The Association between Fever and Prognosis in Lung Cancer Patients with Bone Metastases Receiving Zoledronic Acid.

Zoledronic acid is an established agent used in the management of metastatic bone disease. The administration of zoledronic acid improves overall survival (OS) of lung cancer patients with bone metastases receiving chemotherapy. However, it is currently unknown whether zoledronic acid-induced fever is associated with OS. The purpose of this study was to examine the association between zoledronic acid-induced fever and prognosis in lung cancer patients with bone metastases. We retrospectively analyzed 98 lung cancer patients with bone metastases who had received zoledronic acid. The end point outcome measure was OS. Multivariate analyses were used to estimate the hazard ratio (HR) for OS due to fever after adjusting for covariates. In multivariate analysis, white blood cell (WBC) count, lactate dehydrogenase (LDH) level, fever, chemotherapy, and hypercalcemia were independent prognostic factors, with HRs of 2.834 for WBC count (<10 × 103/µL vs. ≥10 × 103/µL, p < 0.001), 3.044 for LDH level (<250 vs. ≥250 IU/L, p < 0.001), 0.603 for fever (<37.0 vs. ≥37.0°C, p = 0.039), 0.481 for chemotherapy (chemotherapy not administered vs. administered, p = 0.006), and 2.453 for hypercalcemia (<11.0 vs. ≥11.0 mg/dL, p = 0.001). Zoledronic acid-induced fever was the most important prognostic factor in this cohort of lung cancer patients with bone metastases.

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.

[FACTORS ASSOCIATED WITH THE PRESENCE OF ALLERGEN-SPECIFIC IgE RESPONSES IN ASTHMA PATIENTS WHO HAD NO IgE RESPONSES DETECTABLE BY MAST-26].

AIM: To elucidate the characteristics of patients with asthma who have specific IgE responses to inhaled allergens detected by ImmunoCAP, which is not detectable by MAST-26. METHODS: A total of 168 patients with adult asthma who reside in the Kanto region were recruited. Levels of total serum IgE and allergen specific IgE antibodies towards 14 common inhaled allergens (MAST-26) were measured. Among these samples, 48 patients with no detectable allergen-specific IgE (group A) and 44 patients with strong sensitization to Dermatophagoides farinae (group B) were selected for further assessment of their sensitization to inhaled allergens such as cockroach and moth using ImmunoCAP. RESULTS: In group A, ImmunoCAP detected specific IgE responses to some inhaled allergens in 27.1% of the patients. The strongest predictive factor for the presence of allergen-specific IgE responses detected by ImmunoCAP was elevated levels of total serum IgE (p=0.0007). In group B, the presence of IgE responses specific to cockroach or moth by ImmunoCAP were found in 27.8% or 52.3% of the patients, respectively. The predictive factor for the presence of these positive IgE responses was also elevated levels of total serum IgE (p=0.0003). CONCLUSION: Asthma patients with no detectable specific IgE responses to any inhaled allergens by MAST-26 may be still sensitized to common inhaled allergens, including cockroach and moth. Thus, the presence of allergen-specific IgE responses may be re-assessed by ImmunoCAP in patients with asthma, especially when patients have higher levels of total serum IgE.

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.

Acquired resistance L747S mutation in an epidermal growth factor receptor-tyrosine kinase inhibitor-naïve patient: A report of three cases.

The purpose of the present study was to report cases of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-naïve patients carrying a mutation associated with acquired resistance to the drug. Gene alterations in 77 lung carcinoma patients were analyzed by collecting and studying curette lavage fluid at the time of diagnosis. PCRs were performed to amplify mutation hotspot regions in EGFR genes. The PCR products were direct-sequenced and the mutations confirmed by resequencing using different primers. Case 1 was a 78-year-old Japanese male diagnosed with stage IB lung adenocarcinoma who was found to have two EGFR mutations, G719S and L747S. Case 2 was a 73-year-old Japanese male diagnosed with stage IV squamous cell lung carcinoma and bone metastasis who had the EGFR mutation, L747S. Case 3 was an 82-year-old Japanese male diagnosed with hyponatremia due to inappropriate secretion of antidiuretic hormone and stage IIIB small cell lung carcinoma (SCLC) who had the EGFR mutation, L747S. Thus, the EGFR mutation L747S associated with acquired EGFR-TKI resistance was detected in two non-small cell lung carcinoma (NSCLC) patients and one SCLC patient, none of whom had ever received EGFR-TKI. The patients were current smokers with stages at diagnosis ranging from IB to IV, and their initial tumors contained resistant clones carrying L747S. L747S may be associated with primary resistance. To the best of our knowledge, this study is the first report of an EGFR mutation associated with resistance to EGFR-TKI in SCLC patients. The early detection of EGFR-TKI resistance mutations may be beneficial in making treatment decisions for lung carcinoma patients, including those with SCLC.

