Role of potassium channels in the regulation of cytokine release from THP-1 cells

The role of K⁺ channels in the regulation of cytokine release was investigated. Quinine, a non-selective K⁺ channel blocking agent inhibited both TNF and IL-1 release but had non-specific effects on cell function. Glipizide and glibenclamide, inhibitors of ATP-sensitive K⁺ channels, reduced IL-1 release but not TNF, whereas apamin, a selective inhibitor of low-conductance Ca²⁺-sensitive channels, inhibited TNF release, potentiated IL-1 but had no effect on IL-6 or IL-8 release. These results suggest that K⁺ channels may differentially regulate cytokine production by THP-1 cells.     

Role of potassium channels in the regulation of cytokine release from THP-1 cells

The role of K⁺ channels in the regulation of cytokine release was investigated. Quinine, a non-selective K⁺ channel blocking agent inhibited both TNFα and IL-1β release but had non-specific effects on cell function. Glipizide and glibenclamide, inhibitors of ATP-sensitive K⁺ channels, reduced IL-1β release but not TNFα, whereas apamin, a selective inhibitor of low-conductance Ca²⁺-sensitive channels, inhibited TNFα release, potentiated IL-1β but had no effect on IL-6 or IL-8 release. These results suggest that K⁺ channels may differentially regulate cytokine production by THP-1 cells.     

Activated T killer cells induce apoptosis in lung epithelial cells and the release of pro-inflammatory cytokine TNF-alpha

Apoptosis is thought to be involved in lung epithelial cell damage in acute respiratory distress syndrome and interstitial pneumonia. Both the role of apoptosis and its underlying molecular mechanisms in human lung tissue remain unclear. To address these issues, we developed an in vitro assay in which a human lung epithelial cell line and a staphylococcal enterotoxin B (SEB)-reactive human CD8(+) CTL line were co-cultured in the presence of SEB. SEB-stimulated CD8(+) CTL induced apoptosis in the lung epithelial cell line primarily through the perforin/granzyme-mediated pathway. In these cells, apoptosis was initially independent of death receptor pathways. We also tested the effect of IFN-gamma on modulation of apoptosis in lung epithelial cells. In IFN-gamma-pretreated lung epithelial cells, CD95 (APO-1/Fas) activation as well as TNF-related apoptosis-inducing ligand (TRAIL) receptor and TNFR activation led to apoptosis. Furthermore, we found that the interaction of SEB-stimulated CD8(+) CTL with lung epithelial cells induced an increase in TNF-alpha secretion. These results suggest an important role for bacterial superantigen-reactive CD8(+) CTL in induction of lung epithelial cell apoptosis and in modulation of inflammatory processes in lung tissue.     

In vitro studies on the effect of particle size on macrophage responses to nanodiamond wear debris

Nanostructured diamond coatings improve the smoothness and wear characteristics of the metallic component of total hip replacements and increase the longevity of these implants, but the effect of nanodiamond wear debris on macrophages needs to be determined to estimate the long-term inflammatory effects of wear debris. The objective was to investigate the effect of the size of synthetic nanodiamond particles on macrophage proliferation (BrdU incorporation), apoptosis (Annexin-V flow cytometry), metabolic activity (WST-1 assay) and inflammatory cytokine production (qPCR). RAW 264.7 macrophages were exposed to varying sizes (6, 60, 100, 250 and 500 nm) and concentrations (0, 10, 50, 100 and 200 μg ml(-1)) of synthetic nanodiamonds. We observed that cell proliferation but not metabolic activity was decreased with nanoparticle sizes of 6-100 nm at lower concentrations (50 μg ml(-1)), and both cell proliferation and metabolic activity were significantly reduced with nanodiamond concentrations of 200 μg ml(-1). Flow cytometry indicated a significant reduction in cell viability due to necrosis irrespective of particle size. Nanodiamond exposure significantly reduced gene expression of tumor necrosis factor-α, interleukin-1β, chemokine Ccl2 and platelet-derived growth factor compared to serum-only controls or titanium oxide (anatase 8 nm) nanoparticles, with variable effects on chemokine Cxcl2 and vascular endothelial growth factor. In general, our study demonstrates a size and concentration dependence of macrophage responses in vitro to nanodiamond particles as possible wear debris from diamond-coated orthopedic joint implants.     

Distinct cytokine release profiles from human endothelial and THP-1 macrophage-like cells exposed to different amphotericin B formulations

Amphotericin B(AmB) formulations, Fungizone, and Amphotec caused substantially greater proinflammatory cytokine release than AmBisome (L-AMB) and Abelcet in TPA differentiated THP-1 macrophages as determined by antibody based protein arrays. Lipopolysaccharide but not AmB induced significant pro-inflammatory cytokines in human endothelial cells.     

