SH2-domain mutations in STAT3 in hyper-IgE syndrome patients result in impairment of IL-10 function

Autosomal-dominant hyper-IgE syndrome (AD-HIES) is a primary immunodeficiency caused by STAT3 mutations. This inherited condition is characterized by eczema, staphylococcal cold abscesses and recurrent pulmonary infections. Given that STAT3 is involved in IL-10 signaling, we examined the immunoregulatory role of IL-10 in inflammation by studying the effects of IL-10 on monocytes, neutrophils and monocyte-derived DCs from HIES subjects. Analysis of gene expression in PBMCs and neutrophils isolated from HIES patients and stimulated with LPS in the presence of IL-10 showed reduced expression of IL1RN, which encodes IL-1 receptor antagonist (IL-1ra), and SOCS3 mRNA but increased CXCL8 mRNA expression. Moreover, secretion of the anti-inflammatory protein IL-1ra was reduced in AD-HIES patients. DCs from HIES patients secreted higher levels of TNF-α, IL-6 and, to a lesser extent, IL-12 when these cells were cultured in the presence of IL-10. These results suggest that IL-10 activity is affected in myeloid cells (e.g. monocytes, DCs) of HIES patients. Impairment of IL-10 signaling in patients with AD-HIES might result in an altered balance between pro-inflammatory and anti-inflammatory signals and might lead to persistent inflammation and delayed healing after infections.     

Quercetin inhibits LPS-induced delay in spontaneous apoptosis and activation of neutrophils

Objective: To investigate the effects of quercetin, an herbal flavonoid, on LPS-induced delay in spontaneous apoptosis, adhesion molecules (CD62L, CD11b/CD18) expression of neutrophils, and superoxide (O₂⁻) generation by LPS-primed fMLP-induced human neutrophils. Methods: Neutrophils were incubated in the presence or absence of lipopolysaccharide (LPS) at a final concentration of 1 μg/ml for 24 hours. Some wells with neutrophils were pre-treated with quecetin at the final concentration ranging from 0–100 μM for 30 min and then 1 μg/ml LPS was added into the cultures for 24 hours. The apoptosis of neutrophils was evaluated by flowcytometry analysis of propidum iodide (PI)-staining of the nuclei and annexin V staning of phosphatidylserine (PS) in the cell membrane. Agarose gel electrophoresis of low molecular weight DNA was performed to analyze DNA fragmentation. The effects of quercetin on adhesion molecules were detected by using flowcytometry analysis. The generation of O₂⁻ by LPS-primed fMLP-induced neutrophils was determined by reduced cytochrome c assay. Results: LPS markedly inhibited the spontaneous apoptosis of neutrophils, but the inhibitory effect was abrogated after the pre-treatment of neutrophils with quercetin (~40 μM) for 30 min. Quercetin (40 μM) also prevented LPS-induced down-regulation of CD62L expression, up-regulation of CD11b/CD18 expression, and O₂⁻ generation by fMLP-induced neutrophils. Conclusion: As one of the pro-inflammatory factors, LPS aggravates inflammation through priming neutrophils to synthesize/release cytotoxic contents and prolonging functional lifespan of neutrophils by delaying the spontaneous apoptosis. Thus, our data suggest to us that quercetin might decrease the susceptibility of neutrophils to pro-inflammatory factors (e. g. LPS), which could partially explain the anti-inflammatory mechanisms of quercetin. Received 5 May 2005; returned for revision 26 August 2005; accepted by I. Ahnfelt R?nne 5 September 2005     

Anti-inflammatory mechanism of a folk herbal medicine, Duchesnea indica (Andr) Focke at RAW264.7 cell line

This study is to explore the anti-inflammatory mechanism of the ethanol extract of Duchesnea indica (Andr) Focke. An inflammatory cellular model was established by addition of lipopolysaccharide (LPS) on RAW264.7 cell line. The cellular secretion of TNF-alpha, IL-1beta, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-alpha, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, and activation of NF-kappaB were assayed by ELISA, the Griess method, real-time quantitative PCR, and Western blot and immunocytochemistry method, respectively. The ethanol extract of D. indica not only reduced production of pro-inflammatory cytokines and mediators and blocked NF-kappaB activation, but also slightly promoted release of the anti-inflammatory mediator HO-1 and suppressed IL-10 secretion. In conclusion, the anti-inflammatory effects of the extract of D. indica are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-kappaB activation. The extract of D. indica can also slightly promote HO-1 production to reduce inflammation.     

