IL-17F induces IL-6 via TAK1-NFκB pathway in airway smooth muscle cells.

INTRODUCTION: Interleukin (IL)-17F plays a critical role in the pathophysiology of asthma. However, the precise role of IL-17F in airway smooth muscle cells (ASMCs) and its regulatory mechanisms remain to be defined. Therefore, we sought to investigate the expression of IL-6 by IL-17F and the involvement of transforming growth factor ß-activated kinase 1 (TAK1) and nuclear factor (NF)-κB by in ASMCs. METHODS: ASMCs were cultured in the presence or absence of IL-17F. The expression of IL-6 gene and protein was analyzed using real-time PCR and ELISA, and the activation of TAK1 and NF-κB was detected by Western blotting. The effect of TAK1 inhibitor 5Z-7-oxozeaenol and NF-κB inhibitor BAY 11-7082 on the expression of IL-6 was investigated. Finally, the short interfering RNAs (siRNAs) targeting TAK1 and a subunit of NF-κB, p65 were transfected into ASMCs. RESULTS: The expression of IL-6 gene and protein was significantly induced by IL-17F. IL-17F activated TAK1 and NF-κB in ASMCs. Transfection of siRNAs targeting TAK1 abolished IL-17F-induced phosphorylation of p65. Both 5Z-7-oxozeaenol and BAY 11-7082 significantly inhibited IL-17F-induced IL-6 production in a dose-dependent manner. Similarly, transfection of the cells with siRNAs targeting TAK1 and p65 inhibited the expression of IL-6. CONCLUSIONS: Collectively, these results provided evidence supporting the potential importance of the Th17-ASMCs crosstalk via the IL-17F-IL-6 axis in airway inflammation and as a candidate pharmacological target for airway inflammatory diseases such as asthma.

Overexpression of microRNA-155 suppresses chemokine expression induced by Interleukin-13 in BEAS-2B human bronchial epithelial cells.

BACKGROUND: MicroRNAs are non-coding small RNAs that regulate expression of target genes by binding to 3' untranslated regions. In this study, we used bronchial epithelial cells to investigate in vitro the role of the microRNA miR-155 in the expression of chemokines associated with airway inflammation. miR-155 has previously been reported to regulate allergic inflammation. METHODS: BEAS-2B bronchial epithelial cells were cultured and transfected with mimic or inhibitor oligonucleotides to overexpress or downregulate miR-155, as confirmed by real-time PCR. Cells were then stimulated with tumor necrosis factor-alpha, interleukin-13 (IL-13), and a double stranded RNA that binds Toll-like receptor 3. Expression and secretion of the chemokines CCL5, CCL11, CCL26, CXCL8, and CXCL10 were then quantified by real-time PCR and ELISA, respectively. Phosphorylation of signal transducer and activator of transcription 6 (STAT6), a target of the IL-13 receptor, was analyzed by ELISA. RESULTS: miR-155 overexpression significantly suppressed IL-13-induced secretion of CCL11 and CCL26. These effects were specific, and were not observed for other chemokines, nor in cells with downregulated miR-155. miR-155 overexpression also suppressed CCL11 and CCL26 mRNA, but did not affect expression of the IL-13 receptor or phosphorylation of STAT6. CONCLUSIONS: miR-155 specifically inhibits IL-13-induced expression of eosinophilic chemokines CCL11 and CCL26 in bronchial epithelial cells, even though the 3'-untranslated region of these genes do not contain a consensus binding site for miR-155.

Synthetic double-stranded RNA induces interleukin-32 in bronchial epithelial cells.

