Ute Oltmanns

Role of c-jun N-terminal kinase in the induced release of GM-CSF, RANTES and IL-8 from human airway smooth muscle cells

Human airway smooth muscle cells (HASMC) contribute to airway inflammation in asthma by virtue of their capacity to produce several inflammatory mediators including IL‐8, GM‐CSF and RANTES. The intracellular signal pathway underlying the production of these cytokines in HASMC is not entirely elucidated. We examined the role of the mitogen‐activated protein kinase (MAPK) c‐jun N‐terminal kinase (JNK) in TNFα‐ and IL‐1β‐induced GM‐CSF, RANTES and IL‐8 production in HASMC by using a novel specific inhibitor for JNK (SP600125). Confluent HASMC were treated with TNFα or IL‐1β (10 ng ml−1) for 24 h in the presence or absence of SP600125 (1–100 μM). JNK activity was determined by a kinase assay. Phosphorylation of JNK, p38 MAPK and ERK was examined by Western blotting. Culture supernatants were assayed for GM‐CSF, RANTES and IL‐8 content by ELISA. Maximum TNFα‐ or IL‐1β‐induced phosphorylation of JNK in HASMC occurred after 15 min and returned to baseline levels after 4 h. SP600125 inhibited TNFα‐ and IL‐1β‐induced JNK activity in HASMC as shown by the reduced phosphorylation of its substrate c‐jun. Furthermore, GM‐CSF, RANTES and to a lesser extent IL‐8 release from HASMC treated with TNFα and IL‐1β was inhibited dosedependently by SP600125. JNK activation is involved in TNFα‐ and IL‐1β‐induced GM‐CSF, RANTES and IL‐8 production from HASMC. JNK may therefore represent a critical pathway for cytokine production in HASMC.

Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

BackgroundThe elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.MethodsLaser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.ResultsWe provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.ConclusionOur findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle.

BackgroundCigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.MethodsHASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferronis t testResultsCSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.ConclusionCigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.

Mitogen-activated protein kinase signalling pathways in IL-1β-dependent rat airway smooth muscle proliferation

Asthma is associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness. We investigated the role of mitogen‐activated protein kinase (MAPK) pathway in IL‐1β induced ASM proliferation in the rat. Rat tracheal ASM cells were dissociated and maintained in culture. We examined the effect of selective MAPK inhibitors, SB239063 (a p38 MAPK inhibitor), U0126 (a mitogen‐activated and extracellular regulated kinase kinase, MEK‐1, inhibitor which inhibits downstream extracellular regulated kinase, ERK, activity), and SP600125 (a c‐jun N‐terminal kinase, JNK, inhibitor) on IL‐1β‐induced proliferation. Proliferation of ASM cells was significantly increased following exposure to IL‐1β in a dose‐dependent manner. p38, JNK and ERK MAPKs were activated by IL‐1β in a time‐dependent manner, with peak activation time at 30, 60 min and at 6 h, respectively. This activation was inhibited by their respective inhibitors. SP600125 (20 μM) had no effect on IL‐1β‐induced ERK and p38 phosphorylation. SB239063, U0126 and SP600125 dose‐dependently inhibited IL‐1β‐dependent proliferation at doses that inhibit the activities of p38, ERK and JNK MAPKs, respectively. No additive or synergistic effects were observed on proliferative responses with any combination of these compounds. In conclusion, the three major MAPK pathways, ERK as well as the p38 MAPK and JNK pathways, are independent regulators of IL‐1β‐dependent proliferation of rat ASM.

Transition from placental to air breathing stimulates haem-oxygenase-1 expression without functional consequence for pulmonary vascular adaptation in pigs and mice.

1 In systemic vessels, haem‐oxygenase (HO) is induced during oxidative stress and known to modulate vasodilatation and vascular remodelling. At birth, with the transition from placental to air breathing, the pulmonary vessels are exposed to oxidative stress and undergo well‐documented remodelling processes. Thus, we investigated the role of HO in the lung during adaptation to extra‐uterine life using a pig and mouse model. In addition to the novel data presented with regard to one isoform, HO‐1, this study is among the first to describe the pulmonary vascular remodelling in the mouse after birth. 2 We show, for the first time, that another isoform, HO‐2, is present constitutively at birth and HO‐1 protein is induced in the porcine and murine lung after birth in vascular and airway structures, peaking at 14 days in the pig and at about 4 days in the mouse. Furthermore, we show that HO‐1 mRNA declines after birth in the mouse lung. 3 Inhibitors of HO did not modify vasodilator responses in vessels from 14‐day‐old pigs. 4 Moreover, lungs from HO‐1‐deficient mice developed normally after birth. 5 HO‐1 is induced at birth but plays no role in the development of vasodilator responses or remodelling that occurs at this time. These data suggest that HO‐1 expression at birth is a redundant response to oxidative stress in the lungs of healthy mammals. However, it remains possible that this pathway protects if complications occur during or after birth.