, 50, or 100 nM or 1 µM for the indicated times (see Fig. 3 ). The cells were washed twice with ice-cold PBS and lysed in 400 µL of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA; Upstate Biotech, Lake Placid, NY), supplemented with 0.2% sodium dodecyl sulfate (SDS) and a 1% (vol/vol) cocktail of protease inhibitors, serine-threonine phosphatase inhibitors, and tyrosine phosphatase inhibitors (Sigma-Aldrich). After 30 minutes’ incubation on ice, the lysates were centrifuged at 14,000 rpm for 20 minutes at 4°C. Supernatants were transferred to clean microfuge tubes, and the total protein concentration of each sample was measured with a bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL). Cell lysates (30 µg protein) in SDS sample buffer were separated on 7% Tris-acetate gel by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Phosphorylated GR and total GR levels were detected by using the polyclonal anti-p-GR (Ser211; Cell Signaling Technology, Beverly, MA) and anti-GR antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Protein bands were visualized by using a chemiluminescence detection kit (Invitrogen, Carlsbad, CA). The intensity of all bands was estimated by densitometric analysis (ImagePro Plus Software; Media Cybernetics, Silver Spring, MD).


Figure 3
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  		FIGURE 3. Dexamethasone induced phosphorylation of glucocorticoid receptors in HRMPs (A), THP-1 cells (B), and HRECs (C). The phosphorylated glucocorticoid receptor at Ser211 and total glucocorticoid receptor protein levels were determined after the cells had been treated with dexamethasone for 5 to 24 hours. The Western blot images and densitometric analysis presented are representative of those obtained in two independent experiments.

 
Stimulation of Cells by High Glucose, TNF-{alpha}, or IL-1β, with or without Dexamethasone
Confluent HRMPs and HRECs, growing in 100-mm tissue culture plates (BD Labware), and THP-1 cells, growing at a density of 1 x 106 cells/mL in T162 tissue culture flasks (Corning Costar Corp., Encinitas, CA), were washed once with PBS and stepped to 1% FBS-containing medium with antibiotics-antimycotics. The next day, 10 mL of fresh medium was added to the confluent HRMPs and HRECs, and the THP-1 cells were centrifuged, resuspended in fresh medium and plated into a six-well plate in 6 mL of medium per well. Preliminary experiments indicated that the maximum inflammatory response to TNF-{alpha} or IL-1β occurred after 5 (THP-1 cells) or 24 (HRMPs and HRECs) hours. The cells were incubated for 5 or 24 hours with medium alone (control cells), TNF-{alpha} (R&D Systems), or IL-1β (R&D Systems) at 10 ng/mL, with or without dexamethasone (10 nM, 100 nM, or 1 µM). To assess the effects of high glucose, HRMPs and HRECs were cultured with normal glucose (5.5 mM D-glucose) or high glucose (25 mM D-glucose) for 24 or 72 hours, with or without dexamethasone (1 nM to 1 µM). The cells cultured with 5.5 mM D-glucose plus 19.5 mM L-glucose were used as an osmotic control. At the end of treatment, the cell culture supernatants were concentrated approximately 10-fold in conical concentrator tubes (iCON; Pierce Biotechnology). Concentrated supernatants were transferred into clean microfuge tubes, and the total protein concentration of each sample was measured by the bicinchoninic acid protein assay (Pierce Biotechnology). The samples were stored at –80°C until analyzed for inflammatory mediators.