草木犀石油醚提取物对小鼠巨噬细胞促炎介质的影响

目的 研究草木犀石油醚提取物在体外的抗炎作用.方法 采用小鼠巨噬细胞系RAW 264.7建立炎症细胞模型,加入10 μg/L的LPS培养液和不同浓度的草木犀石油醚提取物进行干预.ELISA法检测上清液中TNF-α, IL-1β, IL-6和NO的分泌量;实时荧光定量RT-PCR检测TNF-α, iNOS 和 COX-2的 mRNA表达;Western 印迹法检测COX-2 蛋白的表达.结果 草木犀提取物干预后细胞所分泌的炎性介质(TNF-α, IL-1β, IL-6和NO)与模型组相比均显著降低(P＜0.01),并存在剂量依赖关系;RT-PCR结果显示干预后细胞TNF-α,iNOS和COX-2的mRNA表达水平显著降低(P＜0.01),也存在剂量依赖关系;Western印迹结果显示草木犀石油醚提取物及地塞米松干预后COX-2蛋白水平明显降低(P＜0.01).结论 草木犀的石油醚提取物通过下调LPS诱导的巨噬细胞表达炎性介质而发挥其体外抗炎作用, 且其下调作用呈剂量依赖性.     

LPS的直接诱导对肺微血管内皮细胞IL-8的表达及对PMN趋化作用影响的研究

目的探讨LPS的直接诱导作用对肺微血管内皮细胞(PMVEC)IL -8表达的影响及诱导发生的PMVEC对多形核中性粒细胞 (PMN)趋化作用的影响。方法100ng/mlLPS刺激PMVEC0、1、2、4、6、8h或1、10、100ng/mlLPS刺激6h,ELISA和原位杂交试验分别检测培养液上清中分泌的IL -8及PMVEC内IL-8mRNA的表达 ,同时琼脂糖平板法检测对PMN的趋化作用 ,并通过抗IL -8的抗体抑制试验观察对趋化作用的影响。结果LPS能显著促进PMVEC表达IL -8 ,包括促进IL-8mRNA的表达及IL -8的分泌。在时间上mRNA的表达先于IL -8分泌。LPS能促进PMVEC对PMN的趋化 ,随刺激作用的持续 ,诱导的PMVEC对PMN的趋化作用加强。抗IL -8抗体能显著抑制对PMN的趋化作用(P<0.01)。结论表明细菌致病因子LPS的直接诱导确能促进PMVEC高效表达和分泌IL -8 ,从而为PMN的迁移提供必需的物质条件,发挥对PMN的趋化作用而参与导致肺损伤。     

Anti-Inflammatory Mechanism of a Folk Herbal Medicine,Duchesnea indica (Andr) Focke at RAW264.7 Cell Line

This study is to explore the anti-inflammatory mechanism of the ethanol extract of Duchesnea indica (Andr) Focke. An inflammatory cellular model was established by addition of lipopolysaccharide (LPS) on RAW264.7 cell line. The cellular secretion of TNF-α, IL-1β, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-α, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, and activation of NF-κB were assayed by ELISA, the Griess method, real-time quantitative PCR, and Western blot and immunocytochemistry method, respectively. The ethanol extract of D. indica not only reduced production of pro-inflammatory cytokines and mediators and blocked NF-κB activation, but also slightly promoted release of the anti-inflammatory mediator HO-1 and suppressed IL-10 secretion. In conclusion, the anti-inflammatory effects of the extract of D. indica are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-κB activation. The extract of D. indica can also slightly promote HO-1 production to reduce inflammation.     

