Levels Of Interleukin-6 Secretion Research Articles

Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The resultsindicate that the D2andD3 domainsof FliCEcNhave more complex interactionscomparedtothe D1-D2 domainpair., and thesedomains alsoplay a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 hasmore missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulatehigherlevelsofIL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Anti-Inflammatory Mechanisms of Curcumin and Its Metabolites in White Adipose Tissue and Cultured Adipocytes.

The plant-derived polyphenol curcumin alleviates the inflammatory and metabolic effects of obesity, in part, by reducing adipose tissue inflammation. We hypothesized that the benefits of curcumin supplementation on diet-induced obesity and systemic inflammation in mice occur through downregulation of white adipose tissue (WAT) inflammation. The hypothesis was tested in adipose tissue from high-fat diet-induced obese mice supplemented with or without curcumin and in 3T3-L1 adipocytes treated with or without curcumin. Male B6 mice were fed a high-fat diet (HFD, 45% kcal fat) with or without 0.4% (w/w) curcumin supplementation (HFC). Metabolic changes in these mice have been previously reported. Here, we determined the serum levels of the curcumin metabolites tetrahydrocurcumin (THC) and curcumin-O-glucuronide (COG) using mass spectrometry. Moreover, we determined interleukin 6 (IL-6) levels and proteomic changes in LPS-stimulated 3T3-L1 adipocytes treated with or without curcumin by using immunoassays and mass spectrometry, respectively, to gain further insight into any altered processes. We detected both curcumin metabolites, THC and COG, in serum samples from the curcumin-fed mice. Both curcumin and its metabolites reduced LPS-induced adipocyte IL-6 secretion and mRNA levels. Proteomic analyses indicated that curcumin upregulated EIF2 and mTOR signaling pathways. Overall, curcumin exerted anti-inflammatory effects in adipocytes, in part by reducing IL-6, and these effects may be linked to the upregulation of the mTOR signaling pathway, warranting additional mechanistic studies on the effects of curcumin and its metabolites on metabolic health.

OR14-05 Taking Advantage Of The Interleukin-6-Biology In Differentiated Thyroid Cancer To Stimulate Sodium Iodide Symporter (NIS)-mediated Iodide Uptake In Engineered Mesenchymal Stem Cells

Abstract Disclosure: V.F. Koehler: Speaker; Self; Sanofi. L. Drago: None. M. Hageneier: None. C. Kitzberger: None. N. Schwenk: None. K. Shehzad: None. Y. Han: None. J. Nagarajah: None. P.J. Nelson: None. C. Spitzweg: None. Based on its role in mediating iodide uptake from the blood into thyroid follicular cells, the sodium iodide symporter (NIS) provides the basis for the use of radioiodine (RAI) for diagnostic imaging and therapy of differentiated thyroid cancer (DTC). The loss of functional NIS expression leads to RAI-refractory disease. The tumor-selective delivery of NIS as a transgene using mesenchymal stem cells (MSCs) represents a therapeutic strategy for RAI-refractory DTC. Interleukin-6 (IL-6) is a potent cytokine omnipresent in the inflammatory microenvironment of many solid tumors thought to sustain and promote tumor proliferation, invasion and angiogenesis, and contribute to immune escape. With the aim to selectively drive NIS-transgene expression in the context of DTC, MSCs were stably transfected with a NIS-expressing plasmid controlled by the human IL-6-promoter (IL-6-NIS-MSCs). This approach may represent a new tumor-target gene therapy for RAI-refractory DTC. To confirm the inducibility of the IL-6 promoter in IL-6-NIS-MSCs, the murine cytokines (IL-1 β, TNF-α, IFN-γ) were applied to stimulate promoter activation. The resulting functional NIS expression was analyzed by an 125Iuptake assay in vitro. The invitro IL-6 concentration in the papillary thyroid cancer cells BCPAP and K1either co-cultured with or without IL-6-NIS-MSCs was determined by ELISA. Subsequently, IL-6-NIS-MSCs were subjected to a gradient of serum free tumor cell conditioned medium (CM) and serum free unconditioned medium and its migratory response was assessed using 3D live-cell imaging migration assay.IL-6-NIS-MSCs treated with IL-1 β, IFN-γ and TNF-α revealed an increased125I uptake as compared to single stimulation studies. Asa basis for a future in vivo application, incubation of IL-6-NIS-MSC with CM of BCPAP and K1 containing diverse tumor-derived factors or directly co-cultured with these cell lines resulted in significant increase of 125I uptake as compared to untreated cells. The ELISA assay revealed high levels of IL-6 secretion in K1-CM andIL-6-NIS-MSCs co-cultured with BCPAP and K1, while a lower concentration was measured in BCPAP-CM. IL-6-NIS-MSCssubjected to a gradient between a serum free tumor cell-CM and serum free unconditioned medium, showed an increased migration towards tumor cell-CM over a period of 24 h. Taken together, our results indicate the feasibility of targeting IL-6 induction in the inflammatory-rich tumor environment of RAI-refractory DTC to re-establish functional NIS expression using engineered MSCs as delivery vehicles. These data also show us the potential of cytokine-induced promoters for driving NIS transgene expression for a novel imaging and theranostic NIS gene approach. Presentation: Friday, June 16, 2023