OBJECTIVE: Interleukin (IL)-32 is a novel cytokine and is involved in the pathogenesis of various inflammatory diseases, including asthma and COPD. However, the regulatory mechanisms of IL-32 expression and its precise pathogenic role remain to be defined. Given that viral infections are known to potentially cause and exacerbate airway inflammation, in this study, we investigated the expression of IL-32 induced by synthetic double-stranded (ds) RNA, and its signaling mechanisms involved. METHODS: Bronchial epithelial cells were stimulated with synthetic dsRNA poly I:C. The levels of IL-32 expression were analyzed using real-time PCR and ELISA. The involvement of transforming growth factor ß-activated kinase 1 (TAK1) and a subunit of nuclear factor-κB (NF-κB), p65 was determined by western blot analyses. TAK1 inhibitor, 5Z-7-Oxozeaenol and NF-κB inhibitor, BAY 11-7082 were added to the culture to identify key signaling events leading to the expression of IL-32. Finally, the effect of short interfering RNAs (siRNAs) targeting TAK1 and p65 was investigated. RESULTS: dsRNA significantly induced IL-32 gene and protein expression, concomitant with activation of TAK1 and p65. Pretreatment of 5Z-7-Oxozeaenol diminished dsRNA-induced phosphorylation of NF-κB. Both 5Z-7-Oxozeaenol and BAY 11-7082 significantly abrogated dsRNA-induced IL-32 production. Moreover, transfection of the cells with siRNAs targeting TAK1 and p65 inhibited the expression of IL-32. CONCLUSIONS: The expression of IL-32 is induced by dsRNA via the TAK1-NF-κB signaling pathway in bronchial epithelial cells. IL-32 is involved in the pathogenesis of airway inflammation, and may be a novel therapeutic target for airway inflammatory diseases.

Clinical features and outcomes of aspiration pneumonia compared with non-aspiration pneumonia: a retrospective cohort study.

Pneumonia is a leading cause of death among elderly patients. Although aspiration pneumonia (AP) commonly occurs with aging, its clinical features and outcomes are still uncertain. The aims of this study were to describe the clinical features and outcomes of AP and to assess whether presence of AP affects clinical outcomes in patients with community-acquired pneumonia (CAP) and healthcare-associated pneumonia (HCAP). We retrospectively analyzed patients with CAP and HCAP hospitalized in our institution in Japan from October 2010 to March 2012. We compared clinical features and outcomes between AP and non-AP, and investigated risk factors for recurrence of pneumonia and death. Of 214 consecutive patients, 100 (46.7%) were diagnosed as having aspiration pneumonia. These patients were older and had lower body mass index, more comorbidities, and poorer Eastern Cooperative Oncology Group performance status (ECOG PS) than the patients with non-AP. Patients with AP had more severe disease, required longer hospital stays, and had a frequent recurrence rate of pneumonia and higher mortality. In multivariate analyses, AP, age, and ECOG PS were related to recurrence of pneumonia, and the prognostic factors were CURB-65 score and ECOG PS. AP was not a significant indicator for prognosis but was the strongest risk factor for recurrence of pneumonia. Clinical background and outcomes including recurrence and mortality of AP were obviously different from those of non-AP; therefore AP should be considered as a distinct subtype of pneumonia, and it is important to prevent the recurrence of pneumonia in the patients with AP.

Expression and effects of IL-33 and ST2 in allergic bronchial asthma: IL-33 induces eotaxin production in lung fibroblasts.

BACKGROUND: Interleukin (IL)-33, a new member of the IL-1 cytokine family, has been recognized as a key cytokine that enhances T helper 2-balanced immune regulation through its receptor ST2; however, the function and relationship of the IL-33 and ST2 pathways in bronchial asthma are still unclear. We investigated the cellular origin and regulation of IL-33 and ST2 in allergic bronchial asthma in vivo and in vitro. METHODS: BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Mice were exposed to aerosolized 1% OVA for 30 min a day for 7 days. These mice were then challenged with aerosolized 1% OVA 2 days after the last day of exposure. After the OVA challenge, the mice were sacrificed and their lung tissues were obtained. Mouse lung fibroblasts were cultured and treated with IL-33 or IL-13. RESULTS: The levels of IL-33 mRNA and IL-33 protein in lung tissue increased after the OVA challenge. Most IL-33-expressing cells were CD11c+ cells and epithelial cells, and many ST2-expressing cells were stained lung fibroblasts and inflammatory cells. IL-33 induced eotaxin/CCL11 production in lung fibroblasts. IL-33 and IL-13 synergistically induced eotaxin expression. CONCLUSIONS: IL-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. IL-33 and ST2 may play important roles in allergic bronchial asthma.

