| Objective:to investigate the expression of macrophage colony-stimulating factor (M-CSF)and its receptor(CSF-1R)in diabetic retinas,and the effects of diabetic microenvironment on retinal microglial activation.Methods:In vivo:Sprague-Dawley(SD)rats were rendered diabetic by streptozotocin injection and killed after 2,4,8,12 weeks.The retinas of diabetic and age-matched control rats were studied.Real-time PCR was applied to evaluate M-CSF and its receptor CSF-1R mRNA expression in the retinas.The protein levels of M-CSF and CSF-1R were evaluated by Western blot analysis.Cellular sources of M-CSF and CSF-1R were determined by double-immunofluorescence staining. M-CSF levels in vitreous samples from 22 patients with PDR were measured by ELISA,the vitreous samples from 22 patients with 19 idiopathic macular holes as control.In vitro:Primary cultured retinal microglia was assessed using the monoclonal antibody OX-42(a microglial marker for complement type 3 receptor)and glial fibrillary acidic protein(GFAP,an astrocyte marker).Phase-contrast microscope and fluorescence microscope were used to observe the GA-stimulated retinal microglia,including the change of morphology,expression of Ibal(marker of microglial activation),TNF-αand IL-1βThe release of TNF-α,IL-1βand M-CSF was detected by Enzyme-Linked Immunosorbent Assay(ELISA). Immunofluorescence,Real-time PC,Western blot and immunoprecipitation(IP)were used to detect the expression of CSF-1R by GA-activated retinal microglia.The protein of TNF-αand IL-1βreleased respectively from M-CSF group,M-CSF antibody(Ab)group,CSF-lRAb group,and GA+M-CSF group,GA+M-CSF antibody(Ab)group,GA+CSF-lRAb group,was also assessed by ELISA, microglia stimulated with GA alone is as control.Results:In vivo:M-CSF and CSF-1R mRNA were upregulated in the retinas as early as 2 weeks after the onset of diabetes,and increased over time.A similar pattern was observed for M-CSF/CSF-1R protein expression levels. Double-immunofluorescence staining revealed that increased M-CSF immunoreactivity occurred mainly in the nerve fiber layer in diabetic retinas, co-localizing with glial fibrillary acidic protein.Increased CSF-1R immunoreactivity was observed in OX-42-labeled microglia with constant CSF-1R immunoreactivity in ganglion cells in the ganglion cell layer.The vitreous level of M-CSF was elevated in patients with PDR compared to control subjects.In vitro:GA-stimulated retinal microglia changes from rod-like shape to bigger and rounder,with the appearance of activated cells.Enhanced Ibal expression was observed in GA-stimulated microglia compared with unstimulated cells and translocated from the cytoplasm to membrane ruffles.Following stimulation with GA, a significant increase in the release of TNF-αwas detected,compared with the control group(38.7±3.2vs.19.7±1.7pg/ml;P＜0.05).Moreover,GA induced the dose-dependent release of TNF-α,with a peak at 250μg/ml.The similar pattern was observed in the release of IL-lβ.Increased expression of CSF-1R mRNA or protein by GA-stimulated microglia was observed,however,unstimulated microglia express basal level of CSF-1R.Furthermore,the neutralization of M-CSF or CSF-1R with antibodies suppressed the proinflammatory response.Conversely,this proinflammatory response was augmented by the administration of M-CSF(p＜0.05), however,M-CSF alone could induce release of neither TNF-a nor IL-lβ.Conclusion:1.The early upregulation of MCSF/CSF-1R signaling may be an important regulatory pathway among neurons,microglia,and glia in diabetic retinopathy.Astrocytes are the primary source of the elevated M-CSF,and the activated microglia express increasingly CSF-1R.2.GA can induce microglial activation by stimulating the release of proinflammatory cytokines such as TNF-αand IL-1βin vitro.3.Activated microglia express increasingly CSF-1R,combining with autocrined or paracrined M-CSF,augment the GA-induced microglial inflammation. M-CSF/CSF-1R signaling represents a further link between microglial inflammation and diabetic retinopathy.
|
PDF Full Text Request