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Katharina Bell, Corina Wilding, Sabine Beck, Norbert Pfeiffer, Franz H Grus; Neuroprotective antibodies trigger CNTF secretion of Müller cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2552. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Changes in autoantibody-immunoreactivities have been detected in glaucoma patients. Several antibodies (abs) (against gamma-synuclein, 14-3-3 and glial fibrillary acidic protein (GFAP)) downregulated in glaucoma patients show neuroprotective effects on neuroretinal cells and primary retinal ganglion cells. Previous studies showed that Müller cells participate in the protective ab effect by up-regulating the expression of glutamine synthetase. The aim of this study was to analyse the role of retinal Müller cells in the context of neuroprotective antibodies in more detail.
Primary porcine Müller cells were isolated and incubated with different neuroprotective abs (0.5 µg/ml anti-gamma-synuclein abs, 1 µg/ml anti 14-3-3 abs or 1 µg/ml anti-GFAP abs for 3h) or without added abs. To win conditioned medium containing the secreted proteins the cells were incubated with DMEM/Ham-F12 plus 2% FCS for 16h. The Dylight 649 labelled secreted proteins were analysed via ab-antigen microarray analysis. Abs against several growth factors (BDNF, CTNF, IL6, glutathione, neurotrophin 3, ILG1 and bFGF) were spotted onto a nitrocellulose slide in triplicate (n=10 per group). The spots were scanned (Array scanner; Aviso GmbH) and quantified with ImaGene Software (BioDiscovery, Waltham). Furthermore porcine retinal organ cultures were incubated with the different conditioned media as well as medium containing 10ng/ml CNTF or control medium. Brn3a staining, DAPI and TUNEL staining was performed with the flatmounts (n=4 per group).
Müller cells incubated with either 14-3-3 (p=0.045) or GFAP (p=0.031) abs showed significantly increased CNTF secretion. Rgc/mm2 quantification showed a significant increase of rgcs (1014 rgc/mm2) in explants incubated with medium from Müller cells conditioned with GFAP abs in comparison to control conditioned medium (740 rgc/mm2) (p = 0.04). 910 rgc/mm2 were detected in retinal explants incubated with 14-3-3 ab conditioned medium (p=0.07). 10ng/ml CNTF (770 rgc/mm2) showed a non-significant increase in rgc number (636 rgc/mm2, p=0.45). TUNEL positive cells were reduced in the GFAP and 14-3-3 group (-11%; p = 0.28).
In consideration with our previous studies, these results lead to the conclusion, that the neuroprotective antibodies not only have direct effects on rgc, but also in indirect effect by increasing CNTF secretion and GS in Müller cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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