The expression pattern of the ITIM-bearing lectin CLECSF6 in neutrophils suggests a key role in the control of inflammation

In our study of the modulation of the expression of inflammation-related genes in neutrophils, we have found a gene called CLECSF6 (C-type lectin superfamily 6). CLECSF6 expresses two mRNA species at low levels in resting neutrophils. Here, we describe for the first time the sequence of the short mRNA version. It lacks amino acids that are likely to affect the functionality of its protein product. GM-CSF, IL-3, IL-4, and IL-13 caused an accumulation of the short CLECSF6 mRNA in neutrophils. The surface expression of the CLECSF6 protein was reduced by TNF-alpha, IL-1alpha, LPS, and Matrigel. CLECSF6 bears the immunoreceptor tyrosine-based inhibition motif (ITIM) involved in signal transduction resulting in the inhibition of leukocyte activation. We propose that some neutrophil activators modulate the expression of CLECSF6 at the mRNA (GM-CSF, IL-3, IL-4, and IL-13) or protein (TNF-alpha, IL-1alpha, LPS, and Matrigel) levels in ways that block ITIM-based transduction of anti-inflammatory signals and therefore promote inflammation.     

The intratracheal administration of endotoxin and cytokines. III. The interleukin-1 (IL-1) receptor antagonist inhibits endotoxin- and IL-1-induced acute inflammation.

Endotoxin, a lipopolysaccharide (LPS) component of gram-negative bacteria, induces alveolar macrophages to express interleukin-1 (IL-1). Lipopolysaccharide and IL-1 both cause severe acute neutrophilic inflammation in the lung after intratracheal injection, suggesting that LPS-induced IL-1 expression contributes to the pathogenesis of LPS-induced acute inflammation. In the present study, the role of IL-1 in LPS-induced acute pneumonia was investigated by quantitating the acute inflammation occurring at 6 hours after the intratracheal injection of LPS as compared to the same timepoint after the intratracheal coinjection of LPS and IL-1 receptor antagonist (IL-1ra). The IL-1ra was found to inhibit LPS-induced acute inflammation (P greater than 0.0001) as measured by the number of neutrophils recovered in bronchoalveolar lavage. The LPS-induced emigration of neutrophils was inhibited by as much as 45%. Recombinant IL-1 beta-induced neutrophil emigration into the lung was inhibited by 95% when IL-1ra was coinjected intratracheally with IL-1 beta. Coinjection of recombinant IL-1 beta and LPS increased the neutrophilic exodus as compared to the intratracheal injection of either agent alone. Intratracheal injection of LPS induces a progressive increase in IL-1ra mRNA expression in whole-lung RNA preparations, suggesting that endogenous IL-1ra may play an important role as a negative feedback mechanism to downregulate LPS initiated IL-1-mediated acute inflammation. In conclusion IL-1ra inhibits both LPS- and IL-1-induced neutrophilic inflammation and may therefore prove clinically useful as an anti-inflammatory agent for the therapy of either septic or aseptic IL-1-mediated acute inflammation.     

Chemoattractant-induced release of elastase by lipopolysaccharide (LPS)-primed neutrophils; inhibitory effect of the anti-inflammatory drug nimesulide

Human neutrophils, pre-exposed to low concentrations (1–10 ng/ml) of bacterial LPS in the presence of 1% autologous serum, released elastase activity in response to n-formyl-met-leu-phe (fMLP). Both cell incubation with LPS without subsequent fMLP stimulus and fMLP stimulation without prior exposure to LPS failed to promote significant elastase release. Therefore, LPS primes neutrophils for the subsequent release of elastase in response to fMLP. Compared with fMLP, human recombinant C5a had a slight although not significant activity, whereas other chemoattractants such as IL-8, platelet-activating factor and leukotriene B₄ were ineffective. The fMLP-induced response of LPS-primed neutrophils was susceptible to suppression by the methane-sulphonanilide anti-inflammatory drug nimesulide and RO 20-1724, which selectively inhibit cAMP-catabolizing phosphodiesterase type IV. This suggests that the elastase release by LPS-primed neutrophils is likely to be controlled by intracellular cAMP, and raises the possibility of limiting pharmacologically the elastase-mediated tissue injury during neutrophilic inflammation.     