Collagen-chitosan 3-D nano-scaffolds effects on macrophage phagocytosis and pro-inflammatory cytokine release

Macrophages are effector cells in the innate and adaptive immune systems and in situ exist within three-dimensional (3-D) microenvironments. As there has been an increase in interest in the use of 3-D scaffolds to mimic natural microenvironments in vitro, this study examined the impact on cultured mice peritoneal macrophages using standard 2-D plates as compared to 3-D collagen-chitosan scaffolds. Here, 2-D and 3-D cultured macrophages were evaluated for responses to lipopolysaccharide (LPS), dexamethasone (Dex), BSA (bovine serum albumin), safranal (herbal component isolated from safranal [Saf]) and Alyssum homolocarpum mucilage (A. muc: mixed herbal components). After treatments, cultured macrophages were evaluated for viability, phagocytic activity and release of tumor necrosis factor (TNF)-α and interleukin (IL)-1β pro-inflammatory cytokines. Comparison of 2-D vs 3-D cultures showed that use of either system – with or without any exogenous agent – had no effect on cell viability. In the case of cell function, macrophages cultured on scaffolds had increases in phagocytic activity relative to that by cells on 2-D plates. In general, the test herbal components Saf and A. muc. had more impact than any of the other exogenous agents on nanoparticle uptake. With respect to production of TNFα and IL-1β, compared to the 2-D cells, scaffold cells tended to have significantly different levels of production of each cytokine, with the effect varying (higher or lower) depending on the test agent used. However, unlike with particle uptake, here, while Saf and A. muc. led to significantly greater levels of cytokine formation by the 3-D culture cells vs that by the 2-D plate cells, there was no net effect (stimulatory) vs control cultures. These results illustrated that collagen-chitosan scaffolds could provide a suitable 3-D microenvironment for macrophage phagocytosis and could also impact on the formation of pro-inflammatory cytokines.     

Mycophenolic acid enhanced lipopolysaccharide-induced interleukin-18 release in THP-1 cells via activation of the NLRP3 inflammasome

Abstract

Background: Interleukin (IL)-18 is a pro-inflammatory cytokine that has important functions in host defense. The maturation and secretion of IL-18 has been shown to be regulated by the NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome. Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), in association with lipopolysaccharide (LPS), is able to promote the secretion of IL-18, but the mechanism remains unknown. This study aims to explore the mechanism by which MPA synergizes with LPS to induced IL-18 release.

Methods: THP-1 cells were stimulated with LPS and MPA and treated with or without the inhibitors of caspase-1, Ac-YVAD-cmk or KCl; IL-18 in the supernatants was measured by ELISA. The intracellular protein levels of NF-κB p-p65, pro-IL-18, NLRP3, and cleaved caspase-1(p20) were measured by Western blot.

Results: We found that MPA alone failed to induce IL-18, whereas MPA enhanced LPS-mediated IL-18 release. MPA did not affect the intracellular protein levels of NF-κB p-p65 or pro-IL-18 but activated the NLRP3 inflammasome. Ac-YVAD-cmk or increasing extracellular K⁺ blocked the activation of caspase-1 and attenuated the release of IL-18.

Conclusions: Taken together, MPA synergized with LPS to induce the release of IL-18 via activating the NLRP3 inflammasome and increasing the degradation of pro-IL-18, rather than by enhancing the production of pro-IL-18.     

IM-133N modulates cytokine secretion by RAW264.7 and THP-1 cells

An indigenous herbal extract IM-133N containing extracts of Prosopis glandulosa Torr and Symplocos racemosa Roxb were evaluated for potential immunomodulatory effects using RAW264.7 and THP-1 cells. The incubation of the cells for 24?h with IM-133N over a dose range 0–125?µg/ml did not cause cytotoxicity that exceeded 10%. The results indicated that non-cytotoxic doses of IM-133N effectively up-regulated iNOS, TNFα, IL-6, IL-10, IL-8 and IFNγ gene expression in both the RAW264.7 and THP-1 cells. The results also indicated IM-133N elicited dose-related increases in nitric oxide (NO) and tumor necrosis factor (TNF)-α production by RAW264.7 or THP-1 cells. These results demonstrated that IM-133N could stimulate NO and induced pro-inflammatory cytokine expression by monocytes/macrophages. As clinical studies have shown IM-133N to be an effective immunomodulator without any adverse effects, the results of the present study provide further support for the potential use of this agent as an immunostimulant or as an immunotherapy adjuvant.     

Estrogen regulates cytokine release in human mast cells.

