S-Nitrosylated S100A8: Novel Anti-Inflammatory Properties

Abstract

S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-γ, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.

S100A8 Induces IL-10 and Protects against Acute Lung Injury

“…The S100A8/S100A9 complex was also proposed to protect against LPS injury by directly binding and neutralizing several proinflammatory cytokines (IL-1b, IL-6, and TNF-a), thereby suppressing overproduction of NO (40). In contrast, we show induction of IL-10 by S100A8, reduced mastcell activation that can initiate vascular responses (41), and suppression of proinflammatory genes induced by LPS. S100A8 has a single, highly reactive Cys residue, and care was taken to ensure that the preparation used in this study was in the reduced form.…”

Hsp 70/Hsp 90 organizing protein as a nitrosylation target in cystic fibrosis therapy

“…Transnitrosation reactions are increasingly recognized to be important for cell signaling, and Hop interacts with and regulates the expression and function of a number of proteins that, themselves, are functionally S-nitrosylated. These include Hsp90 itself, protein von Hippel Lindau, S100A class proteins, and others (37)(38)(39)(40). Hop is believed to have an array of important cellular functions that could be affected by GSNO (23,24), an important consideration with regard to potential toxicities of S-nitrosothiol therapies.…”

Calgranulins May Contribute Vascular Protection In Atherogenesis

“…19, 22 In keeping with a role in resolution of inflammation, we find that classically activated macrophages stimulated with interferon-γ (IFN-γ ) plus LPS (M1 type) do not express S100A8 whereas inducible nitric oxide synthase (iNOS) is highly upregulated. 23 On the other hand, macrophages differentiated to the M2 type (mediate resolution), express S100A8 without further stimulation [Hiroshima & Geczy, unpubl. data].…”

“…ulating factor (GM-CSF) express high levels of S100A8 and S100A9 compared with M-CSF differentiated macrophages and, like macrophages, 23 LPS plus interferon (IFN)-γ reduces expression. 25 Several nonmyeloid cell types express calgranulins following activation, including microvascular endothelial cells (EC); 26 GC are similarly synergistic.…”