Enhanced Interleukin-8 Secretion Research Articles

Treponema pallidum recombinant protein Tp47 enhanced interleukin-6 secretion in human dermal fibroblasts through the toll-like receptor 2 via the p38, PI3K/Akt, and NF-κB signalling pathways.

Interleukin-6 (IL-6) is a multi-effective cytokine involved in multiple immune responses. Whether fibroblasts also turn out to be a cytokine IL-6 factory during interaction with Treponema pallidum is not yet understood. To explore whether fibroblasts participate in inflammation due to syphilis, a series of experiments were performed to explore the role of T. pallidum lipoprotein Tp47 in IL-6 production in human dermal fibroblasts. The Toll-like receptor 2 (TLR2) and participating signalling pathways in this process were also evaluated. The results showed that the expressions of IL-6 and the protein levels of TLR2 in fibroblasts were upregulated after stimulation with Tp47, and this effect was impeded by the TLR2 inhibitor C29. In addition, Tp47 promoted the phosphorylation of p38, PI3K/Akt, and nuclear factor-kappaB (NF-κB), and the translocation of NF-κB in fibroblasts. Moreover, p38, PI3K, and NF-κB inhibitors significantly reduced IL-6 production in fibroblasts stimulated with Tp47. Furthermore, the TLR2 inhibitor C29 inhibited the phosphorylation of p38, Akt, and NF-κB, and the translocation of NF-κB in fibroblasts. In conclusion, our results showed that Tp47 enhanced IL-6 secretion in human dermal fibroblasts through TLR2 via p38, PI3K/Akt, and NF-κB signalling pathways. These findings contribute to our understanding of syphilis inflammation.

STAT1- and NFAT-independent amplification of purinoceptor function integrates cellular senescence with interleukin-6 production in preadipocytes.

Senescent preadipocytes promote adipose tissue dysfunction by secreting pro-inflammatory factors, although little is known about the mechanisms regulating their production. We investigated if up-regulated purinoceptor function sensitizes senescent preadipocytes to cognate agonists and how such sensitization regulates inflammation. Etoposide was used to trigger senescence in 3T3-L1 preadipocytes. CRISPR/Cas9 technology or pharmacology allowed studies of transcription factor function. Fura-2 imaging was used for calcium measurements. Interleukin-6 levels were quantified using quantitative PCR and ELISA. Specific agonists and antagonists supported studies of purinoceptor coupling to interleukin-6 production. Experiments in MS1 VEGF angiosarcoma cells and adipose tissue samples from obese mice complemented preadipocyte experiments. DNA damage-induced senescence up-regulated purinoceptor expression levels in preadipocytes and MS1 VEGF angiosarcoma cells. ATP-evoked Ca2+ release was potentiated in senescent preadipocytes. ATP enhanced interleukin-6 production, an effect mimicked by ADP but not UTP, in a calcium-independent manner. Senescence-associated up-regulation and activation of the adenosine A3 receptor also enhanced interleukin-6 production. However, nucleotide hydrolysis was not essential because exposure to ATPγS also enhanced interleukin-6 secretion. Pharmacological experiments suggested coupling of P2X ion channels and P2Y12 -P2Y13 receptors to downstream interleukin-6 production. Interleukin-6 signalling exacerbated inflammation during senescence and compromised adipogenesis. We report a previously uncharacterized link between cellular senescence and purinergic signalling in preadipocytes and endothelial cancer cells, raising the possibility that up-regulated purinoceptors play key modulatory roles in senescence-associated conditions like obesity and cancer. There is potential for exploitation of specific purinoceptor antagonists as therapeutics in inflammatory disorders.

Community-acquired methicillin-resistant Staphylococcus aureus provoked cytokine storm causing severe infection on BALB/c mice