Resveratrol protects human nucleus pulposus cells from degeneration by blocking IL-6/JAK/STAT3 pathway

BackgroundNucleus pulposus cells’ (NPCs’) degeneration is mainly responsible for the intervertebral disc degeneration (IDD), which is closely related to inflammatory response. Among the major proinflammatory factors that are related to NPCs’ degeneration, interleukin-6 (IL-6) and its downstream JAK/STAT3 pathway have received recent attention. The goal of our study is to figure out whether or how resveratrol (RSV) can protect NPCs from degeneration by affecting IL6/JAK/STAT3 pathway.MethodsDifferent concentrations of RSV were added to NPCs’ mediums. Cell viability was measured by MTT assay and crystal violet staining. Cell cycle and apoptosis were analyzed by flow cytometry. Protein expression level was determined by western blot. mRNA expression level was measured by qPCR.ResultsOur study showed that RSV improved NPCs’ cell viability. It also inhibited cell apoptosis and cell cycle arrest, which were accompanied by the increased expression level of heat shock protein 90 (HSP90) and N-Cadherin. What’ more, RSV also improved the NPCs’ degeneration which was reflected in the increase of extracellular matrix (collagen II, Aggrecan). Moreover, RSV significantly attenuated the level of IL-6 secretion, which was accompanied by less phosphorylation of the transcription factors Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3).ConclusionRSV exerted its protective effect on HNPCs’ degeneration by improving cell survival and function. The possible mechanism may be associated with the suppression of JAK/STAT3 phosphorylation and the decreased IL-6 production, which could be explained by a blockage of the positive feedback control loop between IL-6 and JAK/STAT3 pathway.

Cryopreservation Impacts Cell Functionality of Long Term Expanded Adipose-Derived Stem Cells

Objective: Adipose-Derived Stem Cells (ADSCs) are less immunogenic cells and have a heterogenic cytokine secretion profile making them a better candidate for cell-based immunotherapy. Even innately or stimulated, Interleukin-6 (IL-6) and Toll-like Receptor 2 (TLR2) secreted by ADSC were modulated and led to different inflammatory mechanism pathways through different inflammatory factors secretion. These properties can be very useful in the treatment of inflammation-associated chronic diseases. To be used, ex-vivo ADSC expansion is a critical issue prior to transplantation or cryopreservation. Functional cell changes have been reported during culture expansion being susceptible to interact with freezing/thawing effects leading to doubt on cell therapeutic outcomes. The aim of this study is to identify the effect of freezing/thawing at the different time point of expansion culture on IL-6 and TLR2 secretion.Methods: ADSC were collected from young female donors, expanded in culture and cryopreserved in Foetal Bovine Serum (FBS) and Dimethylsulfoxide (DMSO) after each passage for 6 months to a year. ADSC was then tested for proliferation, clonogenicity, cytokine gene expression and assessment before cryopreservation (fresh) and after thawing and cultured until confluence (frozen/thawed). ADSC preserved at Passage 0 (P0) were thawed and tested after confluence at P1.Results: Cryopreserved ADSC as P1 resulted in increased clonogenicity, total RNA and protein secretion compared to the fresh ones. Relative Quantification (RQ) and cytokine assessment of IL-6, IL-10, Tumor Necrosis Factor (TNF)-α and TLR2 revealed a moderate up-regulation of TLR2 while significantly higher IL-6 secretion levels were observed in long term expanded and cryopreserved ADSC.Conclusion: Our results suggested that cryopreserved ADSC long term expanded in culture were functionally different and might have impaired immunosuppressive properties through modulation of the inflammatory responses by IL-6 and TLR2 activation.