Establishment of the Insulin Resistance Induced by Inflammatory Response in 3T3-L1 Preadipocytes Cell Line

In the light of given recent reports, insulin resistance related to inflammation is characterized by increasing a diverse array of pro-inflammatory cytokines. In this study, hypothesizing that 3T3-L1 non-differentiated preadipocytes cell line as a cell model could be used to investigate this linkage, the aim is to determine whether the preadipocytes induced by different inflammatory responses could cause insulin resistance. This paper has determined the time and concentration-dependent effects of insulin on glucose consumption in the 3T3-L1 non-differentiated preadipocytes. Glucose consumption has also been assayed in the preadipocytes which are treated with LPS and CM originated from LPS-activated RAW264.7. Then protein level of each group has been measured by coomassie brilliant blue protein kit. Furthermore, secretion levels of IL-6 and TNF-alpha are measured by ELISA in the supernatant of RAW264.7 and preadipocytes. Finally, the mRNA expressions for IL-6, TNF-alpha and PPARgamma has been assessed by RT-PCR. The results show that administration of LPS and CM both can increase releases of IL-6 and TNF-alpha, as well as gene expression of IL-6 mRNA; this change is accompanied with suppression of PPARgamma mRNA activation in 3T3-L1 undifferentiated preadipocytes. In conclusion, our results suggest that in preadipocytes, pro-inflammatory cytokines can result in insulin resistance, and deserve further investigation to be helpful for treatment and revealing mechanisms of T2DM.     

Dexamethasone Attenuates LPS-induced Acute Lung Injury through Inhibition of NF-κB,COX-2, and Pro-inflammatory Mediators

Dexamethasone (DEX) is a synthetic glucocorticoid with potent anti-inflammatory effects that is widely used to treat inflammatory diseases. The aim of the present study was to investigate the possible protective effect of DEX on the lipopolysaccharides (LPS)-induced acute lung injury (ALI) in a mouse model. Animals were pretreated with DEX (5 and 10 mg/kg, i.p.) for seven days and acute lung injury was induced by intranasal (i.n.) administration of LPS on day 7. In the present study, administration of LPS resulted in significant increase in neutrophils and lymphocytes count whereas a substantial reduction in T cell subsets (CD3⁺ and CD4⁺) and pro-inflammatory (IL-6 and TNF-α) cytokines occurred, which were reversed by DEX treatment. RT-PCR analysis revealed an increased mRNA expression of IL-6, TNF-α, COX-2, iNOS, and NF-κB p65 and decreased IL-10 in the LPS group, which were reversed by treatment with DEX in lung tissues. Western blot analysis revealed an increased expression of COX-2, iNOS and NF-κB p65 in the LPS-group, which was reduced by treatment with DEX. Compared with the LPS group, the DEX treatment also demonstrated a considerable increase in the protein expression level of IL-10 cytokine. Administration of LPS resulted in marked increase in malondialdehyde (MDA) levels and myeloperoxidase (MPO) activity whereas noticeable decrease in glutathione (GSH) content. These changes were significantly reversed by treatment with DEX. The histological examinations revealed protective effect of DEX while LPS group aggravated lung injury. The present findings demonstrate the potent anti-inflammatory action of the DEX against acute lung injury induced by LPS.     