Methicillin-resistant Staphylococcus aureus (MRSA) has become the most important pathogen of hospital-acquired (HA) or community-acquired (CA) infections. However, it is unclear of the cytokines responsible for pathological hyper-inflammation in sepsis related cytokine storm for MRSA infection. In this study, we selected typical HA-MRSA strain (YNSA163: ST239-t030-SCCmecⅢ) and two CA-MRSA isolates (YNSA7: ST59-t439-SCCmecⅣa and YNSA53: ST59-t437-SCCmecⅤb) from our previous research, infected on BALB/c mice, and analyzed the cytokine storm patterns during infection process. The animal experiments revealed the most serious lethal effect on BALB/c mice caused by YNSA7 strain infection, followed by YNSA53, and no BALB/c mice died for YNSA163 infection. Histopathological analyses revealed that lung was the most seriously damaged organs, followed by spleen and kidney, especially for CA-MRSA infection. The severe inflammatory reactions, tissue destruction, and massive exudation of inflammatory mediators and cells could be identified in CA-MRSA strains infected mice. Interleukin-6 (IL-6) and IL-10 were both highly expressed in spleen and lung of YNSA7 and YNSA53 dead cases compared with YNSA53 survived and YNSA163 cases, which demonstrated cytokine storm pattern for CA-MRSA strains infection. The results of IL-6 intervention experiment verified that the enhanced IL-6 secretion was responsible for the host lethality of YNSA7 infection. RNA-sequencing results among three MRSA isolates indicated most of the differentially expressed genes referred to cellular process, metabolism and genetic information processing of bacteria. Specifically, clpP, chp chemotaxis inhibit, fnbB, pathogencity island protein and virulence associated protein E were highly expressed in YNSA7 strain. In general, CA-MRSA strains provoked cytokine storm on BALB/c mice led to severe infection and lethality, the up-regulated of some virulence genes might play important role in pathogenesis.

Platelet Interactions with Liver Sinusoidal Endothelial Cells and Hepatic Stellate Cells Lead to Hepatocyte Proliferation

(1) Background: Platelets were postulated to constitute the trigger of liver regeneration. The aim of this study was to dissect the cellular interactions between the various liver cells involved in liver regeneration and to clarify the role of platelets. (2) Methods: Primary mouse liver sinusoidal endothelial cells (LSECs) were co-incubated with increasing numbers of resting platelets, activated platelets, or platelet releasates. Alterations in the secretion of growth factors were measured. The active fractions of platelet releasates were characterized and their effects on hepatocyte proliferation assessed. Finally, conditioned media of LSECs exposed to platelets were added to primary hepatic stellate cells (HSCs). Secretion of hepatocyte growth factor (HGF) and hepatocyte proliferation were measured. After partial hepatectomy in mice, platelet and liver sinusoidal endothelial cell (LSEC) interactions were analyzed in vivo by confocal microscopy, and interleukin-6 (IL-6) and HGF levels were determined. (3) Results: Co-incubation of increasing numbers of platelets with LSECs resulted in enhanced IL-6 secretion by LSECs. The effect was mediated by the platelet releasate, notably a thermolabile soluble factor with a molecular weight over 100 kDa. The conditioned medium of LSECs exposed to platelets did not increase proliferation of primary hepatocytes when compared to LSECs alone but stimulated hepatocyte growth factor (HGF) secretion by HSCs, which led to hepatocyte proliferation. Following partial hepatectomy, in vivo adhesion of platelets to LSECs was significantly increased when compared to sham-operated mice. Clopidogrel inhibited HGF secretion after partial hepatectomy. (4) Conclusion: Our findings indicate that platelets interact with LSECs after partial hepatectomy and activate them to release a large molecule of protein nature, which constitutes the initial trigger for liver regeneration.

Dexamethasone modifies mitomycin C-triggered interleukin-8 secretion in isolated human Tenon's capsule fibroblasts