Oxaliplatin-induced lung injury with allergic reaction.

A 79-year-old man was diagnosed as stage IV colon cancer and treated with a modified FOLFOX6 (mFOLFOX6) regimen. On the 12th cycle, we observed erythema and dyspnea. Radiographs showed ground grass opacities. Blood tests showed elevated levels of eosinophils and immunoglobulin E. We diagnosed this finding as response to drug allergy and administered high-dose methylprednisolone. The treatment was successful and he was discharged. The drug lymphocyte stimulating test against oxaliplatin was positive, indicating a type I and IV allergic reaction due to oxaliplatin. Regimens including oxaliplatin must be carefully monitored and frequent blood tests and chest radiographs are needed.

Cooperative activation of CCL5 expression by TLR3 and tumor necrosis factor-alpha or interferon-gamma through nuclear factor-kappaB or STAT-1 in airway epithelial cells.

BACKGROUND: CCL5/RANTES contributes to prolonged eosinophilic inflammation and asthma exacerbation after a viral infection. We studied the mechanism of CCL5 expression using viral product double-stranded RNA (dsRNA), a ligand of Toll-like receptor 3 (TLR3), and inflammatory cytokines in airway epithelial cells. METHODS: The airway epithelial cell line BEAS-2B was used in our in vitro study, and the levels of CCL5 mRNA and CCL5 protein expression were determined using real-time PCR and ELISA. The activity of the CCL5 promoter region and nuclear factor (NF)-kappaB was assessed by dual luciferase assay using specific luciferase reporter plasmids. We used actinomycin D to assess the stability of mRNA. Phosphorylation of signal transducer and activator of transcription 1 (STAT-1) was analyzed by Western blot. RESULTS: Synthetic dsRNA up-regulated the expression of CCL5 mRNA and CCL5 protein. Adding TNF-alpha or IFN-gamma to dsRNA further increased the expression of CCL5. The combination of TNF-alpha and dsRNA cooperatively activated the CCL5 promoter region and the NF-kappaB-specific reporter. IFN-gamma did not activate these reporters. However, it increased the stability of CCL5 mRNA induced by dsRNA. IFN-gamma phosphorylated STAT-1, but dsRNA did not. The effects of IFN-gamma were not evident in the cells transfected with short interfering RNA for STAT-1. CONCLUSIONS: Cross-talk between TLR3 signaling and inflammatory cytokines regulates the expression of CCL5 in airway epithelial cells. In this mechanism, TNF-alpha may activate NF-kappaB, in cooperation with TLR3 signaling. IFN-gamma may stabilize CCL5 mRNA up-regulated by TLR3. This mechanism may depend on STAT-1.

[Effects of double-stranded RNA poly(I:C) on matrix metalloproteinase mRNA expression in human nasal polyp epithelial cells].

OBJECTIVE: Chronic rhinosinusitis was often exacerbated by viral infection. A disruption of the mechanisms that regulate the activity of matrix metalloproteinases (MMPs) during viral infection was one possible mechanism responsible for the exacerbation. The purpose of study was to achieve a better understanding of MMP expression in nasal epithelial cells after viral infection. METHODS: Human nasal epithelial cells were isolated from nasal polyp specimens obtained during endoscopic endonasal surgery in chronic rhinosinusitis patients. The expression of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA in primary human nasal polyp epithelial cells after double stranded RNA (ds RNA) stimulation were investigated. RESULTS: Among the genes whose expression was evaluated, only expression of MMP-9 mRNA increased significantly after dsRNA stimulation (22.61 +/- 5.47 fold increase, Z = -2.52, P = 0.012). CONCLUSIONS: The significant up-regulation of MMP-9 mRNA, which was not modulated by TIMP-1, was an additional source of increased proteolytic activity in virus-infected upper airways that might contribute to the exacerbation of chronic rhinosinusitis with nasal polyps.

Involvement of IL-17F via the induction of IL-6 in psoriasis.

