Including Interleukin-8 Expression Research Articles

FGF2-primed 3D spheroids producing IL-8 promote therapeutic angiogenesis in murine hindlimb ischemia

Peripheral artery disease is a progressive, devastating disease that leads to critical limb ischemia (CLI). Therapeutic angiogenesis using stem cell therapy has emerged as a promising approach for its treatment; however, adapting cell-based therapy has been limited by poor cell survival and low treatment efficiency. To overcome unmet clinical needs, we developed a fibroblast growth factor 2 (FGF2)-immobilized matrix that enabled control of cell adhesion to the surface and exerted a priming effect on the cell. Human adipose-derived stem cells (hASCs) grown in this matrix formed a functionally enhanced cells spheroid (FECS-Ad) that secreted various angiogenic factors including interleukin-8 (IL-8). We demonstrated that IL-8 was upregulated by the FGF2-mediated priming effect during FECS-Ad formation. Immobilized FGF2 substrate induced stronger IL-8 expression than soluble FGF2 ligands, presumably through the FGFR1/JNK/NF-κB signaling cascade. In IL-8-silenced FECS-Ad, vascular endothelial growth factor (VEGF) expression was decreased and angiogenic potential was reduced. Intramuscular injection of FECS-Ad promoted angiogenesis and muscle regeneration in mouse ischemic tissue, while IL-8 silencing in FECS-Ad inhibited these effects. Taken together, our data demonstrate that IL-8 contributes to therapeutic angiogenesis and suggest that FECS-Ad generated using the MBP-FGF2 matrix might provide a reliable platform for developing therapeutic agents to treat CLI.

Involvement of Oxidative Stress and the Epidermal Growth Factor Receptor in Diesel Exhaust Particle-Induced Expression of Inflammatory Mediators in Human Mononuclear Cells.

Exposure to diesel exhaust particles (DEPs) has been associated with increased incidence of cardiopulmonary diseases. This study is aimed at examining the proinflammatory effects of DEP on primary human peripheral blood mononuclear cells (PBMC) and the underlying mechanisms using a human mononuclear cell line, THP-1. DEPs were incubated with the PBMC and THP-1 cells for 24 h, respectively. The supernatants were collected and subjected to measurement of proinflammatory mediators including interleukin 8 (IL-8) or tumor necrosis factor α (TNFα) by ELISA. Levels of reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the epidermal growth factor receptor (EGFR) was examined with immunoblotting. Exposure to DEP induced a concentration-dependent increase in the expression of IL-8 and TNFα in the PBMC and THP-1 cells. Further mechanistic studies with THP-1 cells indicated that DEP stimulation increased intracellular levels of ROS, an indicator of oxidative stress, and phosphorylation of the EGFR, indicative of EGFR activation. Pretreatment of THP-1 cells with the antioxidant N-acetyl-L-cysteine (NAC) markedly blunted DEP-induced EGFR phosphorylation, indicating that oxidative stress was involved in DEP-induced EGFR activation. Furthermore, the pretreatment of THP-1 cells with either NAC or a selective EGFR inhibitor significantly blocked DEP-induced IL-8 expression, implying that oxidative stress and subsequent EGFR activation mediated DEP-induced inflammatory response. In summary, DEP stimulation increases the expression of proinflammatory mediators in human mononuclear cells, which is regulated by oxidative stress-EGFR signaling pathway.

HTNV-induced upregulation of miR-146a in HUVECs promotes viral infection by modulating pro-inflammatory cytokine release

Increasing research has shown a link between viruses and miRNAs, such as miRNA-146a, in regulating virus infection and replication. In the current study, the association between miR-146a and hantaan virus (HTNV) infection in human umbilical vein endothelial cells (HUVECs) was investigated, with a focus on examining the expression of pro-inflammatory cytokines. The results showed that HTNV infection promoted the production of miR-146a in HUVECs and activated nuclear factor-κB (NF-κB) signaling, along with the upregulation of pro-inflammatory cytokines, including interleukin 8 (IL-8), C-C Motif Chemokine Ligand 5 (CCL5, also RANTES), interferon-inducible protein-10 (IP-10) and interferon beta (IFN-β). Moreover, miR-146a exhibited a negative regulatory effect on the NF-κB pathway. Accordingly, a miR-146a inhibitor increased the expression of IL-8, CCL5, IP-10 and IFN-β, whereas a miR-146a mimic reduced the levels of these cytokines. Consequently, exogenous transduction of miR-146a significantly enhanced HTNV replication in HUVEC cells. We also discovered that viral proteins (NP/GP) contributed to miR-146a expression via enhancement the activity of miR-146a promoter. In conclusion, these results imply the negative regulation of miR-146a on the production of HTNV-induced pro-inflammatory cytokines contributes to virus replication, which suggest that miR-146a may be regarded as a novel therapeutic target for HTNV infection.

Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C–C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.

MiRNA-181a regulates Toll-like receptor agonist-induced inflammatory response in human fibroblasts.

MicroRNAs (miRNA) regulate the synthesis of cytokines in response to Toll-like receptor (TLR) activation. Our recent microarray study comparing normal and inflamed human dental pulps showed that miRNA-181 (miR-181) family is differentially expressed in the presence of inflammation. Prior studies have reported that the dental pulp, which is composed primarily of TLR4/2+ fibroblasts, expresses elevated levels of cytokines including Interleukin-8 (IL-8) when inflamed. In this study, we employed an in-vitro model to determine the role of the miRNA 181 family in the TLR agonist-induced response in human fibroblasts. TLR4/2+ primary human dental pulp fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (Pg LPS), a known oral pathogen, and IL-8 and miR-181 expression measured. An inversely proportional relationship between IL-8 and miR-181a was observed. In-silico analysis identified a miR-181a binding site on the 3′UTR of IL-8 which was confirmed by dual-luciferase assays. MiR-181a directly binds to the 3′UTR of IL-8, an important inflammatory component of the immune response, and modulates its levels. This is the very first report demonstrating miR-181a regulation of IL-8.

Elevated miR-155 Promotes Inflammation in Cystic Fibrosis by Driving Hyperexpression of Interleukin-8

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ΔF508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.

P125 Application of biomarkers to sentinel lymph node biopsy for the management of breast cancer

Objective Sentinel lymph node in breast cancer is a predictor of the tumor’s metastatic potential.Cytokines emerging from antitumor immune response might also target sentinel lymph node (SLN).In this study,we evaluated the cytokine profile including interleukin-8 (IL-8),interleukin-12 (IL-12),interferon-gamma (IFN-γ),and tumor necrosis factor alpha (TNF-α) for the T-cell response.Methods From January 2008 to August 2010,35 patients with breast mass underwent SLN biopsy in UNIMED Medical Institute.The specimens were formalin-fixed and paraffin-embedded for hematoxylin and eosin staining and immunohistochemistry with following markers:IL-8,IL-12,IFN-γ and TNF-α.The staining intensity was scored according to four grades:negative (-),weak expression (+),moderately positive expression (+ +),strong positive expression (+ + +).The correlation between the expression of cytokines and pathological characteristics were also analyzed using the Spearman Rank Correlation test.Results Out of 35 patients,SLN metastases were observed in 20 patients and among them,75% (15 /20) were strongly stained (+ + +) with IL-8 and moderately stained (+ +) with IL-12,but only 25% (5 /20) were strongly stained (+ + +) with IFN-γ and TNF-α.The expression of IL-8 was significantly associated with all tumor grades (r=0.639,P=0.00),estrogen receptor (r=0.778,P=0.01),progesterone receptor (r=0.759 P=0.00) and HER-2 /neu receptor (r=0.863 P=0.00).Conclusion The strong association between IL-8 expression and SLN metastases might highlight the prognostic significance of IL-8 in SLN.

The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65.

