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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

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

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

Prostaglandin [E.sub.2] inhibits fibroblast chemotaxis. Am J Physiol Lung Cell Mol Physiol 281: L1257-L1263, 2001.--Fibroblasts are the major source of extracellular connective tissue matrix, and the recruitment, accumulation, and stimulation of these cells are thought to play important roles in both normal healing and the development of fibrosis. Prostaglandin [E.sub.2] (PG[E.sub.2]) can inhibit this process by blocking fibroblast proliferation and collagen production. The aim of this study was to investigate the inhibitory effect of PG[E.sub.2] on human plasma fibronectin (hFN)- and bovine bronchial epithelial cell-conditioned medium (BBECCM)-induced chemotaxis of human fetal lung fibroblasts (HFL1). Using the Boyden blind well chamber technique, PG[E.sub.2] ([10.sup.-7] M) inhibited chemotaxis to hFN 40.8 [+ or -] 5.3% (P < 0.05) and to BBEC-CM 49.7 [+ or -] 11.7% (P < 0.05). Checkerboard analysis demonstrated inhibition of both chemotaxis and chemokinesis. The effect of PG[E.sub.2] was concentration dependent, and the inhibitory effect diminished with time. Other agents that increased fibroblast cAMP levels, including isoproterenol ([10.sub.-5] M), dibutyryl cAMP ([10.sub.-5] M), and forskolin (3 x [10.sub.-5] M) had similar effects and inhibited chemotaxis 54.1, 95.3, and 87.0%, respectively. The inhibitory effect of PG[E.sub.2] on HFL1 cell chemotaxis was inhibited by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, which suggests a cAMP-dependent effect mediated by PKA. In summary, PG[E.sub.2] appears to inhibit fibroblast chemotaxis, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of the wound healing response after injury. eicosanoids; adenosine 3',5'-cyclic monophosphate; fibronectin; fibrosis; repair

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-κB 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-κB activation but failed to affect STAT6 activation, almost completely attenuated DEP-induced eotaxin production, whereas these agents failed to attenuate IL-13induced eotaxin production. These findings suggested that DEP stimulated eotaxin gene expression via NF-κB-dependent, but STAT6-independent, pathways. [ABSTRACT FROM AUTHOR]

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)-β1, fibronectin, PGE[sub 2], and TGF-β1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 × 10[sup 5] cell/ml), only 5% CSE augmented contraction in higher-density cultures (3-5 × 10[sup 5] cells/ml). CSE also inhibited fibronectin and TGF-β1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-β1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-β1 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-β1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke. [ABSTRACT FROM AUTHOR]

Assesses the influence of glutathione in preventing smoke inhibition of fibroblast repair. Effects of the antioxidant defense system on pulmonary functions; Imbalance between oxidants and antioxidants in the chronic obstructive pulmonary disease; Alteration of the cellular response of fibroblasts to smoke.

Examines the regulation of type I collagen gel contraction by T helper 2 cytokines. Reduction of fibroblast prostaglandin release by interleukin (IL)-4 and IL-13; Relation between PGE[sub 2] release and cyclooxygenases expression reduction; Inhibition of PGE[sub 2] release by indomethacin.

Evaluates the effect of prostaglandin E[sub 2] on fibroblast chemotaxis. use of purified chemoattractant human plasma fibronectin for chemoattractant; Mechanism of PGE[sub 2]; Effect of PGE[sub 2] on human fetal lung fibroblasts cell chemotaxis.

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-β1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-β1 and reduced several TGF-β1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-β1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-β1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts., (Copyright © 2014 the American Physiological Society.)

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-kappaB 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-kappaB 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-kappaB-dependent, but STAT6-independent, pathways.