Estrogens are important for bone homeostasis and are classified as anti-resorptive agents. In ovariectomized rats, mast cell changes occurred during the activation of resorption. In addition, quantitative changes occurred in mast cell population residing near the site undergoing resorption. Considering these studies, mast cells may play a role in osteoporosis. Therefore, it is of paramount importance to study mast cell cytokine production also in the presence or absence of estrogen. When cultured in the absence of estrogen, human mast cells treated with PMA or A23187 demonstrated significantly greater release of TNF-alpha and IL-6 than cells grown under estrogen-depleted condition. Our results show that treatment of mast cells with estrogen prevented PMA or A23187-stimulated TNF-alpha or IL-6 release. These data provide evidence for a potent inhibition of cytokines by estrogen in human mast cells. This study may help to explain the association between mast cells and osteoporosis.     

The effect of diffusion hardened oxidized zirconium wear debris on cell viability and inflammation--an in vitro study

Wear debris generation in total joint replacement remains a concern because of its association with aseptic loosening, osteolysis, and ultimately implant failure. In a quest to develop new implant materials with reduced wear and improved biocompatibility a new composition of oxidized Zr2.5Nb alloy; diffusion hardened oxidized zirconium (DHOxZr) has been developed. In this study, we have determined the in vitro biocompatibility of the wear debris of this new composition and compared it to wear debris particles of Ti6Al4V, Cobalt, and CoCr. The cytotoxicity of these particles on fibroblast-like cells (L929) and osteoblast-like cells (SaOS2) was assessed using lactate dehydrogenase and DNA quantification assays. The inflammatory response to these particles was assessed by release of interleukin-1 beta and tumor necrosis factor from macrophage-like cells. The results showed that wear debris generated from DHOxZr was less cytotoxic and elicited a reduced inflammatory response as compared to that of Cobalt or CoCr and might therefore offer a benefit as a joint prosthesis.     

Selective permissiveness of TPA differentiated THP-1 myelomonocytic cells for human cytomegalovirus strains AD169 and Towne

Two highly passaged laboratory strains of human cytomegalovirus (HCMV), AD169 and Towne, were tested for their ability to infect and replicate in THP-1 myelomonocytic cells differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment of human THP-1 cells increased the number of cells that expressed HCMV immediate early (IE1) antigen from 0.06% prior to TPA treatment to 12% following cell differentiation. The Towne but not the AD169 strain replicated in differentiated THP-1 cells as determined by HCMV DNA replication and infectious virus production. Major early (HCMVTRL4) mRNA was present in both the abortive and productive THP-1 cell infections.     

内皮细胞在感染性休克炎症性细胞因子释放中的作用

目的 以感染性休克患者血清刺激人原代内皮细胞(HPAEC)和外周血单个核细胞(PBMC),观察内皮细胞在感染性休克细胞因子释放中的作用.方法 采用密度梯度离心法分离健康人PBMC;RT-PCR及免疫印迹检测HPAEC细胞表面标志CD144和血管假性血友病因子(von Will-ebrand factor,vWF)的表达;ELISA检测感染性休克患者和健康对照者血清中的IL-6、TNF-α、MCP-1的水平;两种血清及LPS刺激HPAEC和PBMC后,ELISA检测培养上清中IL-6、TNF-α、MCP-1的水平;流式细胞技术检测休克患者血清和LPS刺激后HPAEC膜表面ICAM-1的表达;S1P1受体激动剂CYM-5442预处理后,休克血清刺激HPAEC培养上清中IL-6、TNF-α、MCP-1的水平.结果 HPAEC表达内皮细胞标志性分子CD144和vWF;感染性休克患者血清中炎症因子IL-6、TNF-α、MCP-1水平明显高于健康对照(P＜0.01);休克血清和LPS处理PBMC和HPAEC后,相对于对照组,均有明显升高(P＜0.01);PBMC的休克组炎症因子与PBMC的LPS组均无明显差异(P＞0.05),而HPAEC的休克组炎症因子相对于HPAEC的LPS组均明显升高(P＜0.01);同样地,LPS刺激两种细胞后,HPAEC的IL-6、TNF-α明显低于PBMC(P＜0.01),MCP-1的水平无明显变化(P＞0.05),但是休克血清刺激后,HPAEC的IL-6、TNF-α与PBMC无明显变化(P＞0.05),MCP-1则明显升高(P＜0.01);休克患者血清刺激S1P1受体特异性激动剂CYM-5442预处理的HPAEC后,培养上清中炎症因子IL-6、TNF-α、MCP-1水平明显降低(P＜0.01).结论 内皮细胞在感染性休克细胞因子释放中起着关键作用.     

The effect of interleukin-4 and amphiregulin on the proliferation of human airway smooth muscle cells and cytokine release

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)- BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.