Intraoperative mitomycin C (MMC) is widely used to prevent pterygium recurrence and glaucoma filtering bleb failure, but it has been shown to induce corneal inflammation and cell death. Postoperative dexamethasone (DEX) is advocated to reduce MMC-related inflammation and cell death in corneal fibroblasts. Nevertheless, its long-term regulation mechanism in Tenon's capsule remains to be explored. The purpose of this study was to investigate how DEX modifies MMC's effects in human Tenon's capsule fibroblasts (HTFs). HTFs isolated from the pterygium surgical patients (n = 6) were treated with MMC at 0, 0.1, 0.2, and 0.4 mg/ml for 5 min and incubated in DEX at 10 μM for 0, 1, 2, and 3 days. Recombinant interleukin-8 (IL-8) was used to verify the effect of MMC-related IL-8 secretion. Cell proliferation of all the treated cells was analyzed by WST-1 assay. The amount of IL-8 secretion in HTFs was determined by enzyme-linked immunosorbent assay. Immunoblotting assay was used to analyze the expression of peroxisome-proliferator-activated receptor gamma (PPARγ) and B-cell lymphoma-extra large (Bcl-xL). Our results revealed that MMC significantly reduced the HTF cell proliferation rate. Additionally, MMC significantly upregulated IL-8 secretion in HTFs concentration-dependently. At 3 days post treatment (dpt), 5-min exposures to 0.1, 0.2, and 0.4 mg/ml MMC resulted in 1.4-fold (p = 0.012), 1.6-fold (p = 0.012), and 2.5-fold (p = 0.001) increases of IL-8 secretion. In contrast, DEX reversed the MMC-retarded cell proliferation rate (p = 0.036) and repressed MMC-related IL-8 secretion by 33.5% at 3 dpt (p = 0.003). Addition of recombinant IL-8 noticeably suppressed HTF cell proliferation in a concentration-dependent manner. DEX stimulated upregulation of both PPARγ and Bcl-xL at 1 dpt in normal HTFs and at 2 dpt in MMC-treated HTFs. PPARγ silencing reduced expression of PPARγ and Bcl-xL, but enhanced IL-8 secretion (p < 0.001). On the other hand, Bcl-xL silencing enhanced IL-8 secretion (p < 0.001), but did not affect PPARγ expression. These revealed that IL-8 secretion in HTFs is modulated by PPARγ-dependent Bcl-xL signaling. We conclude that DEX reversed the MMC-inhibited HTF cell proliferation via diminishing the MMC-induced IL-8 secretion, which resulted from a late-phase upregulation of the PPARγ and Bcl-xL. These long-term effects suggest a beneficial postoperative DEX treatment following intraoperative MMC application.

Abstract 1547: Tumor-derived macrophage Migration Inhibitory Factor and interleukin-6 cooperated in hypoxic accumulation of CD11b+Gr-1+ myeloid cells.

Abstract Recently, myeloid derived suppressor cells (MDSCs) have gained much attention due to their role in tumor immunity suppression as well as promotion of angiogenesis, tumor cell invasion, and metastases. However, the mechanisms that regulate MDSCs recruitment have not been well clarified. Here, we demonstrated that hypoxic tumor microenvironment stimulate the recruitment of MDSCs through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Knock-down (KD) of HIF-1/2α in HNSCC cells decreased MIF level but did not inhibit the MDSCs migration, because HIF-1/2α KD increased NF-κB activity which further enhanced IL-6 secretion. In vivo administration of neutralizing IL-6 antibody and inhibitors against MIF and NF-κB decreased MDSCs accumulation, accompanied by reduced micro-vessel density (MVD), lymphatic vessel density (LVD), and xenograft tumor growth. The supplementation of MDSCs increased MVD, LVD, and xenograft tumor growth. In conclusion, hypoxic HNSCC cells may stimulate the accumulation of MDSCs through secreting MIF and IL-6. This process is mediated by interactions between NF-κB and HIF-1/2α. Strategies targeting hypoxic tumor microenvironment may represent attractive approaches to HNSCC therapy. [G Zhu and Y Tang contributed equally to this paper.] Citation Format: Guiquan Zhu, Yaling Tang, Ling Li, Xinhua Liang. Tumor-derived macrophage Migration Inhibitory Factor and interleukin-6 cooperated in hypoxic accumulation of CD11b+Gr-1+ myeloid cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1547. doi:10.1158/1538-7445.AM2013-1547

CD133+ liver tumor-initiating cells promote tumor angiogenesis, growth, and self-renewal through neurotensin/interleukin-8/CXCL1 signaling

A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). We previously identified a TIC population derived from hepatocellular carcinoma (HCC) that is characterized by membrane expression of CD133. Here, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133(+) liver TICs obtained from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133(+) cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133(+) liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis, and initiate tumors. In further support of these observations, IL-8 repression in CD133(+) liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be preferentially expressed in CD133(+) liver TICs. Addition of exogenous NTS resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, both key components of the mitogen-activated protein kinase (MAPK) pathway. Enhanced IL-8 secretion by CD133(+) liver TICs can in turn activate an IL-8-dependent feedback loop that signals through the MAPK pathway. Further, in its role as a liver TIC marker CD133 also plays a functional part in regulating tumorigenesis of liver TICs by way of regulating NTS, IL-8, CXCL1, and MAPK signaling. CD133(+) liver TICs promote angiogenesis, tumorigenesis, and self-renewal through NTS-induced activation of the IL-8 signaling cascade.