Quantification of Inflammatory Mediators
Eighty-nine inflammatory or angiogenic proteins were analyzed by multianalyte profile (MAP) testing at a certified service laboratory (Rules-Based Medicine, Austin, TX).27 28 29 30 Briefly, supernatant samples were thawed at room temperature, vortexed, and centrifuged at 13,000g for 5 minutes for clarification. A 150-µL sample (undiluted or 1:50 diluted samples) was transferred into a master microtiter plate for MAP antigen analysis. An aliquot of each sample was introduced by automated pipetting into one of the capture microsphere multiplexes of the human antigen MAP. These combined samples and microspheres were mixed and incubated at room temperature for 1 hour. Multiplexed cocktails of biotinylated reporter antibodies were then added robotically and, after mixing, were incubated for 1 hour at room temperature. Multiplexes were developed with an excess of streptavidin-phycoerythrin solution, which was mixed into each multiplex and incubated for 1 hour at room temperature. The volume of each multiplexed reaction was reduced by vacuum filtration and increased by dilution into matrix buffer for analysis (Luminex 100; Luminex, Austin, TX). The resulting data stream was interpreted with proprietary data-analysis software (Qiagen, Inc., Valencia, CA). For each multiplex, calibrators and controls were included on each microtiter plate, eight-point calibrators were run in the first and last column of each plate, and three-level controls were included in duplicate. Testing results were determined first for the high, medium, and low controls for each multiplex, to ensure proper assay performance. Unknown values for each of the analytes localized in a specific multiplex were determined by using the four- and five-parameter, weighted and nonweighted curve-fitting algorithms that were included in the data-analysis software.

Statistical Analysis
Statistical analyses were conducted with commercially available software (Microsoft Excel 2000; Microsoft Corp., Redmond, WA). The results are presented as the mean of three different experiments. Student’s unpaired t-test was used to (1) compare data from treatment (TNF-{alpha} or IL-1β exposure) and control (medium) experiments and (2) to compare data from the dexamethasone- and TNF-{alpha}– or IL-1β–treated groups with results from the TNF-{alpha}– and IL-1β–treated groups, respectively. For all analyses, P < 0.05 was considered sufficient to reject the null hypothesis.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
HRMPs and HRECs Expressed Functional PDGFB-R and VEGFR-2, Respectively
As expected, some, but not all, growth factors were able to stimulate Ca2+ mobilization in the HRMPs and HRECs. A dose-dependent increase in Ca2+ mobilization was evident for the HRMPs exposed to PDGF-BB, but no effect was apparent after exposure to VEGF165; the VEGFR-2-specific ligand, VEGF-E; or the VEGFR-1-specific ligand, PlGF (Fig. 2A) . In HRECs, VEGF165, VEGF121, and VEGF-E caused a dose-dependent increase in Ca2+ mobilization, but no effect was apparent after exposure to PlGF (Fig. 2B) .


Figure 2
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  		FIGURE 2. Calcium mobilization in HRMPs (A) and HRECs (B) in response to growth factors (VEGF165, VEGF121, PlGF, VEGF-E, and PDGF-BB). Changes in intracellular calcium-ion were measured after the cells were treated with various concentrations of growth factors for 30 minutes. Fluorescence intensity is shown in arbitrary units, and each calcium mobilization trace represents 30 seconds of measurement. The data are representative of results in two independent experiments performed in triplicate.

 
Dexamethasone-Induced Phosphorylation of GRs in HRMPs, THP-1 Cells, and HRECs
Dexamethasone induced GR phosphorylation in HRMPs (Fig. 3A) , THP-1 cells (Fig. 3B) , and HRECs (Fig. 3C) in a dose-dependent manner. After 24 hours, Ser211 phosphorylation remained high in all cell types, compared with the control cells. In terms of total GR protein levels, dexamethasone treatment had a minimal effect in all cell types at 6 hours, but caused a dose-dependent decrease after 24 hours (Fig. 3) .

High Glucose–Induced Secretion of Inflammatory Mediators by HRMPs but Not by HRECs
Incubation of HRECs in 25 mM D-glucose for 24 or 72 hours, with or without dexamethasone, had no effect on secretion of inflammatory or angiogenic mediators compared with cells incubated in 5 mM D-glucose (data not shown). However, culture of HRMPs in high glucose for 72 hours resulted in a twofold or greater increase in secretion of eotaxin, G-CSF, IL-8, and RANTES (Table 1) . There was no increase in cytokine and chemokine secretion when 25 mM L-glucose was used instead of D-glucose. This result suggests that the high glucose–induced effects were not due to an increase in osmolarity. Incubation of HRMPs in high glucose for 24 hours had no effect on inflammatory mediator secretion (data not shown). Treatment of HRMPs with dexamethasone for 72 hours, in both normal and high glucose, resulted in a dose-dependent decrease in the secretion of eotaxin, G-CSF, IL-8, and RANTES (Table 1) .


