Abstract
Estrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types. There is now renewed attention on neutrophils and neutrophil serine proteases (NSPs) such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) in inflammation and autoimmunity. In this study, we found that although estrogen treatment significantly reduced total splenocytes number, it markedly increased the splenic neutrophil absolute numbers in estrogen-treated C57BL/6 (B6) mice when compared to placebo controls. Concomitantly, the levels of NSPs and myeloperoxidase (MPO) were highly upregulated in the splenocytes from estrogen-treated mice. Despite the critical role of NSPs in the regulation of non-infectious inflammation, by employing NE-/-/PR3-/-/CG-/- triple knock out mice, we demonstrated that the absence of NSPs affected neither estrogen’s ability to increase splenic neutrophils nor the induction of inflammatory mediators (IFNγ, IL-1β, IL-6, TNFα, MCP-1, and NO) from ex vivo activated splenocytes. Depletion of neutrophils in vitro in splenocytes with anti-Ly6G antibody also had no obvious effect on NSP expression or LPS-induced IFNγ and MCP-1. These data suggest that estrogen augments NSPs, which appears to be independent of enhancing ex vivo inflammatory responses. Since estrogen has been implicated in regulating several experimental autoimmune diseases, we extended our observations in estrogen-treated B6 mice to spontaneous autoimmune-prone female MRL-lpr, B6-lpr and NZB/WF1 mice. There was a remarkable commonality with regards to the increase of neutrophils and concomitant increase of NSPs and MPO in the splenic cells of different strains of autoimmune-prone mice and estrogen-treated B6 mice. Collectively, since NSPs and neutrophils are involved in diverse pro-inflammatory activities, these data suggest a potential pathologic implication of increased neutrophils and NSPs that merits further investigation.
Highlights
Estrogen has been shown to regulate the immune system of both normal and autoimmune individuals either via activation of estrogen receptor α (ERα) and/or ERβ or through ER-independent mechanisms [1,2,3,4,5]
By using Fluorescent-Labeled Inhibitors of Serine Protease (FLISP) Serine Protease Detection Assay, we further demonstrated that there was a significantly higher chymotrypsin-like serine protease activity in living splenocytes from estrogen-treated mice when compared to placebo controls (Fig 1B)
Real-time RT-PCR analysis indicated that mRNA expression levels of all three neutrophil serine proteases (NSPs) (NE, proteinase 3 (PR3), and cathepsin G (CG)) were substantially increased in the freshly isolated splenocytes from estrogen-treated mice when compared to placebo controls (Fig 2A)
Summary
Estrogen has been shown to regulate the immune system of both normal and autoimmune individuals either via activation of estrogen receptor α (ERα) and/or ERβ or through ER-independent mechanisms [1,2,3,4,5]. Estrogen is capable of promoting B cell survival and activation or breakdown of B cell tolerance to induce lupus-related serology and pathology in non-autoimmune mice [10,11,12,13] Together, these data demonstrate a pivotal role of estrogen in the regulation of T and B lymphocyte-mediated inflammation and autoimmune responses. NSPs can regulate inflammation via activation of cell surface receptors such as integrins, protease-activated receptors (PARs), and toll-like receptors (TLRs) [14, 16,17,18] In addition to their primary role in innate immune responses and inflammation, neutrophils are critically involved in the adaptive immune response by attracting T cells to sites of inflammation and/or by priming and engaging T cell activation [19, 20]
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