Recently, the important role of T helper 17 (Th17) cells in psoriasis has been clarified; however, the role of IL-17F produced by Th17 cells is still not fully understood. IL-6 exhibits multiple biologic functions, such as regulation of immunological responses including those in psoriatic reactions. Therefore, we examined the production of IL-6 protein in normal human epidermal keratinocytes (NHEKs) stimulated by IL-17F, TNF-alpha, IL-17A, and IL-17A in combination with TNF-alpha, and PBS control. We then examined the expression of IL-6 mRNA in mouse skin after intradermal injection of IL-17F. Finally, IL-17F expression in skin biopsy specimens from psoriasis patients was examined by immunohistochemistry. The results showed that IL-17F induced production of IL-6 in NHEKs in a time-dependent manner. This could be attenuated by chimeric inhibitor blocking the IL-17 receptor. The amounts of IL-6 stimulated by IL-17F were much higher than those stimulated by TNF-alpha or IL-17A. IL-6 was also significantly upregulated via synergistic stimulation with IL-17A plus TNF-alpha. The expression of IL-6 mRNA 24 h after IL-17F injection in the mouse skin was 3.2-fold higher than that in the control group. Immunohistochemistry of inflammatory cells in the dermis demonstrated a large number of CD4(+) T cells showing IL-17F positivity in psoriatic skin lesions, but few or none in non-lesional psoriatic skin. Our results indicate that IL-17F produced by CD4(+) T cells causes the inflammation in psoriasis partly through induction of IL-6 in keratinocytes.

Expression of antiviral molecular genes in nasal polyp-derived cultured epithelial cells.

CONCLUSION: The results of the present study indicate the existence of natural immunity mechanisms via which viruses are eliminated from nasal and paranasal sinus mucosa. OBJECTIVES: Acute sinusitis and acute aggravation of chronic sinusitis are often caused by bacteria, which are secondary to viral infection of the nose. Antiviral molecules are considered to be expressed and protect the host after viral infection. We investigated the expression of antiviral molecules after viral infection of the nose. MATERIALS AND METHODS: We assessed the expression of antiviral molecules, defensin and interferon mRNA, by the real-time polymerase chain reaction (PCR) technique after stimulating cultured nasal polyp cells with polyinosine-polycytidylic acid (poly(I:C)), which is an analog of double-stranded (ds) RNA. RESULTS: The expression of beta-defensin mRNA significantly increased after the stimulation. On the other hand, expression of both interferon-alpha mRNA and interferon-beta mRNA was recognized, but only the expression of interferon-beta mRNA increased after the stimulation.

Double-stranded RNA poly(I:C) enhances matrix metalloproteinase mRNA expression in human nasal polyp epithelial cells.

CONCLUSION: The significant up-regulation of matrix metalloproteinase (MMP)-9 mRNA, which is not modulated by tissue inhibitor of metalloproteinase (TIMP)-1, is an additional source of increased proteolytic activity in virus-infected upper airways that might contribute to the exacerbation of chronic rhinosinusitis with nasal polyps. OBJECTIVES: Chronic rhinosinusitis is often exacerbated by viral infection. We hypothesized that a disruption of the mechanisms that regulate the activity of MMPs during viral infection is one possible mechanism responsible for the exacerbation. In the present study we attempted to achieve a better understanding of MMP expression in nasal epithelial cells after viral infection. MATERIALS AND METHODS: Human nasal epithelial cells were isolated from nasal polyp specimens obtained during endoscopic endonasal surgery in chronic rhinosinusitis patients. We investigated the expression of MMP-2, MMP-9, and TIMP-1 mRNA in primary human nasal polyp epithelial cells after dsRNA stimulation. RESULTS: Among the genes whose expression was evaluated, only expression of MMP-9 mRNA increased significantly after dsRNA stimulation.

IL-17F-induced IL-11 release in bronchial epithelial cells via MSK1-CREB pathway.