Abstract 2451: CD133+ liver tumor-initiating cells promote tumor growth, angiogenesis and self-renewal through activation of the neurotensin / interleukin-8 signaling cascade

Abstract A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs). We have previously identified a TIC population from hepatocellular carcinoma (HCC) that is marked by a CD133 surface phenotype and bears features that include the abilities to self-renew, differentiate, initiate tumors in vivo and resist standard chemotherapy through AKT pathway dysregulation (Ma et al., Gastroenterology 2007; Oncogene 2008 and Cell Stem Cell in press). Our present study aims to investigate the underlying molecular mechanism by which CD133+ liver TICs mediate tumor growth and maintenance. cDNA microarray approach was employed to compare gene expression profiles between sorted CD133 subpopulations in HCC cell lines Huh7 and PLC8024. A dysregulated interleukin-8 (IL-8) signaling functional network was preferentially identified in CD133+ liver TICs. IL-8 was found to be overexpressed at both genomic and proteomic (endogenous and secretory) levels in CD133+ cells isolated from HCC cell lines and clinical samples; while its expression also correlated positively with CD133 expression in a panel of liver cell lines. Functional studies on sorted CD133 HCC cells found enhanced IL-8 secretion in CD133+ liver TICs to possess a greater ability to self-renew and induce tumor angiogenesis and growth in vitro and in vivo. These observations were further validated when silencing of IL-8 in CD133+ liver TICs by knockdown or neutralizing antibody approach led to abolishment of these effects. Subsequent studies in the IL-8 functional network also found neurotensin (NTS) and chemokine ligand 1 (CXCL1) to be preferentially expressed in CD133+ liver TICs. Their expression also correlated with that of CD133 and IL-8 across a panel of liver cell lines. Upon addition of exogenous NTS, both expression of IL-8 and CXCL1 was dramatically up-regulated, with concomitant activation of phospho-ERK1/2, a key player of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, enhanced IL-8 secretion in CD133+ liver TICs can in turn activate an IL-8 positive feedback loop through MAPK signaling. Lastly, we also provide evidence to show that the transmembrane protein, CD133, apart from serving as a liver TIC marker, also plays a critical functional role in conferring TIC properties. Stable repression of CD133 in CD133+ liver TICs by lentiviral based approach resulted in inactivation of the NTS / IL-8 / CXCL1 / MAPK signaling cascade and attenuation of TIC self-renewal and tumorigenic properties. Taken together, these results suggest that CD133+ liver TICs promote tumor growth, angiogenesis and self-renewal through activation of NTS / IL-8 signaling cascade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2011-2451

Helicobacter Pylori Diversity in Gastric Cancer and GERD Among Singapore Chinese Population

Introduction: H.pylori (Hp) infection leads to divergent clinical outcomes like PUD, chronic gastritis, gastric cancers (GC) and GERD. Bacterial factors, host factors, combination of both and environmental factors play major role in establishing the disease. We hypothesize that diverse genetic factors cause GC and GERD in our population. Aim: To determine various genotypes associated with GC and GERD among Singaporean Chinese population. Materials & Methods: H.pylori was cultured from 182 Chinese patients (52GC, 70 gastritis & 60 GERD) were analyzed in this prospective, on-going study. DNA was extracted, sequence analysis and genotyping was performed using specific gene primers of various virulence factors such as cagPAI, vacAs1, s2, m1, m2, cagE, babA2, sabA and oipA. Results: Univariate and multivariate analysis revealed that cagPAI, cagE, babA2, sabA and oipA were significant among GC when compared with gastritis (p < 0.001). oipA “on” status was significantly associated with expression of cagPAI and suppression of sabA (p < 0.01). Sequence analysis of representative Hp strains indicated that cagPAI, cagE, babA2, sabA and oipA oipA exhibited >92% identity to previously published data. Univariate analysis of GERD revealed that cagE, babA2 and oipA were significant (P < 0.01), whereas, multivariate analysis revealed that none of the genotypes were significant. Conclusion: Our on-going, prospective study indicates that Hp genetic diversity is related to clinical presentation. Virulence factors associated with GC were: CagPAI, CagE and outer membrane proteins (SabA, OipA, babA2). Host factors may have a moderating role in the clinical manifestation of Hp infection. Majority of oipA genotype is detected at functional status. The “on” status was significantly related to gastric cancer. This could probably be associated with enhanced interleukin-8 secretion and inflammation, important at initial stages of disease process. Further studies will be focused on measurement of levels of interleukin, OipA proteins or anti-OipA antibody in patients' sera, pathological assessment & oipA functional status and disease presentation. Hp strains from GERD patients did not significantly express any of the genotypes when compared to NUD patients. Our study suggests these genotypes may protect against the development of GERD in our population.