Analysis of EGFR, KRAS and P53 mutations in lung cancer using cells in the curette lavage fluid obtained by bronchoscopy.

Histopathological samples are commonly used for molecular testing to detect both oncogenes and tumor-suppressor genes in lung cancer. The purpose of this study was to determine the efficacy of using curette lavage fluid for molecular testing to detect EGFR, KRAS and P53 mutations in lung cancer patients. Samples were obtained from 77 lung cancer patients by bronchoscopy at the time of diagnosis, collected by scraping the site of the primary tumor lesion with a curette. DNA was extracted from cells in the curette lavage fluid, and PCRs were performed to amplify mutation hot spot regions in the EGFR, KRAS and P53 genes. The PCR products were direct-sequenced to detect mutations of each gene. The reference sequence of each gene was obtained from GenBank. Overall, 27% (21 of 77) were found with EGFR mutations, 1% (1 of 77) with KRAS mutations, and 36% (28 of 77) with P53 mutations. KRAS mutations were not detected in patients harboring mutations in either EGFR or P53. P53 mutations were identified in 38% (8 of 21) of the patients with EGFR mutations, all of who had advanced lung cancer. Of these patients, a 62-year-old female current smoker was given EGFR-TKI as third-line therapy, with no improvement in clinical symptoms or results of radiographic examination. Multivariate analysis indicated that P53 mutation rates in advanced-stage lung cancer were significantly higher than those in early-stage lung cancer (P=.017). In contrast, EGFR mutation rates were not significantly associated with staging. L747S in EGFR, described as a mutation associated with secondary resistance to EGFR-TKI, was detected in three patients who had never received EGFR-TKI, including one SCLC patient. It is possible to analyze EGFR, KRAS and P53 mutations using curette lavage fluid collected from lung cancer patients. This is useful when sufficient amounts of tumor samples cannot be obtained. Data from the current study suggest that EGFR mutations in concert with P53 mutations accelerate cancer development and lead to evolution of therapeutic resistance.

Azacitidine-induced pneumonitis in a patient with myelodysplastic syndrome: first case report in Japan.

A 74-year-old Japanese man with myelodysplastic syndrome (MDS) received chemotherapy with azacitidine. From the second day after starting the administration, he complained of fever, cough and shortness of breath. Chest roentgenography and computed tomography showed consolidations and ground-glass opacities. His symptoms grew from worse to life-threatening. We diagnosed him with azacitidine-induced pneumonitis and began administering corticosteroids. Thereafter, his symptoms and radiographic abnormalities improved. Azacitidine is a hypomethylating agent that improves the survival of MDS patients. Although this drug is commonly well tolerated and rarely causes severe lung injury, it is important to consider the potentially serious adverse effects of azacitidine-induced pneumonitis.

IL-17A stimulates granulocyte colony-stimulating factor production via ERK1/2 but not p38 or JNK in human renal proximal tubular epithelial cells.