IL-17F is involved in asthma, but its biological function and signaling pathway have not been fully elucidated. IL-11 is clearly expressed in the airway of patients with allergic airway diseases such as asthma and plays an important role in airway remodeling and inflammation. Therefore, we investigated the expression of IL-11 by IL-17F in bronchial epithelial cells. Bronchial epithelial cells were cultured in the presence or absence of IL-17F and/or Th2 cytokines (IL-4 and IL-13) or various kinase inhibitors to analyze the expression of IL-11. Next, activation of mitogen- and stress-activated protein kinase (MSK) 1 by IL-17F was investigated. Moreover, the effect of short interfering RNAs (siRNAs) targeting MSK1 and cAMP response element binding protein (CREB) on IL-17F-induced IL-11 expression was investigated. IL-17F induced IL-11 expression, whereas the costimulation with IL-4 and IL-13 augmented this effect even further. MEK inhibitors PD-98059, U0126, and Raf1 kinase inhibitor I, significantly inhibited IL-11 production, whereas overexpression of a Raf1 dominant-negative mutant inhibited its expression. IL-17F clearly phosphorylated MSK1, whereas PD-98059 inhibited the phosphorylation of IL-17F-induced MSK1. Both MSK1 inhibitors Ro-31-8220 and H89 significantly blocked IL-11 expression. Moreover, transfection of the cells with siRNAs targeting MSK1 inhibited activation of CREB, and the siRNAs targeting MSK1 and CREB blocked expression of IL-11. These data suggest that IL-17F may be involved in airway inflammation and remodeling via the induction of IL-11, and RafI-MEK1/2-ERK1/2-MSK1-CREB is identified as a novel signaling pathway participating in this process. Therefore, the IL-17F/IL-11 axis may be a valuable therapeutic target for asthma.

Functional characterization of IL-17F as a selective neutrophil attractant in psoriasis.

IL-17F is known to be involved in many inflammatory diseases, but its role in skin diseases has not been fully examined. Because IL-8 is involved in many skin diseases such as psoriasis, we investigated the production of IL-8 in normal human epidermal keratinocytes (NHEKs) stimulated by IL-17F, tumor necrosis factor-alpha (TNF-alpha), IL-17A, and control using real-time PCR and ELISA. The results showed that IL-17F induced production of IL-8 in NHEKs in a time-dependent manner. Interestingly, the amounts of IL-8 stimulated by IL-17F were much higher than those stimulated by TNF-alpha or IL-17A. Next, we confirmed that selective mitogen-activated protein kinase kinase inhibitors significantly inhibited IL-17F-induced IL-8 production. Moreover, mouse skin intradermally injected with IL-17F expressed high level of IL-8 mRNA and induced ERK1/2 phosphorylation. Histological examination of mouse skin that was injected with IL-17F revealed marked neutrophilia in dermis and the infiltration was significantly inhibited by anti-IL-8 antibody. Finally, IL-17F expression in skin biopsy samples from psoriasis patients were examined by western blotting and ELISA. IL-17F was upregulated in lesional psoriatic skin compared with nonlesional skin. These results indicate that IL-17F may be involved in psoriasis via, in part, the activation of ERK1/2 and the induction of IL-8 in keratinocytes.

Differential regulation of eotaxin expression by dexamethasone in normal human lung fibroblasts.

Lung fibroblasts are a major source of several cytokines including CC chemokine eotaxin. We aimed to study the regulation of eotaxin-1/CCL11 production by dexamethasone and analyze its molecular mechanisms in human lung fibroblasts. Normal human lung fibroblast cells were exposed to IL-4 (40 ng/ml) and/or dexamethasone (10(-6)-10(-9) M), and eotaxin mRNA expression and production was evaluated. Mechanisms of transcriptional regulation were assessed by Western blotting and dual luciferase assay for eotaxin promoter. The effects of dexamethasone on suppressor of cytokine signaling (SOCS)-1 and eotaxin mRNA expression in the cells transfected with expression vector (pAcGFP1-C1) or short interfering RNA (siRNA) for SOCS-1 were also investigated. Within 24 hours, dexamethasone inhibited IL-4-induced eotaxin mRNA expression and protein production, while eotaxin production was markedly increased at 48 and 72 hours after coincubation with IL-4 and dexamethasone. IL-4-induced eotaxin promoter activity was inhibited by dexamethasone at 8 hours, but enhanced at 48 hours after coincubation. Dexamethasone suppressed SOCS-1 mRNA expression but enhanced IL-4-induced STAT6 phosphorylation at 36 to 48 hours after coincubation. Enhanced expression of eotaxin mRNA by dexamethasone 48 hours after coincubation was completely diminished in the cells transfected with either expression vector or siRNA for SOCS-1. These results indicated that dexamethasone, depending on the exposure duration, can either inhibit or enhance IL-4-induced expression and production of eotaxin in the lung fibroblasts. The mechanisms of later enhanced production may depend on the prolonged transcriptional activity of the eotaxin gene, in part due to inhibition of SOCS-1 expression.

