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J.V. Jester, N. Morishige, B. Holguin, I. Bettahi, L. BenMohamed, G.–C. Perng; Stimulated Corneal Derived Macrophages Induce Activation of Rabbit Keratocytes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1811.
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© ARVO (1962-2015); The Authors (2016-present)
Recent studies have identified a novel population of bone marrow–derived monocytic cells showing dynamic migratory and surveillance activity in the stroma. The purpose of this study was to further characterize the corneal monocytic cell population and determine whether monocytic/macrophages interact with corneal keratocytes.
Stromal cells were isolated from rabbit eyes by collagenase digestion and monocytic cells separated from keratocytes using magnetic bead separation with antibodies specific to rabbit CD45. CD45 cells were further characterized by flow cytometry using anti–rabbit specific antibodies to CD4, CD8, CD25, CD11c and CD11b. Isolated CD45 cells were then allowed to attach to plastic culture dishes to collect stromal macrophages and then stimulated with LPS, LTA and IL–1α at various concentrations. Serum–free keratocyte cultures were then exposed to stimulated, un–stimulated macrophages and conditioned media. Cells were then evaluated by western blotting, live cell imaging and staining for actin, α–smooth muscle actin, vinculin, and fibronectin.
On average 2% of the stromal cell population from collagenase digests were CD45 positive, comprised predominantly of CD11c (80%) and CD11c/11b (10%) cells. Isolated stromal macrophages co–cultured with keratocytes showed similar migratory and surveillance behavior as detected ex vivo using live cell imaging but did not result in activation of keratocytes. Treatment of keratocytes with LPS, LTA and IL–1α also did not result in keratocyte activation consistent with the absence of Toll–like receptor expression by keratocytes identified by western blotting. However, keratocytes co–culture with stimulated macrophages or their conditioned medium resulted in fibroblast differentiation as detected by cell elongation, actin filament assembly and fibronectin organization.
Stimulated corneal–derived macrophages are capable of activating keratocytes under serum–free conditions. These finding support the hypothesis that resident corneal macrophages perform a surveillance function involving early activation of corneal keratocytes in response to injury or infection.
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