Expression of functionally active NOD1 and NOD2 in folliculostellate TtT/GF pituitary cells

The follicullostellate (FS) cells of the pituitary share properties with dendritic cells and also with macrophages. A contingent of FS cells express MHC class I and MHC class II molecules and also several lymphoid markers and lymphatic markers. Therefore, FS cells were found to regulate the secretion of hormones by endocrine cells inside of the pituitary and to be a source of growth factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGF), as well as, cytokines as leukaemia inhibitor factor (LIF) and interleukin-6 (IL-6). Functionally, it was suggested that FS cells are capable of initiating immune responses and act as part of a neuroendocrine immune regulation system. Understanding of the way macrophages and/others cells respond to endotoxins and development of innate immunity, a major step was archived with the discovery of Toll-like receptors (TLRs). Toll receptors are members of a membrane-anchored proteins that upon stimulation recruits proteins kinases via a adaptor MyD88 inducing the activation of nuclear factor κB (NF-κB). Recently, nucleotide-binding oligomerization domain (NOD) proteins, NOD1 and NOD2 were found to represent an intracellular pathogen sensing system identified in both immune and non-immune cell types. The ligand of NOD2 is muramyl dipeptide (MDP), a component of bacterial cell-wall peptidoglycan present in most bacterial species. NOD2 oligomerization after bind in MDP activates RICK, an effector molecule that leads to phosphorylation of IKB and release of NF-κB for translocation in the nucleus. Considering characteristics of FS cells and the function of NOD molecules, we decided to investigate the expression and function of NOD in FS cells. Our preliminary RT-PCR data demonstrated the presence of NOD1 and NOD2 transcripts in FS cells. Moreover, NOD2 protein expression was increased in FS cells after 24 hours stimulation with MDP. We also observed significantly enhanced IL-6 secretion in the supernatant of FS cell cultures after stimulation with MDP. The MDP-induced and NOD-mediated IL-6 secretion by FS cells may play a role in modulating hormone secretion (e.g. stimulation of ACTH) during bacterial infections.

The interleukin-6 −174 promoter polymorphism is associated with extrapulmonary bacterial dissemination in Streptococcus pneumoniae infection

Interleukin-6 (IL-6) is required for the clearance of bacteria in pneumococcal pneumonia. The abundance of endogenous IL-6 production on infectious stimuli is associated with genotypic differences in the −174 promoter region of IL-6 (−174 G→C), showing increased IL-6 levels in patients carrying the GG genotype. One hundred patients with culturally proven pneumococcal disease were analyzed for distribution of the G-/C-alleles in the IL-6 −174 promoter region in comparison to 50 age-matched controls. Extrapulmonary pneumococcal dissemination, including septic metastasis, endocardial and meningeal infection, was used as parameter for impaired clearance of the bacteria. No significant differences in the allele distribution were observed between patients and controls. Within the patient group, the interleukin-6 GG homozygous carriers were less likely to develop extrapulmonary pneumococcal infection (10.3% versus 30.9%; OR 0.26, 95% CI 0.07–0.94, p = 0.04). The IL-6 GG genotype, encoding for enhanced IL-6 secretion on bacterial stimuli, reduces the risk of bacterial spread to extrapulmonary sites in pneumococcal infection, possibly due to a more effective clearance of the pathogen from the blood and the respiratory tract.