Synergistic induction of CX3CL1 by TNF alpha and IFN gamma in osteoblasts from rheumatoid arthritis: involvement of NF-kappa B and STAT-1 signaling pathways.

To explore the regulation of CX3CL1 in inflammatory bone diseases, CX3CL1 expression by osteoblasts (OB) was examined. Human OB isolated from rheumatoid arthritis (RA) patients, osteoarthritis patients, and normal individuals were incubated in the presence of cytokines. Soluble CX3CL1 levels were determined with an enzyme-linked immunosorbent assay. Expression of CX3CL1 mRNA was examined using quantitative real-time polymerase chain reaction. Although tumor necrosis factor (TNF)-α or interferon (IFN)-γ alone RA OB induced negligible CX3CL1 secretion, the combination of TNF-α and IFN-γ induced dramatic increases in both soluble CX3CL1 protein and mRNA transcripts. This synergistic effect was more pronounced in OB from RA than in OB from either osteoarthritis or normal individuals. The expression of CX3CL1 was markedly reduced by specific inhibitors of the nuclear factor-κB (NF-κB) or STAT-1 transcription factor. These findings suggest that osteoblasts are an important cellular source of CX3CL1 and may play roles in inflammatory bone/joint diseases.

Role of RIG-I, MDA-5, and PKR on the expression of inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells.

BACKGROUND: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and reported that dsRNA stimulates expression of inflammatory chemokines through a receptor of dsRNA Toll-like receptor (TLR) 3 in airway epithelial cells. In this study, we focused our study on the role of other receptors for dsRNA, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and double-stranded RNA-dependent protein kinase (PKR). METHODS: Airway epithelial cell BEAS-2B was cultured in vitro. Expression of target RNA and protein were analyzed by PCR and ELISA. To analyze the role of receptors for dsRNA, knockdown of theses genes was performed with short interfering RNA (siRNA). RESULTS: We first investigated the effects of chloroquine, an inhibitor of lysosomal acidification, on the expression of chemokines. Preincubation with 100 microM chloroquine significantly inhibited the expression of mRNA for RANTES, IP-10, and IL-8, stimulated by poly I:C, indicating that poly I:C may react with a receptor expressed inside the cells. RIG-I, MDA-5, and PKR are supposed to be expressed inside the airway epithelial cells. However, the expression of chemokines stimulated with poly I:C was not significantly inhibited for these putative receptors in the cells which were transfected with siRNA. CONCLUSIONS: Synthetic dsRNA poly I:C stimulates the expression of inflammatory chemokines in airway epithelial cells, but the putative receptors for dsRNA such as RIG-I, MDA-5, or PKR may not play pivotal roles in this process. TLR3 may play a major role as reported previously.

Expression of interleukin-17F in a mouse model of allergic asthma.

