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N. Kawanaka, A. W. Taylor; Molecular Characterization of Retinal Pigmented Epithelial (RPE) Mediated Induction of Suppressor Macrophages. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2508.
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We previously demonstrated that healthy retinas have suppressor macrophages (MØ) detected by the simultaneous expression of nitric oxide synthase 2 (NOS2) and Arginase 1. In addition, we found that in wounded retinas the MØ are either inflammatory MØ (only expressing NOS2), or wound repairing MØ (only expressing Arginase1). To understand how this change can happen, we need to identify the soluble factors produced by RPE cells responsible for regulating macrophage functionality in the healthy retina.
Our source of primary MØ were from resident peritoneal exudate cells of C57BL/6J mice. The MØ were treated with the conditioned media (CM) of RPE-eyecups as described previously; however, this time the RPE-CM were absorbed of several suspected neuropeptides and cytokines (CGRP, α-MSH, NPY, PEDF, SOM, and TGF-β) using their corresponding specific antibodies and Protein-G coated beads. The treated MØ were incubated for 24 hours, formalin fixed, and stained with antibodies against the enzymes NOS2 and Arginase1. We also documented the MØ morphology, and assayed nitric oxide (NO) production by the MØ.
The MØ incubated with CM were morphologically activated, co-expressed Arginase 1 and NOS2, with enhanced NO production. Changes in enhanced NO production occurred only when we absorbed the CM of α-MSH or NPY. When α-MSH was absorbed, the CM treated MØ expressed only small amounts of Arginase 1 and NOS2, and were round like inactivated MØ. When NPY was absorbed, the treated MØ expressed only Arginase 1, and were morphologically similar to activated MØ. When both were absorbed, the treated MØ expressed only small amounts of NOS2, and were round like inactivated MØ.
These findings show that RPE mediates the expression of NOS2 and Arginase in primary MØ through at least α-MSH and NPY. The results suggest that in the healthy retina α-MSH is an activator of MØ, and with NPY makes the MØ differentiate into suppressor MØ.
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