Tumour necrosis factor-α: prognostic role and relationship with interleukin-8 and endothelin-1 in non-small cell lung cancer

Cytokines mediate numerous physiological and immune reactions, which are manifested in various biological effects, including tumouricidal activity. We evaluated the expression of the pleiotropic cytokine, tumour necrosis factor-alpha (TNF-alpha), by competitive PCR technique in 47 non-small cell lung cancer (NSCLC) cases and the impact of TNF-alpha on their clinical behaviour. Using univariate analysis, our study demonstrated a positive correlation between high TNF-alpha expression and favourable prognosis in NSCLC in terms of overall survival and disease free interval (p=0.03 and 0.04, respectively) and TNF-alpha maintained its independent role in multivariate analysis. TNF-alpha can stimulate the expression of many molecules, including interleukin-8 (IL-8) and endothelin-1 (ET-1); in our study, the expression of TNF-alpha was significantly associated with high IL-8 mRNA levels (p=0.008) and ET-1 mRNA positivity (p=0.03). We suggested that TNF-alpha can induce ET-1 mRNA expression in NSCLC, similarly to IL-8 expression. Our study may also contribute to advancing the knowledge of the molecular relationship between cytokines and endothelial functions in NSCLC.

Reactive oxygen and nitrogen species differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and interleukin-8 expression.

Toll-like receptor 4 (TLR4) has been identified as a transmembrane protein involved in the host innate immune response to gram-negative bacterial lipopolysaccharide (LPS). Upon activation by LPS recognition, the TIR domain of TLR4 signals through MyD88 to activate the nuclear factor kappa B (NF-kappa B) pathway, a critical regulator of many proinflammatory genes, including interleukin-8 (IL-8). Emerging evidence suggests that reactive oxygen species (ROS) can contribute to diverse signaling pathways, including the LPS-induced cascade. In the present study we investigated the role of ROS in TLR-mediated signaling. Purified Escherichia coli LPS, a highly specific TLR4 agonist, elicited an oxidative burst in the monocyte-like cell line THP-1 in a time- and dose-dependent manner. This oxidative burst was shown to be dependent on the presence of TLR4 through transfection studies in HEK cells, which do not normally express this protein, and with bone marrow-derived macrophages from C3H/HeJ mice, which express a mutated TLR4 protein. LPS-stimulated IL-8 expression could be blocked by the antioxidants N-acetyl-L-cysteine and dimethyl sulfoxide at both the protein and mRNA levels. These antioxidants also blocked LPS-induced IL-8 promoter transactivation as well as the nuclear translocation of NF-kappa B. These data provide evidence that ROS regulate immune signaling through TLR4 via their effects on NF-kappa B activation.

Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells.

Shiga toxins made by Shiga toxin-producing Escherichia coli (STEC) are associated with hemolytic uremic syndrome. Shiga toxins (Stxs) may access the host systemic circulation by absorption across the intestinal epithelium. The effects of Stxs on this cell layer are not completely understood, although animal models of STEC infection suggest that, in the gut, Stxs may participate in both immune activation and apoptosis. Stxs have one enzymatically active A subunit associated with five identical B subunits. The A subunit inactivates ribosomes by cleaving a specific adenine from the 28S rRNA. We have previously shown that Stxs can induce multiple C-X-C chemokines in intestinal epithelial cells in vitro, including interleukin-8 (IL-8), and that Stx-induced IL-8 expression is linked to induction of c-Jun mRNA and p38 mitogen-activated protein (MAP) kinase pathway activity. We now report Stx1 induction of both primary response genes c-jun and c-fos and activation of the stress-activated protein kinases, JNK/SAPK and p38, in the intestinal epithelial cell line HCT-8. By 1 h of exposure to Stx1, mRNAs for c-jun and c-fos are induced, and both JNK and p38 are activated; activation of both kinases persisted up to 24 h. Stx1 enzymatic activity was required for kinase activation; a catalytically defective mutant toxin did not activate either. Stx1 treatment of HCT-8 cells resulted in cell death that was associated with caspase 3 cleavage and internucleosomal DNA fragmentation; this cytotoxicity also required Stx1 enzymatic activity. Blocking Stx1-induced p38 and JNK activation with the inhibitor SB202190 prevented cell death and diminished Stx1-associated caspase 3 cleavage. In summary, these data link the Stx1-induced ribotoxic stress response with both chemokine expression and apoptosis in the intestinal epithelial cell line HCT-8 and suggest that blocking host cell MAP kinases may prevent these Stx-associated events.

Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression.

Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. We and others showed that several cytokine levels, including interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFalpha), are elevated in the peritoneal fluid of women with endometriosis compared with those in women without endometriosis. We also demonstrated that the addition of IL-8 to the culture medium stimulated the proliferation of cultured endometriotic stromal cells. TNFalpha is a multipotent cytokine that induces IL-8 production in various cell types. Therefore, we hypothesized that TNFalpha may also contribute to the pathogenesis of endometriosis by inducing the production of IL-8. To test this hypothesis, we analyzed the peritoneal fluid concentrations of IL-8 and TNFalpha using enzyme-linked immunosorbent assay (ELISA). We observed a significant correlation between the levels of TNFalpha and IL-8 in the peritoneal fluid of endometriosis patients. We also obtained the endometriotic stromal cells from chocolate cyst linings of the ovary. The expression of the receptors for TNFalpha (TNFR) was examined by RT-PCR. We observed the expression of both TNFR-I and TNFR-II genes in endometriotic stromal cells. The expression of IL-8 gene and protein was analyzed by Northern blot hybridization and enzyme-linked immunosorbent assay, respectively. TNFalpha induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. The addition of TNFalpha promoted the proliferation of the endometriotic stromal cells, and the stimulatory effects of TNFalpha were abolished by adding anti-IL-8 antibody. We demonstrated for the first time that TNFalpha stimulated proliferation of endometriotic stromal cells through induction of IL-8 gene and protein expression. We concluded that the TNFalpha may be one of the essential factors for the pathogenesis of endometriosis.

Proinflammatory Potential of Cytomegalovirus Infection

We observed the effects of antiviral therapy on CMV and/or oxidative-stress-induced stimulation of proinflammatory molecules including interleukin-8 (IL-8), melanoma growth stimulatory activity-α (GRO-α) and intercellular adhesion molecule 1 (ICAM-1) using human foreskin fibroblasts. Ganciclovir, foscarnet or cidofovir completely suppressed virus replication, as demonstrated by CMV late (L) antigen production. These drugs did not influence CMV immediate-early (IE) antigen expression and had no effects on CMV-induced cellular changes in IL-8, GRO-α and ICAM-1 levels. Phosphorothioate oligonucleotide (ISIS 2922) suppressed both CMV IE and L antigen by 99%. ISIS 2922 completely suppressed CMV-induced upregulation of both chemokines and ICAM-1. Induction of oxidative stress by H₂O₂ upregulated IL-8 expression. Oxidative stress and CMV infection showed synergistic effects on IL-8 expression. ISIS 2922 only partially inhibited the upregulation of IL-8 in infected cells treated with H₂O₂, whereas cotreatment with ISIS 2922 and antioxidants inhibited the upregulation almost completely. The results showed that inhibition of CMV IE expression alone or in combination with antioxidants is promising for the treatment of CMV disease.

Role of recruited neutrophils in interleukin-8 production in dog trachea after stimulation with Pseudomonas in vivo.

Cell-free supernatant of Pseudomonas aeruginosa (PA) recruits neutrophils into the airways indirectly by inducing the production of chemotactic factors, including interleukin-8 (IL-8). PA products stimulate IL-8 expression selectively in surface airway epithelium, gland ducts, serous cells, and recruited neutrophils. To examine the relative contribution of neutrophils in IL-8 release in the airway lumen, we studied the effect of inhibition of neutrophil recruitment on IL-8 concentration in tracheal fluid after introduction of PA supernatant into the dog trachea in vivo. Tracheal superfusion with PA supernatant caused neutrophil recruitment and increased the IL-8 concentration in the tracheal lumen; NPC 15669 inhibited both effects. To study whether migration of neutrophils into the airway lumen per se induces their expression of IL-8, we compared effects of local introduction of IL-8 and of PA supernatant into the trachea on IL-8 expression in neutrophils recruited into the trachea. PA supernatant, but not exogenous IL-8 alone, induced IL-8 mRNA expression in neutrophils recruited into the trachea. To determine what product(s) of PA stimulate IL-8 expression in neutrophils, we examined neutrophils isolated from peripheral blood. PA supernatant induced IL-8 production in neutrophils, an effect reproduced by PA lipopolysaccharide and inhibited by polymyxin B. These results suggest that neutrophils recruited into the airway lumen play a major role in local IL-8 production in airways in response to bacteria such as PA, depending on the presence of stimuli such as lipopolysaccharide.