Abstract
Purpose: :
Ciliary neurotrophic factor (CNTF) has been reported to exhibit multiple effects on development, differentiation and survival of retinal neurons. CNTF appears to induce transcriptional activation of chemokine genes that are involved in the activation and migration of microglia and macrophages. To understand the biological roles of microglia/macrophages in the retina, we have examined molecular changes in these cell types from CNTF-treated retina.
Methods: :
Fluorescence activated cell sorting using CD11b and CD45 antibodies was employed to obtain microglia/ macrophage populations from CNTF-injected and PBS-injected eyes. Microarray analysis was carried out using established protocols. Differential expression was validated by qPCR and ELISA.
Results: :
Flow cytometry showed a five-fold increase in the number of CD11b+, CD45+ cells at three days after CNTF treatment. Microarray data showed that 631 transcripts were changed at least two-fold (P<05), at 1 day following CNTF treatment. Among these, 431 transcripts were upregulated 2- to 14-fold, and 200 transcripts were downregulated 2- to 7-fold. Ingenuity Pathway Analysis of the microarray data showed that differentially regulated genes fell into several functional types including growth factors, cytokines, G-protein coupled receptors, kinases, transporters, and ion channels. Three cytokines, CCl13, CCl7 and CCl2 were upregulated, but only two to three-fold. We did not find upregulation of proinflammatory cytokines such as interleukins or TNF. There was no change in iNOS transcript levels. At three days after CNTF treatment, only 19 transcripts had changed; none involving cytokines.
Conclusions: :
Our studies lead to the surprising finding that CNTF treatment leads to influx of macrophages into the retina and that infiltrating macrophages do not express proinflammatory cytokine genes. Whether the activated retinal microglia/macrophages have a role in CNTF-mediated neuroprotection or in retinal cellular repair remains to be established.
Keywords: retina • microglia • flow cytometry