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Q. Liu, D. Gagen, A. M. McDermott; Nerve Growth Factor Induces Migration and Differentiation of Mouse Monocytes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1950.
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Nerve growth factor (NGF) has been shown to regulate immune cell function in addition to its neurotrophic effect. The purpose of the present study is to determine whether NGF induces migration and differentiation of murine monocytes (isolated from peripheral blood and resident in the cornea) into dendritic cells.
C57BL/6 mice (8 to 12 weeks of age) were used for the experiments. Monocytes were isolated from peripheral blood with a two-step density gradient procedure: a single density Ficoll (1.088 g/ml) and then a continuous Percoll gradient. The effect of NGF (10ng/ml, 100ng/ml) on monocyte migration was assessed using migration chambers with 8µm micropore membranes. The migration membranes were also immunostained for the dendritic cell marker CD11c to determine if differentiation occurred. Expression of CD11c by the isolated monocytes was assessed with flow cytometry after culturing with NGF (100ng/ml). For in vivo experiments, NGF (1µg/ml, 2µl) was applied by intrastromal injection, and then the expression of CD11c was assessed 16 hrs later by immunostaining.
The purity of the isolated monocytes was greater than 95%, as assessed by observing cell attachment to culture dishes and non-specific esterase staining. About 60% of the isolated cells were CD115 and CD204 positive by flow cytometry. NGF induced migration of monocytes (44 ± 2.54 cells/field for 100ng/ml, 32.3 ± 5.6 cells/field for 10ng/ml) in vitro within 3hrs. The effect of 100ng/ml NGF was close to that of fibronectin (53.6 ± 5.72 cells/field) the positive control. Also the migrated cells developed dendrites and expressed CD11c by 12hrs as detected by immunostaining. An elevated level of CD11c expression was also detected with flow cytometry. NGF intrastromal injection increased the number of CD11c positive cells in the center and periphery of the stroma as determined by immunostaining both wholemount cornea and cryosections.
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