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R. D. Vicetti Miguel, T. L. Cherpes, K. C. McKenna; Aqueous Humor Selectively Inhibits Inducible Nitric Oxide Synthase (NOS2) Activity in Intratumoral Macrophages Through a Post-Translational Mechanism. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5167.
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© ARVO (1962-2015); The Authors (2016-present)
Determine mechanisms that inhibit NOS2 activity in macrophages infiltrating intraocular tumors.
Ovalbumin (OVA) specific CD8+ CTL were intravenously transferred into C57Bl/6 mice 6 or 10 days after injection of OVA-expressing tumors (E.G7-OVA) in the anterior chamber or skin respectively. Six days later intratumoral macrophages were isolated and analyzed for tumoricidal activity and nitric oxide (NO) production. Flow cytometry was used to determine expression of NOS2 and Arginase I (Arg1). Peritoneal and RAW 264.7 macrophages were stimulated in vitro with IFN-γ and TNF or LPS in the presence or absence of aqueous humor (AqH). Nitrite concentration was then determined as an indirect measure of NO production. NOS2 activity in cell lysates was measured by conversion of L-arginine to L-citrulline. RT-PCR for NOS2, GTP cyclohydrolase, CAT2B, Arg1 and Arg2 mRNAs was performed. Flow cytometry was employed to determine protein expression of NOS2, Arg1, CD40, PDL-1, MHCII, IL-12 and TNF.
Intratumoral macrophages isolated from ocular tumors but not skin tumors of CTL transferred mice were impaired in their ability to produce NO despite NOS2 protein expression. Similarly, peritoneal macrophages and RAW 264.7 cells stimulated in the presence of AqH produced significantly less NO despite NOS2 protein expression. Decreased NO production was the result of inhibited NOS2 activity which was not due to increased Arg1 activity or inhibited GTP cylohydrolase activity because mRNAs for these molecules were not affected by AqH and supplementation of cultures with L-arginine and tetrahydrobiopterin did not overcome AqH-mediated NO suppression. TGF-β2, CGRP and α-MSH alone or in combination did not recapitulate the suppressive effect of AqH on NO production by macrophages. Macrophage expression of CD40, PDL-1, MCH-II, IL-12 and TNF were increased upon stimulation and were not affected by AqH, indicating selective inhibition of NOS2 activity.
Reduced NO production by ocular tumor associated macrophages was due to inhibition of NOS2 activity rather than decreased NOS2 protein expression. A similar phenomenon was observed when macrophages were stimulated in the presence of AqH suggesting that the ocular microenvironment is capable of inhibiting macrophage effector function. The effect of AqH on NOS2 activity was not mediated by TGF-β2, CGRP or α-MSH challenging current dogma.
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