Effect of atorvastatin on high glucose-stimulated expressions of MALAT1 and inflammatory factors in endothelial cells

Objective To investigate the effect of atorvastatin on the expressions of long non-coding RNA(LncRNA)metastasis-associated lung adenocarcinoma transcript 1(MALAT1)and inflammation factors in human umbilical vein endothelial cells(HUVECs)stimulated by high glucose. Methods The expression of MALAT1 in HUVECs incubated with high glucose(30 mmol/L)for different time periods were detected by real-time PCR. Under high glucose condition, the expressions of MALAT1, interleukin-6(IL-6), and interleukin-8(IL-8)in HUVECs were detected after MALAT1 was silenced by siRNA or atorvastatin was added. Results (1)After HUVECs were incubated with high glucose for different time periods, the expressions of MALAT1 were increased to some extent(P<0.05), with the peak at 12h(P<0.01). The levels of IL-6 and IL-8 expression and secretion were increased after HUVECs were stimulated by high glucose for 12h(P<0.05). (2)The silence of MALAT1 markedly suppressed high glucose-stimulated expression and secretion of IL-6 and IL-8(P<0.05). (3)Atorvastatin significantly inhibited high glucose-stimulated expressions of MALAT1, IL-6, and IL-8(all P<0.05). Conclusions High glucose induces the secretion of inflammatory factors by stimulating MALAT1 expression in endothelial cells. Atorvastatin significantly inhibits high glucose-stimulated MALAT1 expression and decreases inflammatory reaction. (Chin J Endocrinol Metab, 2017, 33: 330-334) Key words: Atorvastatin; High glucose; Metastasis-associated lung adenocarcinoma transcript 1; Inflammatory reaction

Ga(III) Nanoparticles Inhibit Growth of both Mycobacterium tuberculosis and HIV and Release of Interleukin-6 (IL-6) and IL-8 in Coinfected Macrophages.

ABSTRACTTreatment of individuals coinfected with human immunodeficiency virus (HIV) type 1 and Mycobacterium tuberculosis is challenging due to the prolonged treatment requirements, drug toxicity, and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are one of the natural reservoirs for both HIV and M. tuberculosis. Here, the treatment of HIV and M. tuberculosis coinfection was studied by preloading human macrophages with MP-targeted gallium (Ga) nanoparticles to limit subsequent simultaneous infection with both HIV and M. tuberculosis. Ga nanoparticles provided sustained drug release for 15 days and significantly inhibited the replication of both HIV and M. tuberculosis. Addition of Ga nanoparticles to MP already infected with M. tuberculosis or HIV resulted in a significant decrease in the magnitude of these infections, but the magnitude was less than that achieved with nanoparticle preloading of the MP. In addition, macrophages that were coinfected with HIV and M. tuberculosis and that were loaded with Ga nanoparticles reduced the levels of interleukin-6 (IL-6) and IL-8 secretion for up to 15 days after drug loading. Ga nanoparticles also reduced the levels of IL-6 and IL-8 secretion by ionomycin- and lipopolysaccharide-induced macrophages, likely by modulating the IκB kinase-β/NF-κB pathway. Delivery of Ga nanoparticles to macrophages is a potent long-acting approach for suppressing HIV and M. tuberculosis coinfection of macrophages in vitro and sets the stage for the development of new approaches to the treatment of these important infections.

Abstract A57: Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer.