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  		TABLE 1. Dexamethasone Inhibition of High Glucose–Induced Secretion of Inflammatory Mediators in HRMPs after 72 Hours

 
TNF-{alpha}– and IL-1β–Induced Secretion of Inflammatory Mediators by THP-1 Cells, HRECs, and HRMPs
Compared with control conditions, 5 (THP-1 cells) or 24 (HRECs and HRMPs) hours of exposure to TNF-{alpha} or IL-1β resulted in a significant increase in the secretion of a wide range of proteins, including several inflammatory and angiogenic mediators (Table 2) . There was a fivefold or greater increase in 11 (THP-1 cells), 14 (HRECs), and 33 (HRMPs) proteins after the cells were exposed to TNF-{alpha} and a fivefold or more increase in 13 (THP-1 cells), 17 (HRECs), and 29 (HRMPs) proteins after the cells were exposed to IL-1β. For the THP-1 cells, the chemokine MIP-1β showed the greatest increase after TNF-{alpha} exposure (356-fold; P = 0.0001) or IL-1 β exposure (108-fold; P = 0.0473). For HRECs and HRMPs, the chemokine RANTES showed the greatest increase after TNF-{alpha} exposure (181-fold; P = 0.0396 and 6,248-fold; P = 0.0001 in HRECs and HRMPs, respectively), and the growth factor GM-CSF showed the greatest increase after IL-1 β exposure (1,756-fold; P = 0.0006 and 39,820-fold; P = 0.0009 in HRECs and HRMPs, respectively).


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  		TABLE 2. Mediators Significantly Induced by TNF-{alpha} or IL-1β Treatment in Three Human Cell Lines

 
Dexamethasone Inhibition of TNF-{alpha}– and IL-1β–Induced Secretion of Inflammatory Mediators by THP-1 Cells
Dexamethasone significantly and differentially inhibited TNF-{alpha}–induced secretion of five inflammatory mediators by THP-1 cells (Figs. 4A 4B) . The inhibitory action of dexamethasone was particularly strong (>80% inhibition) against MCP-1 and IL-1β, resulting in an IC50 of 3 and 7 nM, respectively. Dexamethasone partially blocked (≤70% inhibition) the secretion of the three mediators IL-8, MIP-1{alpha}, and MIP-1β, with IC50s of 55, 59, and 34 nM, respectively.


Figure 4
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  		FIGURE 4. Dexamethasone differentially blocks secretion of inflammatory mediators in THP-1 cells in response to TNF-{alpha} (A, B) or IL-1β (C). The cells were treated with TNF-{alpha} (10 ng/mL) or IL-1β (10 ng/mL) in the presence or absence of dexamethasone (10 nM, 100 nM, or 1 µM) for 5 hours. Because of the number of mediators tested, the results are displayed in three graphs (A–C). The data are representative of results of three independent experiments.

 
Dexamethasone also significantly and differentially inhibited IL-1β–induced secretion of three mediators by THP-1 cells (Fig. 4C) . The inhibitory action of dexamethasone was particularly strong (>80% inhibition) against MCP-1, with an IC50 of 3 nM. Dexamethasone partially blocked the secretion of IL-7 and MIP-1{alpha}, with an IC50 of 58 and 332 nM, respectively.

Dexamethasone had no significant effect on mediator secretion by THP-1 cells when incubated alone with the cells for 5 hours (data not shown).

Effect of Dexamethasone on TNF-{alpha}– and IL-1β–Induced Secretion of Inflammatory Mediators by HRECs
In contrast to the results obtained with HRMPs (described in the next section) and THP-1 cells, dexamethasone did not inhibit either the TNF-{alpha}– or IL-1β–induced release of inflammatory mediators by HRECs (data not shown).

Effect of Dexamethasone on TNF-{alpha}– and IL-1β–Induced Secretion of Inflammatory Mediators by HRMPs
Dexamethasone significantly and differentially inhibited TNF-{alpha}–induced secretion of 17 inflammatory mediators by HRMPs (Fig. 5) . The inhibitory action of dexamethasone was particularly strong (>80% inhibition) for G-CSF, GM-CSF, MIP-1{alpha}, the IL-1 receptor antagonist IL-6, and RANTES, with the IC50 for these mediators ranging between 2 and 6 nM. Several mediators, including IL-1β, IL-8, MMP-3, VEGF165, and ICAM-1, were partially blocked by dexamethasone. The IC50 of these mediators ranged between 44 and 995 nM.


