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Byline: Fu-Qiang Wen (1), Tadashi Kohyama (1), C. Magnus Skold (2), Yun Kiu Zhu (3), Xiangde Liu (1), Debra J. Romberger (1), Julie Stoner (4), Stephen I. Rennard (1,5) Keywords: glucocorticoids; transforming growth factor-[beta] (TGF-[beta]); Smads; AP-1 complex; fibroblast Abstract: TGF-[beta] is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF-[beta] isoforms affect TGF-[beta] production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-[beta]1, -[beta]2, or -[beta]3. Post-culture media were collected for ELISA assays of TGF-[beta]1, -[beta]2, and -[beta]3 . TGF-[beta] mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-[beta]2 and -[beta]3 stimulated TGF-[beta]1 production significantly (p &lt 0.01 relative to control). TGF-[beta]1 stimulated TGF-[beta]2 production (p &lt 0.01 relative to control). TGF-[beta]3 was undetectable. Glucocorticoids significantly inhibited TGF-[beta]1 and TGF-[beta]2 production and reduced expression of the up-regulated TGF-[beta]1 and TGF-[beta]2 mRNA induced by exogenous TGF-[beta]1, -[beta]2, or -[beta]3 (p &lt 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-[beta]-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-[beta] isoforms may stimulate production of various TGF-[beta] isoforms in the lung. Glucocorticoids then may block TGF-[beta] production by modulating mRNA levels and c-Jun. Author Affiliation: (1) Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (2) Department of Medicine, Karolinska Institute, Stockholm, Sweden (3) Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China (4) Preventive and Societal Medicine Section, University of Nebraska Medical Center, Omaha, Nebraska (5) The Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, Nebraska, 68198-5125, USA Article History: Registration Date: 19/10/2004

Cigarette smoke exposure has been associated with a variety of diseases, including emphysema. The current study evaluated the interaction of cell density and cigarette smoke extract (CSE) on fibroblast contraction of collagen gels. Protein levels of transforming growth factor (TGF)-[beta]1, fibronectin, PG[E.sub.2], and TGF-[beta]1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 x [10.sup.5] cell/ml), only 5% CSE augmented contraction in higher-density cultures (3-5 x [10.sup.5] cells/ml). CSE also inhibited fibronectin and TGF-[beta]1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-[beta]1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-[beta]1 mRNA expression was inhibited in high-density cultures by 10% CSE, expression was increased in the presence of 5% CSE. These results suggest that CSE-induced inhibition of low-density fibroblast contraction is due to inhibition of fibronectin production, whereas CSE's stimulatory effect on high-density cells is the result of increased release of TGF-[beta]1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke. cigarette smoke extract; collagen gels; transforming growth factor-[beta]; lung

ABSTRACT Chronic obstructive pulmonary disease (COPD) is disabling and often fatal, and acute exacerbations are a primary reason for ICU admission and use of emergency department services. Prompt restoration of [...]

Byline: Fu-Qiang Wen (1), Tadashi Kohyama (1), C. Magnus Skold (2), Yun Kiu Zhu (3), Xiangde Liu (1), Debra J. Romberger (1), Julie Stoner (4), Stephen I. Rennard (1) Keywords: glucocorticoids; transforming growth factor-[beta] (TGF-[beta]); Smads; AP-1 complex; fibroblast Abstract: TGF-[beta] is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB-[beta] isoforms affect TGF-[beta] production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-[beta]1, -[beta]2, and -[beta]3. TGF-[beta] mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-[beta]2 and -[beta]3 stimulated TGF-[beta]1 production significantly (p &lt 0.01 relative to control). TGF-[beta]1 stimulated TGF-[beta]2 production (p &lt 0.01 relative to control). TGF-[beta]3 was undetectable. Glucocorticoids significantly inhibited TGF-[beta]1 and -[beta]2 production and reduced expression of the upregulated TGF-[beta]1 and -[beta]2 mRNA induced by exogenous TGF-[beta]1, -[beta]2 or -[beta]3 (p &lt 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-[beta]-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-[beta] isoforms may stimulate production of various TGF-[beta] isoforms in the lung. Glucocorticoids then may block TGF-[beta] production by modulating mRNA levels and c-Jun. Author Affiliation: (1) Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (2) Department of Medicine, Karolinska Institute, Stockholm, Sweden (3) Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China (4) Preventive and Societal Medicine Section, University of Nebraska Medical Center, Omaha, Nebraska Article History: Registration Date: 12/10/2004

The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PG[I.sub.2]) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PG[I.sup.2] on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PG[I.sub.2] analog carbaprostacyclin ([10.sup.-6] M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 [micro]g/ml) 58.0 [+ or -] 13.2% (P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 [+ or -] 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PG[I.sub.2] analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both [10.sup.-6] M), significantly inhibited fibroblast migration to fibronectin. In summary, PG[I.sub.2] appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling. prostaglandin [I.sub.2]; protein kinase; tissue repair; inflammation