Abstract Purpose: While chemotherapy may succeed initially at decreasing the number of ovarian cancer (OC) cells, it leaves behind tumors enriched in ovarian cancer stem cells (OCSCs), which most likely drive chemoresistance and tumor relapse. An emerging model indicates that non-OCSCs may dedifferentiate and acquire stem cell properties under certain circumstances. Recent studies in OC implicate a critical role for aberrant cytokine interleukin-6 (IL-6) secretion and the IL-6 signaling pathway may play a critical role in converting non-CSCs to CSC. In the current study, we investigated the mechanism contributing to OCSC enrichment after platinum treatment in vivo. We hypothesize that platinum-induced IL-6 secretion activates STAT3 signaling-mediated expression of the cancer stem cell marker ALDH1A1, resulting in CSC enrichment. In addition, based on our previous study in OCSCs (Wang et al., 2014, Cancer Res.), we sought to examine the mechanism by which guadecitabine (SGI-110), a second generation hypomethylating agent HMA, inhibits OCSCs in OC xenograft residuals after platinum treatment. Methods: OC cells (OVCAR4 and A2780) were cultured alone or co-cultured with normal omental fibroblasts (NOFs) or guadecitabine (100nM, 3days)-treated NOFs in the starving condition for 24h and then treated with 3h cisplatin (CDDP; respective IC50 doses of 27.4 and 14.7μM), guadecitabine, IL-6 (100ng/ml), neutralizing antibody (Nab) against IL-6 (IL-6-Nab, 200ng/mL) or IL-6+IL-6-Nab. IL-6 secretion levels were measured by ELISA. FACS was used to analyze OCSC population marked as aldehyde dehydrogenase (ALDH)+ cells, and western blot was used to examine ALDH1A1 and pSTAT3 levels in cells treated as described above. An ALDH1A1 promoter-luciferase reporter (pGL3-ALDH1A1-Luc; -1 to -1031) assay was used to determine whether IL-6 transactivates ALDH1A1 expression in OC cells. OC cell migration under the described conditions was determined by transwell migration assays. Results: We found that CDDP induced (P&amp;lt;0.05) IL-6 secretion by OCs up to 48h post-treatment and up to 96h by NOFs or by co-cultured cells, suggesting a role of the tumor microenvironment in platinum-induced IL-6 secretion. Guadecitabine inhibited (P&amp;lt;0.05) CDDP-induced IL-6 secretion by NOFs alone or in co-culture with OVCAR4, suggesting an inhibitory role of the HMA on IL-6 signaling. By assaying FACS sorted ALDH+/- cells, we determined that ALDH+ cells express increased (P&amp;lt;0.05) levels of the IL-6 receptor and secrete higher (P&amp;lt;0.05) levels of IL-6 in the conditioned media compared to ALDH- cells. Treatment of OC cells with IL-6 enriched the percentage of ALDH+, which was inhibited by IL-6-Nab. Luciferase assay results revealed that IL-6 transactivated (p&amp;lt;0.05) ALDH1A1 reporter gene expression in ALDH- cells, which was blocked by IL-6-Nab. Further, we observed that IL-6- or CDDP-treated OC cells increased (P&amp;lt;0.05) ALDH1A1 and pSTAT3 protein expression, and guadecitabine treatment inhibited (P&amp;lt;0.05) expression of ALDH1A1 and pSTAT3 in OCs. ALDH+ cells showed greater migration potential than ALDH- cells, and guadecitabine inhibited (P&amp;lt;0.05) migration of both ALDH+ and ALDH- cells. Guadecitabine, alone or combination with IL-6-Nab, inhibited (P&amp;lt;0.05) CDDP-induced OVCAR4 migration in co-culture with NOFs. Conclusion: Our data indicate that IL-6 is a potent regulator of ALDH1A1 expression and the OCSC phenotype and further suggest an inhibitory effect of guadecitabine on the conversion of non-OCSCs to OCSCs. The data support a role for IL-6 in OCSC enrichment after platinum treatment and suggest that a combination approach of IL-6 neutralizing antibody with guadecitabine could represent a novel maintenance strategy after chemotherapy for eradicating OCSCs and preventing tumor recurrence. Citation Format: Yinu Wang, Anirban Kumar Mitra, Kenneth P. Nephew. Platinum induces IL-6-signaling mediated activation of ALDH1A1 and enriches the cancer stem cell population in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A57.