Helicobacter pyloriInduces Interleukin-8 Secretion by Toll-Like Receptor 2- and Toll-Like Receptor 5-Dependent and -Independent Pathways

Helicobacter pylori is an important human pathogen that causes gastritis and is strongly associated with gastric ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphomas. In response to H. pylori, interleukin-8 (IL-8) is secreted from host cells to attract components of the innate and adaptive immune systems to the site of infection. Toll-like receptor 2 (TLR2) and TLR5 have been shown to recognize H. pylori and to initiate signaling pathways that result in enhanced activation of NF-kappaB. Here, we evaluated the contribution of mitogen-activated protein kinase signaling pathways to TLR2-dependent and TLR5-dependent secretion of IL-8. Secretion of IL-8 from H. pylori-infected HEK293 cells was augmented by the expression of TLR2 or TLR5. While H. pylori infection resulted in the activation of ERK, JNK, and p38, the enhanced IL-8 secretion from TLR2- and TLR5-expressing cells coincided with increased p38 activation and phosphorylation of the transcription factor ATF2. When p38 activity was inhibited in TLR2- or TLR5-expressing cells, H. pylori-dependent IL-8 secretion returned to the level observed in infected parental HEK293 cells that did not express TLR2 or TLR5; inhibition of p38 had no effect on IL-8 secretion from infected parental HEK cells. In contrast, inhibition of JNK and/or ERK resulted in substantially less IL-8 secretion from infected cells, independent of TLR2 or TLR5 expression. Based on these data, we propose that H. pylori induces IL-8 secretion through a dual mechanism that includes a TLR2/5-independent component involving the activities of JNK and ERK and a TLR2/5-dependent component that requires p38 activity.

Candida albicans and Saccharomyces cerevisiae induce interleukin-8 production from intestinal epithelial-like Caco-2 cells in the presence of butyric acid

Intestinal epithelial cells (IEC) are important in initiation and regulation of immune responses against numerous foreign substances including food, microorganisms and their metabolites in the intestine. Since the responses of IEC against yeasts have not yet been well understood, we investigated the effects of Candida albicans, Saccharomyces cerevisiae, and their cell wall components on interleukin-8 (IL-8) secretion by the IEC-like Caco-2 cells. Live cells of both yeast species stimulated Caco-2 cells to produce IL-8 only in the presence of butyric acid, which is a metabolite produced by intestinal bacteria. S. cerevisiae zymosan and glucan also enhanced IL-8 secretion. Treatment of Caco-2 cells with butyric acid increased the expression of mRNAs coding for Toll-like receptor 1 (TLR1), TLR6 and dectin-1, which recognize zymosan. C. albicans induced more IL-8 secretion and also decreased transepithelial electrical resistance more rapidly than S. cerevisiae. These results suggest that both yeasts in the intestine stimulate the host's mucosal immune systems by interacting with IEC.

Human prostatic carcinoma cells produce an increase in the synthesis of interleukin-6 by human osteoblasts.

The aim of this work was to evaluate the effect produced by conditioned medium from human prostatic carcinoma cell (PC-3) culture on human osteoblast (HOB) interleukin 6 (IL-6) synthesis. PC-3 cells were cultured in Ham's F12K medium with 10% fetal calf serum (FCS) up to confluence. Medium was changed by Dulbecco modified Eagle medium (DMEM)/F12K (1:1) with 0.1% bovine serum albumin. Cells were cultured for 24 hr, and medium (PC-3-CM) was collected. HOBs were cultured up to confluence, and after 48 hr without FCS, medium was removed and PC-3-CM was added to the wells. After 24 hr, supernatant was collected for the determination of IL-6. In another experiment, HOBs were cultured up to confluence in Petri dishes, and after 48 hr without FCS, PC-3-CM or DMEM/F12K (1:1) was added. After different periods of time, medium was removed, and total RNA was extracted. IL-6 mRNA was quantified using reverse transcription polymerase chain reaction. PC-3-CM significantly enhanced IL-6 secretion into HOB culture supernatants (between 1,812% and 372%, depending on the osteoblastic line) with respect to HOBs cultured in DMEM/F12K. PC-3-CM also produced an increase in IL-6 mRNA levels in HOBs. Prostate carcinoma cells (PC-3) produce a factor or factors that enhance the synthesis and release of IL-6, a known activator of bone resorption.

Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle.

