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Abstract
Background and aims Oestrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types and has been implicated in lupus development.
Methods To determine the regulatory role of oestrogen on neutrophil development and function, we treated B6 mice with placebo- or oestrogen implants for 6–8 weeks, and then analysed splenic neutrophil serine proteases (NSPs, such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG)) and myeloperoxidase (MPO) expression by qRT-PCR. NE-/-/PR3-/-/CG-/- triple knockout mice and in vitro depletion of neutrophils approaches were performed to determine the role of NSPs and neutrophils in estrogen-mediated inflammatory responses. The splenic neutrophil and NSPs expression in lupus-prone MRL-lpr, B6-lpr and NZB/WF1 and their respective controls were also analysed.
Results Although oestrogen reduced total splenocytes number, it markedly increased the splenic neutrophil numbers, NSPs and MPO expression in B6 mice (Figure 1). Splenic neutrophils, NSPs and MPO were also significantly increased in MRL-lpr, B6-lpr and NZB/WF1 mice (Figure 2). Despite of the critical role of NSPs and neutrophils in inflammation, depletion of NSPs in vivo did neither affect oestrogen’s ability to increase in splenic neutrophils nor the induction of inflammatory mediators from ex vivo activated splenocytes, and depletion of splenic neutrophils in vitro had also no obvious effect on NSPs expression (due to the increase of NSPs in cells other than neutrophils) and LPS-induced IFNg and MCP-1 (Figure 3).
Estrogen treatment increasesneutrophil number,NSP and MPO expression in wild type B6 mice .(A) Flow cytometry analysis of splenic neutrophil percentage in placebo-and estrogen-treated B6 mice.(B) The total splenocytes count in placebo- and estrogen-treated mice.(C)The total CD11b*GR1* splenic neutrophilcounts in placebo- and estrogen-treated mice.(D and E) Real-time RT-PCR analysis the expressoin of NSPs (D), and MPO (E) in splenocytes from placebo- and estrogen- treated mice. The graphs show means±SEMs (n≥ 4).Unpaired students ttests(placebo vs estrogen) were performed.*, p< 0.05;**, p< 0.01; and ***, p<0.001.
Increased neutrophil percentages , NSPs and MPO expression in the spleen cells of three different murmine lupus strains.The graphs on left panel show the summary of flow analysis of neutrophil percentage in the spleens of MRL-lpr (A),B6-lpr (B), and NZBWFI (C).The graphs on right panel summarized the real-time RT-PCR analysis of the relative expression levels of NE, PR3, CG, and MPO in the splenocytes of these lupus mice. The graphs represented means±SEMs (n≥ 4).Unpaired student t test (MRL vs MRL-lpr; and B6-lpr, NZW vs NZB/WFI); *, p< 0.05; **, p< 0.01; and ***, p< 0.001.
Either depletion of NSPs in vivo or depletion splenic neutrophil in vitro has obvious effect on estrogen-mediated promotion of inflammatory molecules.(A-E): The splenocytes from placebo and estrogen-treated wild type (WT) and NSP−/− knock out mice were simulated with LPS to measure the production of IFN ϒ(A, LPS 6 hours ), IL-6 (B, LPS 24hours), MCP-1(C, LPS 24 hours), and inflammatory molecule NO (D,LPS 48 hours). The expression iNOS protein levels in activated splenocytes (LPS placebo-and estrogen treated B6 mice were treated with anti-mouse Ly6G antibody to deplete neutrophils, and then simulated with LPS for 24 hours to measure IFNϒ(F), IL-6 (G), and MCP-1 (H) production.The graphs show means±SEMs (n≥ 3).Unpaired student t tests (No Ab vs anti-Ly6G) were performed; *,p< 0.05; **,p< 0.01; and ***, p< 0.001.
Conclusions Overall, we demonstrated a remarkable commonality with regards to the increase of neutrophils and NSPs in the spleens of autoimmune-prone mice and estrogen-treated B6 mice.