Abstract 635: Apolipoprotein A-I Inhibits Lipopolysaccharide-Induced Inflammation and Activation of Major Stress Signaling Kinases in 3T3-L1 Adipocytes in an ABCA1-Dependent Manner

Obesity is characterised by adipose tissue inflammation and increased secretion of pro-inflammatory adipokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). A recently suggested mechanism linking obesity with its co-morbidities is an increase in stress signalling pathways, including p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK). Apolipoprotein A-I (apoA-I), the main lipoprotein of high density lipoproteins, has potent anti-inflammatory properties. It is also the primary acceptor of cholesterol that is exported from cells expressing the ATP-binding cholesterol transporter A1 (ABCA1). The role of apoA-I and ABCA1 in the inhibition of adipocyte inflammation is unknown. Aim: To determine if apoA-I inhibits inflammation and activation of stress signalling pathways in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes. Methods: Mature 3T3-L1 adipocytes were pre-incubated for 16 h with or without apoA-I (1 mg/mL), then stimulated for 6 h with LPS (100 ng/mL). MCP-1 and IL-6 secretion and mRNA levels were quantified by ELISA and qPCR, respectively. 3T3-L1 adipocytes were also pre-incubated with apoA-I, then stimulated with LPS for 10, 20, 30 and 60 min prior to quantification of ERK1/2, p38MAPK and JNK1/2 phosphorylation by western blotting. ABCA1 expression was silenced by transfection with ABCA1siRNA (50 nM) for 24 h. Results: Pre-incubation of 3T3-L1 cells with apoA-I reduced the LPS-mediated secretion of MCP-1 from 2666±216 to 983±65 pg/mg cell protein, while IL-6 secretion decreased from 120±22 to 55±11 pg/mg cell protein (p&lt;0.05 for both). MCP-1 and IL-6 mRNA levels decreased by 43- and 5-fold, respectively, (p&lt;0.05 for both). These effects of apoA-I were attenuated in 3T3-L1 cells in which ABCA1 expression was silenced. LPS induced phosphorylation of ERK1/2, p38MAPK and JNK1/2 in 3T3-L1 cells. Pre-treatment of the LPS-stimulated cells with apoA-I inhibited phosphorylation of ERK1/2, p38MAPK and JNK1/2 by 66.2±4%, 58.8±10.7% and 33.2±14% respectively (p&lt;0.05 for all). Conclusion: ApoA-I exerts anti-inflammatory effects and inhibits activation of MAP kinases in 3T3-L1 adipocytes in an ABCA1-dependent manner.

Circadian rhythmicity, variability and correlation of interleukin-6 levels in plasma and cerebrospinal fluid of healthy men.

Interleukin-6 (IL-6) is a cytokine with pleiotropic actions in both the periphery of the body and the central nervous system (CNS). Altered IL-6 secretion has been associated with inflammatory dysregulation and several adverse health consequences. However, little is known about the physiological circadian characteristics and dynamic inter-correlation between circulating and CNS IL-6 levels in humans, or their significance. Simultaneous assessment of plasma and cerebrospinal fluid (CSF) IL-6 levels was performed hourly in 11 healthy male volunteers over 24h, to characterize physiological IL-6 secretion levels in both compartments. IL-6 levels showed considerable within- and between-subject variability in both plasma and CSF, with plasma/CSF ratios revealing consistently higher levels in the CSF. Both CSF and plasma IL-6 levels showed a distinctive circadian variation, with CSF IL-6 levels exhibiting a main 24h, and plasma a biphasic 12h, circadian component. Plasma peaks were roughly at 4 p.m. and 4 a.m., while the CSF peak was at around 7 p.m. There was no correlation between coincident CSF and plasma IL-6 values, but evidence for significant correlations at a negative 7-8h time lag. This study provides evidence in humans for a circadian IL-6 rhythm in CSF and confirms prior observations reporting a plasma biphasic circadian pattern. Our results indicate differential IL-6 regulation across the two compartments and are consistent with local production of IL-6 in the CNS. Possible physiological significance is discussed and implications for further research are highlighted.