We investigated the potential role of IL-17A in the induction of granulocyte colony-stimulating factor (G-CSF), a critical granulopoietic growth factor, in human renal proximal tubular epithelial cells. Human renal proximal tubular cells (HK-2, ATCC) were used to characterize the effects of IL-17A or IL-17F on G-CSF production, using ELISA, real-time RT-PCR, and immunoblotting. The cell surface expression of IL-17 receptors (IL-17Rs) was analyzed by flow cytometry. IL-17A stimulation of proximal tubular cells led to a dose- and time-dependent increase in secreted G-CSF. This effect was dependent on mRNA transcription and protein translation. Real-time RT-PCR demonstrated that G-CSF mRNA expression reached a maximum level at 6 h following IL-17A stimulation and that this increase was dose dependent. Both IL-17RA and IL-17RC were expressed on proximal tubular cells. IL-17A also enhanced TNF-α- or IL-1ß-mediated G-CSF secretion from cells. Additionally, IL-17A induced MAPK (ERK1/2 but not p38 MAPK or JNK) activation, and pharmacological inhibitors of MEK1/2 (U0126) but not of p38 MAPK (SB203580) or JNK (SP600125), significantly blocked the IL-17A-mediated G-CSF release. We demonstrated the potential ability of IL-17A to induce G-CSF in renal proximal tubular cells. It is proposed that IL-17A may play an important role in neutrophil transmigration and activation via stimulation of G-CSF in tubular injury.

IL-17A and IL-17F stimulate chemokines via MAPK pathways (ERK1/2 and p38 but not JNK) in mouse cultured mesangial cells: synergy with TNF-alpha and IL-1beta.

We investigated the role of IL-17 family members IL-17A and IL-17F in the induction of chemokines in mouse cultured mesangial cells (SV40 MES 13 cells). We evaluated the expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) by ELISA and real-time RT-PCR (Q-PCR). Activation of MAPK was assessed by immunoblotting. IL-17RA and IL-17RC were inhibited by small interfering RNA (siRNA). We found that IL-17A or IL-17F stimulation of mesangial cells led to both a dose- and time-dependent increase in MCP-1 and MIP-2 release. This effect was dependent on mRNA transcription and protein translation. Both also enhanced TNF-alpha- and IL-1beta-mediated MCP-1 and MIP-2 release in the cells. Additionally, we observed that IL-17A and IL-17F induced MAPK (p38 MAPK, ERK1/2, and JNK) activation and that pharmacological inhibitors of p38 MAPK (SB203580) and ERK1/2 (U0126), but not JNK (SP600125), blocked the IL-17A/IL-17F-mediated MCP-1 and MIP-2 release. Mesangial cells expressed IL-17RA and IL-17RC, and the IL-17A-mediated MCP-1 and MIP-2 release was significantly blocked by soluble IL-17RA. Furthermore, inhibition of either IL-17RA or IL-17RC expression via siRNA led to significant reduction of IL-17A/IL-17F-stimulated chemokine production. We conclude that IL-17A and IL-17F induce the production of chemokines MCP-1 and MIP-2 via MAPK pathways (p38 MAPK and ERK1/2), as well as mRNA transcription and protein translation and have synergistic effects with TNF-alpha and IL-1beta in cultured mesangial cells.

[A case of MALT lymphoma simultaneously associated with gastric and pulmonary involvement].

A 52-year-old man had been treated by hemodialysis because of IgA nephropathy since 1994. Gastric MALT lymphoma was diagnosed in January 2007. Radiation therapy was performed for 4 weeks (40Gy) and the response was complete remission (CR) by September 2007. He was admitted to our hospital in February 2008 because of an abnormal chest shadow. Chest CT showed multiple cystic lesions with calcification and consolidation. Transbronchial lung biopsy from the area of consolidation (left S5) showed pulmonary invasion of small lymphoid cells. PCR analysis showed clonal rearrangement of the heavy chain of the immunoglobulin gene. Accordingly, MALT lymphoma was diagnosed. Rituximab infusion was performed, because CD20 immunostaining was positive and he had been treated by hemodialysis. The abnormal chest shadow was presented since gastric MALT lymphoma was diagnosed. We considered that MALT lymphoma occurred simultaneously in the stomach and lung.

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.

[A case of lung adenocarcinoma of pancoast type successfully treated with concurrent chemoradiotherapy].

