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Abstract Background Extended culture of monocytes and fibroblasts in three-dimensional collagen gels leads to degradation of the gels (see linked study in this issue, "Fibroblasts and monocytes contract and degrade three-dimensional collagen gels in extended co-culture"). The current study, therefore, was designed to evaluate production of matrix-degrading metalloproteinases by these cells in co-culture and to determine if neutrophil elastase could collaborate in the activation of these enzymes. Since co-cultures produce prostaglandin E2 (PGE2), the role of PGE2 in this process was also evaluated. Methods Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Matrix metalloproteinases (MMPs) were assessed by gelatin zymography (MMPs 2 and 9) and immunoblotting (MMPs 1 and 3). The role of PGE2 was explored by direct quantification, and by the addition of exogenous indomethacin and/or PGE2. Results Gelatin zymography and immunoblots revealed that MMPs 1, 2, 3 and 9 were induced by co-cultures of fibroblasts and monocytes. Neutrophil elastase added to the medium resulted in marked conversion of latent MMPs to lower molecular weight forms consistent with active MMPs, and was associated with augmentation of both contraction and degradation (P < 0.01). PGE2 appeared to decrease both MMP production and activation. Conclusion The current study demonstrates that interactions between monocytes and fibroblasts can mediate tissue remodeling.

Abstract Background Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. Methods Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Results Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P < 0.01). However, with extended co-culture, contraction of the collagen gels was greatly augmented (P < 0.01). In addition, with extended co-culture, degradation of collagen in the gels occurred. The addition of neutrophil elastase to the medium augmented both contraction and degradation (P < 0.01). Prostaglandin E2 production was significantly increased by co-culture and its presence attenuated collagen degradation. Conclusion The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture.

Fine particles derived from diesel engines, diesel exhaust particles (DEP), have been shown to augment gene expression of several inflammatory cytokines in human airway epithelial cells in vitro. However, it remains unclear whether or not DEP have any effect on the expression and production of eotaxin, an important chemokine involved in eosinophil recruitment into the airways. We studied the effects of DEP by using a conventional suspended DEP and by a recently established in vitro cell exposure system to diesel exhaust (Abe S, Takizawa H, Sugawara I, and Kudoh S, Am J Respir Cell Mol Biol 22: 296-303, 2000). DEP showed a dose-dependent stimulatory effect on eotaxin production by normal human peripheral airway epithelial cells as well as by bronchial epithelial cell line BET-1A as assessed by specific ELISA. mRNA levels increased by DEP were shown by RT-PCR. DEP showed an additive effect on IL-13-stimulated eotaxin expression. DEP induced NF-[kappa]B activation by EMSA as previously reported but did not induce signal transducer and activator of transcription (STAT) 6 activation according to Western blot analysis. Finally, antioxidant agents (N-acetyl cysteine and pyrrolidine dithiocarbamate), which inhibited NF-[kappa]B activation but failed to affect STAT6 activation, almost completely attenuated DEP-induced eotaxin production, whereas these agents failed to attenuate IL-13-induced eotaxin production. These findings suggested that DEP stimulated eotaxin gene expression via NF-[kappa]B-dependent, but STAT6-independent, pathways. airway epithelium; signal transduction; interleukin-13

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

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

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