Interleukin 8 (IL-8) is a chemotactic cytokine involved in the recruitment and activation of neutrophils as well as in cell proliferation and angiogenesis. Because these events are essential components of folliculogenesis, ovulation, and subsequent repair of the ruptured follicle, the presence and regulation of IL-8 in the human follicle of the menstrual cycle was investigated. The concentrations of IL-8 were higher in follicular fluids from dominant follicles of late follicular/ovulatory phase compared with those of midfollicular phase. IL-8 was detected in the media from cultured granulosa and theca cells, with 10-fold higher levels in the theca cell cultures. Exposure to FSH and LH increased the IL-8 secretion from granulosa cells, but no effect was seen in theca cell cultures. Estradiol and progesterone did not affect IL-8 secretion from any cell type. The cytokines IL-1alpha and IL-1beta, but not tumor necrosis factor alpha, enhanced IL-8 secretion from both cell types. IL-8 levels in cultures of granulosa-lutein cells from hyperstimulated in vitro fertilization cycles were not affected by either gonadotropins or steroids. These data provide evidence that ovarian IL-8 is gonadotropin and cytokine induced and may be involved in the hormonally regulated stages of follicular development and ovulation.

Effects of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 production of murine B cells and B cell lines

The effect of T-cell-independent B cell mitogen, a pectic polysaccharide, bupleuran 2IIc, from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 (IL-6) production of murine B cells and B cell lines was investigated in order to clarify the mechanism of enhanced immunoglobulin (Ig) secretion from B cells. Bupleuran 2IIc enhanced IgM secretion from highly purified murine normal B cells. When normal B cells from murine spleen were cultured with bupleuran 2IIc in the presence of anti-IL-6 neutralizing antibody, the enhanced IgM secretion by bupleuran 2IIc was reduced. When B cells were stimulated with bupleuran 2IIc, their IL-6 secretion and the transcription of IL-6 mRNA were enhanced. The enhanced IL-6 secretion by bupleuran 2IIc was also observed in B cell line, Y16 cell. When bupleuran 2IIc was digested with endo-α-(1→4)-d-polygalacturonase, the resulting enzyme resistant carbohydrate portion, “ramified” region (PG-1) composed of rhamnogalacturonan core containing neutral sugar side chains showed potent IL-6 secretion-enhancing activity. These results suggest that the “ramified” region of bupleuran 2IIc stimulates the secretion of IL-6 as the active site, and the resulting IL-6 may partially contribute the enhancement of IgM secretion as an autocrine and/or paracrine mechanism.

Regulation of gingival fibroblast interleukin-6 secretion by cyclosporine A.

Cyclosporine A (CsA) is a widely used immunosuppressant, with clinical applications ranging from organ transplants to chronic inflammatory diseases. One of the side effects associated with CsA treatment is the development of gingival overgrowth. Exuberant growth of connective tissue within the periodontium can result from hyperactivity of resident fibroblasts. Fibroblasts are capable of secreting interleukin-6 (IL-6), which has been shown to enhance proliferation as well as collagen and glycosaminoglycan synthesis by these cells. We tested the hypothesis that one of the pathogenetic mechanisms underlying CsA-induced fibrosis is an enhanced IL-6 secretion by gingival fibroblasts (GF) in response to this drug. The ability of CsA to upregulate GF IL-6 secretion alone or in combination with bacterial challenge or other inflammatory cytokines was tested in an in vitro system. Fibroblast cultures were established from systemically healthy gingival tissue donors and were challenged with CsA in the absence or presence of bacteria, IL-1beta, or tumor necrosis factor (TNF) alpha as co-stimulants. Nifedipine and phenytoin were also tested to further support findings with CsA. After 72 hours of incubation, culture supernatants were collected and analyzed for IL-6 content by ELISA. We have shown that GF respond to CsA with an increase in IL-6 secretion. The magnitude of this response varies among cultures derived from different tissue donors. We have also demonstrated that GF IL-6 responses to bacterial challenge or TNFalpha are downregulated by CsA. However, CsA synergizes with IL-1beta to further upregulate IL-6 secretion, and this effect is shared by phenytoin and nifedipine. We conclude that one of the pathogenetic mechanisms underlying drug-induced gingival overgrowth may be enhanced secretion of IL-6 by GF in response to these medications. This is the first report on direct and indirect effects of gingival overgrowth-related medications on GF IL-6 metabolism. This work will lay the foundation for future studies directed towards the development of prevention or treatment modalities for gingival overgrowth based on blocking the fibrogenic activities of IL-6 at the cellular level.