We reported a case of lung adenocarcinoma of Pancoast type that was successfully treated with chemoradiotherapy. A 66-year-old man was admitted to our hospital because of back pain. Chest computed tomography (CT) showed a Pancoast tumor on the left side. Using transbronchial needle aspiration, we diagnosed lung adenocarcinoma (cT3N0M0). The patient received chemoradiotherapy simultaneously(carboplatin AUC5 and irinotecan 60 mg/m2). There are no findings of tumor recurrence 8 years after chemoradiotherapy. This patient was successfully treated with concurrent chemoradiotherapy, which is suggested to be a useful therapy for Pancoast tumor.

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.

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.

The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells.

BACKGROUND: IL-17F is involved in airway inflammation, but its biologic activity and signaling pathway remain incompletely defined. Interferon-gamma-inducible protein 10 (IP-10) is widely expressed and plays a role in airway inflammatory diseases. OBJECTIVE: We sought to investigate the functional linkage between IL-17F and IP-10 expression in bronchial epithelial cells. METHODS: Bronchial epithelial cells were cultured in the presence or absence of IL-17F, and/or a T(H)1 cytokine, T(H)2 cytokines, proinflammatory cytokines, various kinase inhibitors, or a Raf1 dominant-negative mutant to analyze the expression of IP-10. Moreover, the involvement of p90 ribosomal S6 kinase (p90RSK) and cyclic AMP response element-binding protein (CREB) in IL-17F-induced IP-10 expression were investigated. RESULTS: IL-17F induces the gene and protein expression of IP-10. The addition of IFN-gamma, IL-1beta, and TNF-alpha augmented IL-17F-induced IP-10 expression. The mitogen-activated protein kinase kinase (MEK) inhibitors PD98059, U0126, and Raf1 kinase inhibitor I significantly inhibited its production. In contrast, a p38 inhibitor, a JNK inhibitor, protein kinase C inhibitors, and a phosphatidylinositol 3-kinase inhibitor, showed no inhibitory effect. Furthermore, overexpression of a Raf1 dominant-negative mutant inhibited its expression. Of interest, IL-17F phosphorylated p90RSK and CREB, and transfection of the cells with a short interfering RNA for p90RSK or CREB inhibited its expression, suggesting p90RSK and CREB as novel signaling molecules of IL-17F. CONCLUSION: IL-17F is a potent inducer of IP-10 in bronchial epithelial cells through the activation of the Raf1-MEK1/2-extracellular signal-regulated kinase 1/2-p90RSK-CREB pathway, supporting its regulatory role in airway inflammation. CLINICAL IMPLICATIONS: The IL-17F-IP-10 axis might be a novel and critical therapeutic target for airway inflammatory diseases.

Induction of granulocyte-macrophage colony-stimulating factor by a new cytokine, ML-1 (IL-17F), via Raf I-MEK-ERK pathway.

BACKGROUND: ML-1 (IL-17F) is a recently discovered cytokine, and its function remains elusive. GM-CSF is a crucial cytokine for the maturation of various cell types and regulates allergic airway inflammation. OBJECTIVE: The functional effect of ML-1 in the expression of GM-CSF was investigated. METHODS: The levels of gene and protein expression in normal human bronchial epithelial cells (NHBEs) in the presence or absence of various kinase inhibitors or, in some cases, of a Raf1 dominant-negative mutant were determined by RT-PCR and ELISA, respectively. Western blotting was performed to investigate kinase activation. RESULTS: The results showed first that ML-1 induces, in a time-dependent and dose-dependent manner, the gene and protein expression for GM-CSF NHBEs, which are associated with activation of Raf1 and MAP kinase kinase (MEK) kinases. Selective MEK inhibitors, PD98059 and U0126, and Raf1 kinase inhibitor I significantly inhibited ML-1-induced GM-CSF production. Furthermore, overexpression of Raf1 dominant-negative mutants inhibited IL-17F-induced GMCSF expression. The combination of PD98059 and Raf1 kinase inhibitor I completely blocked GM-CSF production, whereas 2 protein kinase C inhibitors, Ro-31-7549 and GF109203X, and a phosphatidylinositol 3-kinase inhibitor, LY294002, showed no inhibitory effect. CONCLUSION: These findings suggest that ML-1 induces GM-CSF expression through the activation of the Raf1-MEK-extracellular signal-regulated kinase 1/2 pathway.