E5564 (Eritoran) inhibits lipopolysaccharide-induced cytokine production in human blood monocytes

Objective and design In this ex vivo laboratory study, we investigated the effects of E5564 (eritoran), a toll-like receptor 4-directed endotoxin antagonist, on intracellular expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated human monocytes assessed by flow cytometry. Material and method Whole blood samples from 10 healthy volunteers (average age: 32 ± 2 years) were pre-incubated with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 10 ng/ml E5564 for 45 min and after this stimulated with LPS (0.2 ng/ml), a dose we found to be the most effective for stimulation. Samples were incubated for 3 h at 37°C and 5% CO₂. Intracellular expression of IL-6 and TNF-α was assessed by flow cytometry. Results Our investigation showed that E5564 (0.03 ng/ml up to 10 ng/ml) caused a dose-dependent inhibitory effect on IL-6 and TNF-α production in LPS-stimulated human monocytes. Conclusions The results of this investigation led us to conclude that E5564 has a remarkable LPS inhibitory activity manifested via down-regulation of the intracellular generation of pro-inflammatory cytokines IL-6 and TNF-α in human monocytes. Received 8 April 2006; returned for revision 13 June 2006; accepted by G. Wallace 7 July 2006     

Metallic but not ceramic wear particles increase prostaglandin E2 release and interleukin-1beta gene expression in human blood monocytes in vitro

In this study the potential of clinically relevant alumina ceramic and metal wear particles to induce an in vitro inflammatory response was assessed in human monocytes and lymphocytes isolated from healthy donors by measuring prostaglandin E2 (PGE2) levels and mRNA expression of various pro-inflammatory cytokines. Bacterial lipopolysaccharide (LPS) was used as positive control. LPS significantly increased PGE2 levels in the incubation medium of monocyte cultures after 24 h. Alumina had no effect on PGE2 production, whereas metals induced a concentration-dependent increase in PGE2 release, that was statistically significant at the dose of 0.1 mg/ml. In lymphocytes, LPS elicited a weak but significant increase in PGE2 release, whereas both alumina and metals did not modify PGE2 amounts at any of the concentrations tested. The gene expression of a number of pro- and anti-inflammatory cytokines was assessed in monocytes and lymphocytes exposed to LPS, 0.1 mg/ml alumina or 0.1 mg/ml metals for 24 h. In monocytes, LPS caused a 2-fold increase in interleukin-1beta (IL-1beta) mRNA levels. The exposure of monocytes to metals resulted in a selective increase in IL-1beta mRNA accumulation (+48% compared to control). By contrast, alumina did not modify IL-1beta mRNA levels. None of the test substances elicited any response on purified lymphocyte population. These findings suggest that PGE2 production and IL-1 mRNA expression are a reliable marker to study the pro-inflammatory effects of wear debris in vitro. The lower activity of alumina compared to metals suggests that the former should be preferred in implants for its favorable biological and mechanical behavior.     

LPS刺激诱导人卵巢癌细胞株Toll样受体4表达及细胞免疫调节

目的 探讨脂多糖(LPS)刺激对体外培养的人卵巢癌细胞株SKOV3的生长、Toll样受体4(TLR4)的表达、细胞活性氧(ROS)表达及6种炎性细胞因子分泌水平变化的影响.方法 用流式细胞仪测定不同浓度LPS刺激SKOV3 4 h后TLR4的表达水平;用LPS分别刺激SKOV3细胞不同时间后,MTT法检测细胞增殖情况,流式细胞仪分析TLR4表达、细胞周期分布、ROS表达水平以及细胞因子分泌水平.结果 TLR4表达与LPS作用浓度之间存在浓度依赖性和最大量效曲线关系;LPS刺激组与正常组的细胞增殖、细胞周期PrI值(细胞增殖指数,S+G_2/M)、ROS表达水平、细胞因子分泌水平均有显著性差异.结论 LPS具有诱导卵巢癌细胞TLR4表达、活性氧表达、炎症因子分泌以及细胞增殖和抑制的作用.     