Cigarette smoke, the major risk factor for the development of emphysema, contains over 4,700 chemical compounds, including free radicals and other oxidants ([10.sup.14]/puff). An imbalance between oxidants and antioxidants has been proposed in the pathogenesis of chronic obstructive pulmonary disease. Inhibition of repair processes has been suggested to be one pathway contributing to the development of emphysema. We hypothesized that cigarette smoke inhibition of repair might result from a shift of the oxidant/antioxidant balance in favor of oxidants. To evaluate this hypothesis, N-acetyl-L-cysteine (NAC), which serves as a substrate for glutathione (GSH) production, and buthionine sulfoximine (BSO), which inhibits GSH production, were incubated in the presence and absence of cigarette smoke extract (CSE) with fibroblasts in three-dimensional collagen gels. Neither agent alone altered gel contraction. CSE inhibition of gel contraction, however, was mitigated by NAC and potentiated by BSO. Parallel effects were observed on cigarette smoke inhibition of fibronectin production and mRNA expression as well as by changes in intracellular GSH content. Pretreatment of fibroblasts with NAC or BSO resulted in similar effects, suggesting that neither agent was acting directly on smoke but, rather, was altering cellular response to smoke. In conclusion, smoke inhibition of fibroblast repair, as reflected by collagen gel contraction and fibronectin production, may be modulated by intracellular GSH levels. chronic obstructive pulmonary disease; oxidants

Bronchial epithelial cell migration is required for the repair of damaged airway epithelium. We hypothesized that bronchial epithelial cell migration during wound repair is influenced by cAMP and the activity of its cyclic nucleotide-dependent protein kinase, protein kinase A (PKA). We found that, when confluent monolayers of bronchial epithelial cells are wounded, an increase in PKA activity occurs. Augmentation of PKA activity with a cell-permeable analog of cAMP, dibutyryl adenosine 3',5'-cyclic monophosphate, isoproterenol, or a phosphodiesterase inhibitor accelerated migration of normal bronchial epithelial cells in in vitro wound closure assays and Boyden chamber migration assays. A role for PKA activity was also confirmed with a PKA inhibitor, KT-5720, which reduced stimulated migration. Augmentation of PKA activity reduced the levels of active Rho and the formation of focal adhesions. These studies suggest that PKA activation modulates Rho activity, migration mechanisms, and thus bronchial epithelial repair mechanisms. bronchi; cell movement; cAMP; epithelial cells

Asthma is characterized by chronic inflammation of the airway wall with the presence of activated T helper 2 (Th2) lymphocytes. The current study assessed the ability of Th2 cytokines to modulate fibroblast-mediated contraction of collagen gels to determine if Th2 cytokines could contribute to tissue remodeling by altering mesenchymal cell contraction. Human fetal lung fibroblasts, human adult bronchial fibroblasts and human airway smooth muscle cells were cast into native type I collagen gels and allowed to contract in the presence or absence of IL (interleukin)-4, IL-5, IL-10, or IL-13. IL-4 and IL-13 but not IL-5 and IL-10 augmented collagen gel contraction in a concentration-dependent manner. Neither IL-4 nor IL-13 altered fibroblast production of transforming growth factor-[beta] or fibronectin. Both, however, decreased fibroblast prostaglandin (PG) [E.sub.2] release. Decreased PG[E.sub.2] release was associated with a decreased expression of cyclooxygenase 1 and 2 protein and mRNA. Indomethacin completely inhibited PG[E.sub.2] release and also augmented contraction. IL-4 and IL-13, however, added together with indomethacin further augmented contraction suggesting both a PGE-dependent and a PGE-independent effect. These findings suggest that IL-4 and IL-13 may modulate airway tissue remodeling and, therefore, could play a role in the altered airway connective tissue which characterizes asthma. asthma; interleukin; prostaglandin [E.sub.2]; cyclooxygenase; T helper 2

Byline: Fu-Qiang Wen (1,2), C. Magnus Skold (3), Xiang-Der Liu (1,2), Ronald F. Ertl (1,2), Yun Kui Zhu (1,2), Tadashi Kohyama (1,2), Hangjun Wang (1,2), Stephen I. Rennard (1,2) Keywords: glucocorticoids; transforming growth factor-[beta] (TGF-[beta]); prostaglandin; collagen gel contraction; fibroblast Abstract: TGF-[beta] plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF-[beta] and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF-[beta] in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF-[beta] for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E.sub.2 (PGE.sub.2) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P &lt 0.01) and dose-dependently inhibited PGE.sub.2 release by HFL-1 fibroblasts. TGF-[beta] significantly augmented gel contraction but also induced a 30% increase in PGE.sub.2 release and increased the expression of COX-1. Glucocorticoids inhibited TGF-[beta]1 induced-PGE.sub.2 release, and enhanced TGF-[beta] augmented gel contraction without significantly affecting TGF-[beta] augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF-[beta] augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF-[beta] in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE.sub.2 production. Such interactions between glucocorticoids and TGF-[beta] may account, in part, for the lack of response of fibrotic diseases to glucocorticoids. Author Affiliation: (1) Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985125, Nebraska (2) Nebraska Medical Center, Omaha, Nebraska, 68198-5125 (3) Department of Medicine, Karolinska Hospital, Stockholm, Sweden Article History: Registration Date: 17/10/2004