Figure 5
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  		FIGURE 5. Dexamethasone differentially blocked secretion of inflammatory mediators in HRMPs in response to TNF-{alpha}. The cells were treated with TNF-{alpha} (10 ng/mL) in the presence or absence of dexamethasone (10 nM, 100 nM, or 1 µM) for 24 hours. Because of the number of mediators tested, the results are displayed in four graphs (A–D). The data are representative of results of three independent experiments.

 
Dexamethasone also significantly and differentially inhibited IL-1β–induced secretion of mediators by HRMPs; 13 mediators were sensitive to dexamethasone (Fig. 6) . The inhibitory action of dexamethasone was strong (>80% inhibition) against G-CSF, GM-CSF, MIP-1{alpha}, IL-6, RANTES, eotaxin, MMP-2, MMP-3, and VEGF165, with the IC50 of these mediators ranging between 12 and 294 nM. It partially blocked the secretion of several mediators, including ENA-78, TNF RII, MIP-1β, and ICAM-1. The IC50 for these mediators ranged between 500 and 600 nM.


Figure 6
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  		FIGURE 6. Dexamethasone differentially blocked secretion of inflammatory mediators in HRMPs in response to IL-1β. The cells were treated with IL-1β (10 ng/mL) in the presence or absence of dexamethasone (10 nM, 100 nM, or 1 µM) for 24 hours. Because of the number of mediators tested, the results are displayed in three graphs (A–C). The data are representative results of three independent experiments.

 
Dexamethasone had no significant effect on mediator secretion by HRMPs when incubated alone with HRMPs for 24 hours (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study is the first to demonstrate that dexamethasone can inhibit, in a selective and dose-dependent manner, the release of inflammatory mediators from human retinal microvascular pericytes. It also provides new data on the identity and quantity of inflammatory mediators released by pericytes cultured in high glucose or exposed to TNF-{alpha} or IL-1β, as well as human monocytes and human retinal endothelial cells, after exposure to these proinflammatory cytokines. These results suggest that pericytes are a key therapeutic target in many retinal inflammatory diseases, including diabetic retinopathy, and that high levels of glucocorticoid (~1 µM) in the retina may inhibit a broad range of pathologic mediators.

The development of diabetic retinopathy has been associated with chronic subclinical inflammation, characterized by increased cytokine production and retinal capillary permeability, upregulation of adhesion molecules, and leukostasis.3 31 Our results showed that high glucose increased the secretion of chemokines in HRMPs. Two of these chemokines, RANTES and eotaxin, belong to the human β-chemokine subfamily of proteins, which primarily chemoattract monocytes and T lymphocytes.32 33 Indeed, TNF-{alpha} and IL-1β are two powerful mediators of inflammation primarily produced by activated monocytes or macrophages. The methodology used in our study allowed us to measure a wide range of inflammatory mediators, and we have shown that both TNF-{alpha} and IL-1β can stimulate the in vitro secretion of a large number of inflammatory mediators from cells present in the human retina. These mediators have been shown to be increased in the vitreous or plasma of patients with diabetic retinopathy and included: sICAM-1 and sVCAM-1,34 35 TNF-{alpha} and IL-1β,13 11 VEGF165,35 36 IL-8, MCP-1 (CCL2), RANTES (CCL5),37 and IL-6.5 Based on the profile of mediators released from pericytes in response to TNF-{alpha} or IL-1β, we can speculate that the adhesion molecules sICAM-1 and sVCAM-1 influence leukostasis and that the inflammatory cell chemoattractants IL-8, MCP-1, and RANTES may stimulate diapedesis. IL-6 and VEGF elicit a broader range of cellular effects including vascular permeability, cell proliferation, cell differentiation, and angiogenesis.