Interleukin-6 secretion by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro

Interleukin-6 (IL-6) is a pluripotent cytokine that may play a role in pulmonary defense against bacterial pathogens. We have quantitated the response of bovine alveolar macrophages (bAM) to bacterial lipopolysaccharide (LPS; E. coli 055:B5) in vitro using the IL-6 sensitive 7TD1 cell line. Bacterial LPS in the absence of serum induced IL-6 secretion from bAM (1 × 10 6 ml −1) over a range of LPS concentrations from 10 ng ml −1 to 10 μg ml −1. This resulted in IL-6 levels ranging from approximately 5 to over 200 U ml −1. IL-6 secretion by LPS-stimulated bAM was increased by 24 h poststimulation, and continued to increase up to 72 h after stimulation. Fetal bovine serum (FBS, 1% vol/vol; 320 μg ml −1) enhanced IL-6 secretion from macrophages in the presence of LPS by approximately 10-fold compared with LPS alone. A bovine serum fraction (1 μg ml −1 protein) prepared using ion-exchange chromatography also markedly enhanced IL-6 secretion versus LPS alone. The stimulatory effect of IL-6-like activity in the bAM supernatants was neutralized by an anti-human IL-6 polyclonal antibody. Northern blot analysis revealed increased IL-6 mRNA at 2 h poststimulation with LPS + FBS, peak levels at 4 h, and levels were decreased by 6 h poststimulation. Results suggest that IL-6 is secreted by bovine alveolar macrophages, and that bacterial LPS and serum components synergize to produce this response.

Effects of Cryopreservation on Immune Responses: VII. Freezing Induced Enhancement of IL-6 Production in Human Peripheral Blood Mononuclear Cells

To better understand the effects of freezing on various immunocompetent cell functions, the interleukin-6 (IL-6)-producing activities of frozen peripheral blood mononuclear cells (PBMCs) from healthy subjects were determined. Frozen, lipopolysaccharide (LPS)-activated PBMCs produced significantly larger quantities of IL-6 than fresh cells. Although elimination of radiosensitive, CD8 + suppressor T cells had no significant effect on PHA-induced IL-6 production by T cells, elimination of CD4 + Leu-8 + suppressor T cell subsets resulted in a significantly enhanced IL-6 secretion. Exogenous addition of prostaglandin E-2 to frozen PBMCs and monocytes inhibited LPS-induced IL-6 production. The results suggest that functional inactivation of a subset of cryosensitive, PGE-2secreting monocytes is associated with an increase in IL-6 production by the other subset. They also indicate that a subset of CD4 + Leu-8 + T cells might be involved in feedback inhibition of PHA-induced T cell-mediated IL-6 production. The results provide further evidence that the presence of larger quantities of IL-6 in conjunction with increased amounts of IL-1 and IL-2 secreted by the frozen cells may be responsible for the previously reported enhanced immunoglobulin-producing abilities of frozen cells from clinically healthy subjects and from patients with lung cancer.

Effects of Interferon-α on a Reduced Release of Interleukin-8 from Latently HIV-1-Infected Monocytic Cell Line U937 Cells

The effects of human interferon-alpha (IFN-alpha) on the release of an antimicrobial interleukin, interleukin-8 (IL-8), from human immunodeficiency virus type 1 (HIV-1)-infected myelomonocytic cell line, U937, were studied in vitro to evaluate the potential of IFN-alpha in the management of acquired immunodeficiency syndrome (AIDS)-associated opportunistic diseases. The latently HIV-1-infected U937 cells (U937/HIV-1(L)) showed a marked reduction of IL-8 secretion as compared to uninfected U937 cells, whereas IL-8 release from productively HIV-1-infected U937 cells was comparable to uninfected cells. The IFN-alpha recovered partially the reduced IL-8 level from U937/HIV-1(L) cells in a dose-dependent manner. Any significant inhibition of IFN-alpha-augmented IL-8 secrement by anti-IL-1 antibody was not observed, suggesting that the enhanced IL-8 secretion occurred without augmenting IL-1 production. The IFN-alpha-augmented IL-8 secretion from latently HIV-1-infected U937 cells may suggest a beneficial potential of IFN-alpha in a treatment of bacterial or fungal infection frequently seen in patients with progressive stages of HIV-1 infection.