BACKGROUND: Interleukin (IL)-17F is a recently discovered cytokine and is derived from a panel of limited cell types, such as activated CD4+ T cells, basophils, and mast cells. IL-17F is known to induce several cytokines and chemokines. However, its involvement in airway inflammation has not been well understood. To this end, the expression of IL-17F and the inhibitory effects of glucocorticoids on its expression in a mouse model of asthma were examined. METHODS: Five-week-old BALB/c male mice were sensitized by intraperitoneal injection (i.p.) of ovalbumin (OVA) with alum, and challenged by daily inhalation of aerosolized 1% OVA. 24 h after last challenge (OVA/OVA), the expression of IL-17F was examined in lung tissues by immunohistochemistry and reverse-transcription polymerase chain reaction. Control mice were sensitized and challenged with saline (Sham/Sham). In addition, a group OF OVA-sensitized mice received i.p. injection of water-soluble dexamethasone (DEX) in saline 1 h before ova challenge (OVA/DEX). RESULTS: In sham-challenged mice, IL-17F was not expressed in the lungs, while, in contrast, IL-17F was predominantly expressed in bronchial epithelial cells in addition to the infiltrating inflammatory cells in OVA/OVA mice. Further, the expression of IL-17 F was significantly attenuated by the treatment of mice with DEX. CONCLUSION: These results suggest that bronchial epithelium-derived IL-17F may represent a new pharmacological target for glucocorticoids and may play a role in allergic asthma.

Differential regulation of chemokine expression by Th1 and Th2 cytokines and mechanisms of eotaxin/CCL-11 expression in human airway smooth muscle cells.

BACKGROUND: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells. METHODS: Human ASM cells were cultured in vitro and treated with IL-4, interferon-gamma (IFNgamma), and tumor necrosis factor-alpha (TNFalpha). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay. RESULTS: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNgamma stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter. CONCLUSIONS: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Involvement of Toll-like receptors in the immune response of nasal polyp epithelial cells.

Recognition systems employed by airway epithelial cells to respond to microbial exposure include the action of Toll-like receptors (TLRs). We investigated the presence and function of TLR2, 3, and 4 in primary cultures of human nasal polyp epithelial cells. dsRNA stimulation significantly enhanced the expression and secretion of RANTES, IP-10, IL-8, and GM-CSF. LPS also exhibited stimulatory action, but it was much weaker than dsRNA. Peptidoglycan had no significant stimulatory action on the genes. Flow cytometry showed that the nasal polyp epithelial cell mainly expressed TLR3 in an intracellular compartment, but expression of TLR2 and TLR4 was very low on both the cell surface and in the cell. The immune response of primary nasal polyp epithelial cells induced by TLR3 could not be blocked by anti-TLR3 antibody. Among the TLR ligands evaluated, dsRNA, the ligand for TLR3, mediated the strongest pro-inflammatory effects in primary nasal polyp epithelial cells.

Expression of angiopoietin-1 in osteoblasts and its inhibition by tumor necrosis factor-alpha and interferon-gamma.

Angiogenesis is a crucial component of bone remodeling under both normal and pathophysiological conditions. Among the various mediators that regulate the angiogenic process is the angiopoietin (Ang) family of growth factors. Ang-1 stabilizes new blood vessels by recruiting surrounding mesenchymal cells and promoting their differentiation into vascular smooth muscle cells, whereas Ang-2 is a natural antagonist of Ang-1 and can inhibit angiogenesis. The expression of Ang-1 and Ang-2 in human osteoblasts (hOBs) isolated from rheumatoid arthritis (RA) and osteoarthritis (OA) patients and from healthy individuals has been examined. After incubation in the presence or absence of tumor necrosis factor-alpha (TNF-alpha) and/or interferon-gamma (IFN-gamma), the culture supernatants were assayed for Ang using an enzyme-linked immunosorbent assay. In addition, expression of Ang protein and mRNA was examined using immunohistochemical techniques and quantitative real-time polymerase chain reaction, respectively. It was found that hOBs expressed Ang-1 but not Ang-2 protein, and cultured hOBs from RA and OA patients and from healthy individuals all spontaneously secreted significant amounts of Ang-1 in the absence of any stimulation. Although stimulation with TNF-alpha or IFN-gamma had little or no effect on Ang-1 secretion, costimulation with IFN-gamma plus TNF-alpha dose- and time-dependently diminished secretion of Ang-1 from hOBs. This inhibitory effect was mediated in part by nuclear factor-kappa B via upregulated expression of inducible nitric oxide synthase and enhanced synthesis of nitric oxide. Taken together, these findings suggest that OBs are an important cellular source of Ang-1 and may modulate bone remodeling through regulation of angiogenesis.