AB0070 Galectin-3 inhibition attenuates interleukin-6 secretion induced by toll-like receptor-stimulation in fibroblast-like synoviocytes

BackgroundGalectin-3 is a β-galactoside-binding lectin that plays an important role in the modulation of immune responses. Galectin-3 levels are increased in rheumatoid arthritis (RA) synovial tissue, synovial fluid and peripheral...

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL-6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well-studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll-like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down-regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up-regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS-treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin-6 (IL-6), a potent stimulator of cell migration. Interestingly, the levels of IL-6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental-derived progenitor cells in sites of periodontal infection.

Effects of Mycophenolic Acid on the Proliferation and Endothelin-1 and Interleukin-6 Secretion of Rat Pulmonary Microvascular Endothelial Cells

Objective: To investigate the effect of mycophenolic acid (MPA) on the proliferation of rat pulmonary microvascular endothelial cells (PMVECs) and on the secretion of endothelin-1 (ET-1) and interleukin-6 (IL-6) by these cells. Methods: Rat PMVECs were treated with five different final concentrations of MPA (0, 0.1, 1, 10 and 100 μM). The fetal bovine serum-induced proliferation of the PMVECs was detected using the Cell Counting Kit-8 (CCK-8). The levels of ET-1 and IL-6 secretion were assessed by radioimmunoassays. Results: At the 24 h time point, the optical density (OD) values at 450 nm for the five groups showed that there were no significant differences between the groups treated with 0 and 0.1 μM MPA (P=0.388) or between the groups treated with 10 and 100 μM MPA (P=0.292), but the OD values were significantly different between all other pairs of groups (P<0.001). At the 48 h time point, the OD values at 450 nm for the five groups showed that there was no significant difference between the groups treated with 0 and 0.1 μM MPA (P=0.094), but the OD values were significantly different between all the other groups (P<0.001). At the 72 h time point, the OD values at 450 nm for the five groups showed that there was no significant difference between the groups treated with the final concentrations of 0 and 0.1 μM MPA (P=0.931), but the OD values were significantly different between all other pairs of groups (10 μM MPA vs 100 μM MPA: P=0.037; 0 μM MPA vs 1 μM MPA: P=0.006; 1 μM MPA vs 10 μM MPA: P=0.005; all other comparisons: P<0.001). There were no significant differences in ET-1 concentration between the groups treated with 0 and 0.1 μM MPA (P=0.156) or the groups treated with 10 and 100 μM MPA (P=0.262), but there were significant differences between all other pairs of groups (P<0.001). By contrast, there were no significant differences in IL-6 concentrations between any pair of groups (P>0.05). Conclusions: MPA inhibits fetal bovine serum-induced rat PMVEC proliferation and ET-1 secretion in a dose-dependent manner.

CORRELATION BETWEEN CLINICAL MANIFESTATIONS OF OSTEOARTHRITIS AND FLUCTUATING BLOOD LEVELS OF INTERLEUKIN 6

Objective — to evaluate associations between fluctuating interleukin 6 (IL6) levels and clinical manifestations of osteoarthritis (OA). Material and methods. Totally 81 patients aged 47—73 y.o. with OA diagnosis, meeting ACR criteria, were evaluated. Anthropometric measurements, duration of the disease, degree of functional disability, IL6 levels and joints radiographs were assessed in all patients. Correlation between different IL6 secretion levels and the severity of OA clinical course and of pain syndrome was also analyzed. Results and discussion. Elevated IL6 levels were associated with more pronounced radiographic structural damage, greater degree of functional disability, presence of synovitis and more severe pain syndrome. Therefore, elevated IL6 levels are associated with unfavorable clinical course of OA.