Anti-Inflammatory Effect of Apigenin on LPS-Induced Pro-Inflammatory Mediators and AP-1 Factors in Human Lung Epithelial Cells

Apigenin is one of the plant flavonoids present in fruits and vegetables, acting as an important nutraceutical component. It is recognized as a potential antioxidant, antimicrobial, and anti-inflammatory molecule. In the present study, the mechanism of anti-inflammatory action of apigenin on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and activator protein-1 (AP-1) factors in human lung A549 cells was investigated. The anti‐inflammatory activity of apigenin on LPS-induced inflammation was determined by analyzing the expression of pro-inflammatory cytokines, nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and different AP-1 factors. Apigenin significantly inhibited the LPS-induced expression of iNOS, COX-2, expression of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, and TNF-α), and AP-1 proteins (c-Jun, c-Fos, and JunB) including nitric oxide production. Study confirms the anti-inflammatory effect of apigenin by inhibiting the expression of inflammatory mediators and AP-1 factors involved in the inflammation and its importance in the treatment of lung inflammatory diseases.     

MLL3 Inhibits Apoptosis of Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Promotes Secretion of Inflammatory Factors by Activating CCL2 and the NF-κB Pathway

Rheumatoid arthritis (RA) remains the most common inflammatory arthritis and a major cause of disability. This study investigated the mechanism of MLL3 in fibroblast-like synoviocyte (FLS) apoptosis and inflammatory factor secretion in RA. Expression of MLL3 in synovial tissue of RA patients and patients with bone trauma was detected. FLS was isolated and identified by flow cytometry. Expressions of TNF-α, IL-1β, IL-8, and IL-10 and apoptosis were measured by MTT, flow cytometry, and ELISA. Western blot and qRT-PCR were performed to detect MLL3 and CCL2 expressions, H3K4me3 level, and NF-κB pathway-related proteins in rat joints. MLL3 was highly expressed in the synovial tissue of RA patients, and silencing MLL3 in FLS-RA promoted apoptosis, inhibited pro-inflammatory factors TNF-α, IL-1β, and IL-8 secretion, and promoted anti-inflammatory factor IL-10 secretion. Inhibition of MLL3 suppressed intracellular H3K4me3 and CCL2 expressions. CCL2 activated the NF-κB pathway to promote pro-inflammatory factors TNF-α, IL-1β, and IL-8, inhibit anti-inflammatory factor IL-10, and inhibit apoptosis in FLS-RA. Inhibition of MLL3 expression in RA rats reduced joint redness, swelling, and intra-articular inflammation, but increasing H3K4me3 level reversed the ameliorative effects of sh-MLL3 on RA rats. Collectively, MLL3 activated the NF-κB pathway by increasing H3K4me3 modification in the CCL2 promoter region in FLS-RA, thereby inhibiting apoptosis and promoting pro-inflammatory factors of FLS-RA.

     

Induction of Interleukin 1α (IL-1α) and IL-1β mRNA Expression and Cellular IL-1 Production by Anti-HLA-DR Antibodies in Human Monocytes

We studied the role of HLA class II antigens in the regulation of interleukin 1 (IL-1) production in human monocytes. Monocytes were cultured with monoclonal anti-HLA-DR antibodies for 24 h after which cellular (i.e. intracellular and membrane-associated) IL-1 production, IL-1 secretion, and the expression of IL-1 alpha and IL-1 beta mRNA were determined. One of the anti-HLA-DR antibodies tested (anti-HLA-DR, Becton Dickinson) clearly induced IL-1 alpha and IL-1 beta mRNA expression and cellular IL-1 production. The other anti-HLA-DR antibody tested (OKIa1, Ortho) had no effect on IL-1 production. The stimulatory effect of anti-HLA-DR was enhanced by IFN-gamma in both fresh and aged monocytes. A synergistic effect by anti-HLA-DR and suboptimal doses of LPS (1 ng/ml) on both cellular IL-1 production and secretion was also demonstrated. The possibility of contaminating LPS causing the IL-1-inducing effect of anti-HLA-DR was excluded by the inability of polymyxin B to abolish the anti-HLA-DR-induced IL-1 production.