Dexamethasone inhibition of trabecular meshwork cell phagocytosis and its modulation by glucocorticoid receptor beta.
Glucocorticoid treatment can lead to the development of glaucomatous ocular hypertension and a secondary open-angle glaucoma due to increased aqueous humor outflow resistance that is associated with morphological and biochemical changes in the trabecular meshwork (TM). The cellular responses of glucocorticoids are achieved by binding to the glucocorticoid receptor alpha (GRalpha), a ligand-activated transcription factor. An alternatively spliced variant, glucocorticoid receptor beta (GRbeta), has dominant negative activity on GRalpha and has been implicated in a variety of steroid-resistant diseases. We previously showed that GRbeta can block dexamethasone (DEX) responsiveness in TM cells. TM cells are actively phagocytic and function in the removal of debris, pigment and other materials from the aqueous outflow drainage pathway. A decrease in phagocytic activity has been proposed in the pathogenesis of glaucoma and glucocorticoid-induced glaucoma. In this study, we investigated the effect of DEX and GRbeta on phagocytosis in normal and glaucomatous TM cells. Human transformed normal NTM-5 and primary normal NTM174-00 cells, which express relatively high amounts of GRbeta, and transformed glaucomatous GTM-3 and primary glaucomatous GTM520-05 cells, which have lower GRbeta expression, were treated with 100 nM DEX or vehicle control for 24h. NTM cells also were transfected with a control or GRbeta expression plasmid to examine the effect of GRbeta on phagocytic activity. The cells were incubated with Alexa 488 conjugated Staphylococcus aureus bioparticles opsonized with rabbit IgG for 1h, followed by fixation and incubation with Alexa 633 conjugated goat anti-rabbit IgG to distinguish ingested from extracellular bioparticles. DAPI nuclear staining was used to quantify cell numbers. Cells and bioparticles were visualized by confocal microscopy. We found that NTM-5 cells ingested more bioparticles than GTM-3 cells. DEX treatment significantly decreased the phagocytosis of bioparticles in NTM-5 and GTM-3 cells, while GTM-3 cells were more responsive to DEX, compared to NTM-5 cells. In primary cell culture, NTM174-00 also engulfed more bioparticles than GTM520-05 cells. DEX treatment significantly decreased the phagocytic activity in GTM520-05, but not in NTM174-00 cells. Transient transfection of pCMX-hGRbeta plasmid increased the expression of GRbeta and consequently maintained the phagocytotic activity of NTM-5 cells in the presence of DEX. Our data demonstrated that the expression level of GRbeta in TM cells can regulate DEX-induced suppression of phagocytotic activity. The lower expression of GRbeta in glaucomatous TM cells may contribute to the altered phagocytic function of TM cells, and may lead to the increased aqueous humor outflow resistance mediated by glucocorticoids.
Zhang
Xinyu
X
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA.
Ognibene
Cherie M
CM
Clark
Abbot F
AF
Yorio
Thomas
T
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D005822:Genetic Vectors; D005901:Glaucoma / Q000473:pathology*; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D010587:Phagocytosis / Q000187:drug effects*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects*; D014162:Transfection
D000893:Anti-Inflammatory Agents; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; C121943:glucocorticoid receptor beta; D003907:Dexamethasone