Atorvastatin Decreases C‐Reactive Protein‐Induced Inflammatory Response in Pulmonary Artery Smooth Muscle Cells by Inhibiting Nuclear Factor‐κB Pathway

C-reactive protein (CRP) is well-known inflammatory marker, and recognized as a risk predictor of pulmonary arterial diseases. Although statins have a beneficial effect in animal models and patients with pulmonary arterial hypertension (PAH), the underlying mechanisms of their actions have less been investigated. The aims of this study was to examined the effects of CRP on expressions of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the possible mechanisms of atorvastatin on CRP-induced IL-6 and MCP-1 production in cultured human pulmonary artery smooth muscle cells (PASMCs). In a preliminary study, the human PASMCs were stimulated by a variety of concentrations of CRP (5-200 microg/mL) at different time points (0, 3, 6, 9, 12, 18 and 24 h) for the purpose of determining the dose- and time-dependent effects of CRP on inflammatory response of the cells. Then, the cells were pre-incubated for 2 h with atorvastatin (0.1-10 micromol/L) in the presence of CRP. The supernatant levels of both IL-6 and MCP-1 secretion were examined by ELISA. The cellular mRNA expressions of IL-6 and MCP-1 and nuclear factor-kappaB (NF-kappaB) activity were determined by real-time reverse transcription and polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assay (EMSA), respectively. CRP resulted in elevated IL-6 and MCP-1 secretion and mRNA expression in a dose- and time-dependent manner. In addition, CRP also significantly activated the NF-kappaB pathway. Preincubation with 0.1-10 micromol/L of atorvastatin significantly decreased the secretions of IL-6 and MCP-1 induced by CRP. Moreover, 10 micromol/L of atorvastatin completely abrogated CRP-induced increase in IL-6 and MCP-1 by attenuating the activation of NF-kappaB. The present study demonstrated that inhibiting effect of atorvastatin on CRP-induced inflammatory response in cultured PASMCs was associated with NF-kappaB pathway. This pathway might represent a promising target for controlling CRP-induced inflammatory response in pulmonary arterial diseases.

CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

High serum levels of interleukin-6 in endometrial carcinoma are associated with uterine serous papillary histology, a highly aggressive and chemotherapy-resistant variant of endometrial cancer

Purpose. To evaluate and compare autocrine expression and production of interleukin-6 (IL-6), a pleiotropic cytokine involved in the resistance to cytotoxic agents and inhibition of anti-tumor immune function in endometrial carcinoma in vitro as well as in vivo. Patients and methods. IL-6 gene expression levels were evaluated in twenty-four primary endometrial tumors including 14 endometrioid carcinomas (EC) and 10 uterine serous papillary carcinoma (USPC) as well as in normal control endometrial cells (NEC) by real-time PCR. Secretion of IL-6 protein by 6 primary endometrial tumor cultures including USPC and EC was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) in vitro. Finally, IL-6 concentration in 71 serum samples including 20 apparently healthy women, 19 women with benign abdominal diseases, 19 women with primary EC, and 13 USPC patients was studied. Results. IL-6 gene expression levels were significantly higher in USPC when compared to EC (mean copy number by RT-PCR = 313 ± 55 vs. 53 ± 11, USPC vs. EC, respectively: P < 0.01). IL-6 serum concentrations between normal healthy females (range 0.01–21.23 pg/ml; mean 3.1 pg/ml) and benign disease patients (range 0.01–95.77 pg/ml; mean 13.07 pg/ml) were not statistically different. In contrast, significantly higher levels of IL-6 were detected in both patients with EC (range 2.86–82.13 pg/ml; mean 20.43 pg/ml) and patients with UPSC (range 16.3–500.1 pg/ml; mean 125.7 pg/ml) when compared to the healthy females ( P < 0.01), with a mean serum IL-6 level in USPC patients 6.1-fold higher when compared to EC patients ( P < 0.03). Accordingly, higher levels of IL-6 secretion were noted in primary USPC cell lines (mean 3121 pg/ml, range between 1099 and 5017 pg/ml/10 5 cells/48 h) when compared to primary EC (mean 88, range between 19 and 112 pg/ml/10 5 cells/48 h) ( P < 0.01) in vitro. Conclusions. IL-6 is highly expressed in USPC, and it is released in high concentration in the serum of USPC patients. IL-6 may be a novel biomarker for USPC. Drugs used to inhibit the expression of IL-6 or the IL-6 signal transduction pathway may potentially be highly beneficial in USPC.