Consistent with the potential role that pericytes may have in driving the ocular inflammation associated with diabetic retinopathy, these cells may also serve as a rational cellular target for anti-inflammatory agents. We have shown that dexamethasone was able to inhibit high glucose– and cytokine-induced secretion of inflammatory mediators by pericytes and that this inhibition occurred in a selective and dose-dependent manner. The release of inflammatory mediators from human monocytes was also inhibited by dexamethasone in a selective and dose-dependent manner, although fewer mediators were strongly suppressed in monocytes than in pericytes. Our results extend the findings from other studies that have shown that dexamethasone can differentially inhibit the release of inflammatory mediators from human fibroblasts38 and human airway39 40 and human vascular smooth muscle cells.41 The molecular mechanisms by which dexamethasone inhibits cytokine-induced inflammatory mediators from human retinal pericytes and monocytes are not well defined. Previous studies on other cell types have shown that dexamethasone reduces, directly or indirectly, both RNA and protein levels of proinflammatory mediators in response to TNF-{alpha} or IL-1β.38 42 43 The cross-talk between cytokine-induced transcription factors, such as NF-{kappa}β, activating protein-1, cAMP-responsive element binding protein, NF-AT, Ets proteins, and GR involves both genomic and nongenomic actions and is thought to be the major mechanism by which glucocorticoids suppress cytokine synthesis and action.19 44 45

In contrast to the effects of dexamethasone on pericytes and monocytes, we found that dexamethasone did not suppress TNF-{alpha}– or IL-1β–induced secretion of mediators from HRECs, despite the presence of an active GR. The mechanism of resistance could be multifactorial.46 47 First, retinal endothelial cells may overexpress the human GR (hGR)-β isoform, a splicing variant of the classic receptor hGR{alpha}, which functions as a dominant-negative inhibitor of hGR{alpha}.48 49 Second, endothelial cells may express a GR with a point mutation in the ligand binding domain, which has been associated with glucocorticoid resistance.50 We intend to investigate the mechanisms of dexamethasone resistance in HRECs.

Our finding that high doses of dexamethasone were necessary to inhibit most of the inflammatory mediators could have clinical implications for the development and delivery of anti-inflammatory agents used to treat retinal diseases. Indeed, a dose-dependent effect, in terms of both the extent and duration of improved visual acuity changes, was evident in a recent study on the use of intravitreous triamcinolone acetonide for the treatment of diabetic macular edema.51 Moreover, in patients with severe chronic asthma, 360 mg triamcinolone acetonide, given intramuscularly, produced a longer exacerbation-free period than did 120 mg.52 Given the need to minimize the risks associated with repetitive intravitreous injections, questions remain regarding the optimal injection frequency, dose, and vitreous pharmacokinetics of glucocorticoids.

In conclusion, our study has shown that high glucose, TNF-{alpha}, and IL-1β can induce the release of a broad range of inflammatory mediators from human retinal pericytes and that the release of these mediators can be inhibited by dexamethasone, in a selective and dose-dependent manner. We speculate that pericytes may be an important source of the inflammatory mediators implicated in the pathogenesis of diabetic retinopathy and other retinal diseases and, as such, may be an important target for pharmacotherapy. A better understanding of the pathologic inflammatory targets in retinal disease, coupled with advances in genomic screening of glucocorticoid effects on cells associated with outflow facility53 and cataract,54 could help accelerate the discovery of optimized ocular glucocorticoids that retain their therapeutic benefit and minimize side effects.


   Acknowledgements
 
The authors thank Anne P. McLaughlin and Tracy M. Nicholson for excellent technical assistance with the FLIPR Ca2+ assays and the independent medical writing assistance provided by ProScribe Medical Communications (www.proscribe.com.au), funded by Allergan Inc. ProScribe’s services complied with international guidelines for Good Publication Practice.


   Footnotes
 
Supported by Allergan, Inc. In compliance with the Uniform Requirements for Manuscripts, established by the International Committee of Medical Journal Editors, the sponsor of this study did not impose any impediment, directly or indirectly, on the publication of the study’s results.

Submitted for publication March 5, 2007; revised July 2 and October 23, 2007; accepted March 7, 2008.

Disclosure: A. Nehmé, Allergan, Inc. (E); J. Edelman, Allergan, Inc. (E)

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

Corresponding author: Jeffrey Edelman, Department of Biological Sciences, Allergan Inc., 2525 Dupont Drive, Irvine, CA 92612; edelman_jeffrey{at}allergan.com.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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