Expression of Interleukin 6 and Its Receptor in Human Gastric and Colorectal Cancers

Interleukin 6 (IL-6) is a pleiotropic cytokine with many physiological functions. The present study was designed to determine the expression of IL-6 and its receptor (IL-6R) in human gastric and colorectal cancers. Nine gastric- and nine colorectal cancer cell lines were analysed. The IL-6 gene was expressed in two gastric cancer cell lines and one colorectal cancer cell line; however, most of the cancer cell lines studied expressed the IL-6R gene. The level of IL-6 secretion in the gastric cancer cell lines correlated with the level of soluble IL-6R secretion, and was significantly higher (< approximately 100 pg/ml) than the level of IL-6 secretion in the colorectal cancer cell lines (< approximately 50 pg/ml). These results suggest that IL-6 may act in a paracrine fashion rather than an autocrine fashion in these cell lines.

Interleukin‐6 polymorphism (–634C/G) in the promotor region and the progression of diabetic nephropathy in Type 2 diabetes

Interleukin-6 (IL-6) is a multifunctional cytokine produced by many different cell types, including glomerular mesangial cells. Recently, a novel C/G polymorphism at position -634 in the promotor region of the IL-6 gene has been reported. The aim of this study was to investigate whether the -634C/G polymorphism is associated with an increased risk for progression to diabetic nephropathy as well as elevated levels of IL-6 secretion by peripheral blood mononuclear cells. The frequency of the -634C/G polymorphism was determined in Japanese patients with Type 2 diabetes and either normoalbuminuria (n = 162), microalbuminuria (n = 138), or macroalbuminuria (n = 154) by polymerase chain reaction-restriction fragment length polymorphism analysis. The level of IL-6 secretion in relation to genotype was assessed in lipopolysaccharide or advanced glycation end products-stimulated IL-6 secretion by peripheral mononuclear cells. The frequency of the -634G/G genotype and -634*G allele was significantly increased in the patients with macroalbuminuria compared with patients with normoalbuminuria (genotype: chi2 = 6.787, Pc = 0.0368; allele: chi2 = 9.080, Pc = 0.0104). Stepwise multiple regression analysis in these patients showed that hypertension (F = 40.48) and IL-6-634 gene polymorphism (F = 5.48) were the relevant variables for the progression of Type 2 diabetic nephropathy. Analysis of the IL-6 secretion data revealed that individuals carrying the -634*G allele had a higher IL-6 secretion capacity than those without the *G allele (P < 0.05). These results suggest that the IL-6-634C/G polymorphism may be a possible genetic susceptibility factor for the progression of diabetic nephropathy.

Elevated Interleukin-6 Expression in Keloid Fibroblasts

Keloids are characterized by a net accumulation of collagen. To date, the role of growth factors and various cytokines in the pathogenesis of these lesions has not been fully characterized. Interleukin-6 (IL-6) is an important immunoregulatory cytokine that has been implicated in a number of fibrotic autoimmune diseases such as scleroderma, interstitial nephritis, and pulmonary interstitial fibrosis. However, the role of IL-6 in the development of keloids has yet to be defined. This study demonstrates increased expression of the IL-6 gene in fibroblasts isolated from patients with keloids when compared with control fibroblasts using the ribonuclease protection assay. Subsequent detection of increased levels of IL-6 secretion by keloid fibroblasts is also demonstrated under unstimulated and stimulated conditions using serum and interferon γ (IFN-γ) (unstimulated: 0.3694 + 0.2499 pg/cell vs 0.0662 + 0.0786 pg/cell, P = 0.0137; serum: 1.066 + 0.513 pg/cell vs 0.233 + 0.231 pg/cell, P = 0.0027; serum and IFN-γ: 1.286 + 0.395 pg/cell vs 0.244 + 0.199 pg/cell, P < 0.0001). These results suggest that IL-6 may play a significant role in the pathogenesis of keloids.