Phosphodiesterase Inhibitor Attenuation of Fibroblast Chemotaxis Depends on Endogenous Prostaglandin Production

oxide (NO) is an intracellular messenger moleN itric cule involved in modulating cell proliferation and

apoptosis in a variety of cells. The present study was designed to determine if NO modulated the proliferation of human lung fibroblasts. Human fetal lung fibroblasts (HFL-1) were maintained in cell culture in Dulbecco’s Modified Eagle’s Medium in the presence of 4% fetal calf serum. Cell proliferation was determined by enumerating cells with a Coulter counter. Inhibition of endogenous NO production by N-nitro-L-arginine methylester had no effect on HFL-1 cell proliferation. Three different NOgenerating compounds were evaluated. Sodium nitroprusside (SNP) and diethylenetetra-amine NONOate were both found to inhibit cell proliferation in a concentration dependent manner. In contrast, 5-amino-3-3(4-morpholiny)-1,2.3-oxadiazolium chloride (SIN-1) had no effect on HFL-1 cell proliferation (Table 1). NO can induce signals by several mechanisms including the activation of guanylyl cyclase to produce soluble cyclic guanosine monophosphate (cGMP). In order to determine if this mechanism was responsible for the effect of SNP and NONOate, two experiments were performed. First, 1H-[1,2,4)oxadiazolo[4,3-a]quinoxalin-1 (10 ␮mol/L), a specific inhibitor of soluble guanylyl cyclase, was not able to alter the antiproliferative effect of SNP or NONOate. Second, 8-(4-chlorophenulthio)guanosine 3⬘:5⬘-cyclic monophosphate (100 ␮mol/L), an analog of cGMP, did not affect HFL-1 proliferation. These data suggest that a cGMP-independent mechanism may be responsible for the growth inhibition observed. SIN-1 differs from the other NO donors in that it can generate both NO and peroxynitrite. Whether this accounts for its lack of effect on HFL-1 proliferation is unknown. This study, however, suggests that the NO donors SNP and NONOate can modulate fibroblast proliferation.

Table 1—Cell Number 7 Days After Plating, 2 ⴛ 104 Cells per Well* Control

SNP

NONOate

SIN-1

40.3 ⫾ 0.1

13.7 ⫾ 0.2†

10.3 ⫾ 0.5†

39.5 ⫾ 0.6‡

*Data are presented as mean ⫾ SD. †p ⬍ 0.01 compared to control by t test. ‡Not significant.

Phosphodiesterase Inhibitor Attenuation of Fibroblast Chemotaxis Depends on Endogenous Prostaglandin Production* Tadashi Kohyama, MD; Xiangder Liu, MD; Yunkui Zhu, MD; Fu-Qiang Wen, MD, PhD; Hangjun Wang, MD; and Stephen I. Rennard, MD, FCCP Abbreviation: PDE4 ⫽ phosphodiesterase 4

(CHEST 2001; 120:14S–15S) are the major source of extracellular conF ibroblasts nective tissue matrix, and the recruitment and activa-

tion of these cells are thought to play an important role in wound healing and in the development of fibrosis. Agents that could block fibroblast accumulation could play a therapeutic role in modulating fibrosis. Previous studies have demonstrated that the phosphodiesterase 4 (PDE4) inhibitor SB207499 (Ariflo; Smith Kline Beecham; King of Prussia, PA) can attenuate fibroblast chemotaxis toward the chemoattractant fibronectin. Since the activity of a PDE4 inhibitor should be dependent on endogenous cyclic adenosine monophosphate levels, the current study was designed to determine if endogenous prostaglandin production rendered cells susceptible to the effects of Ariflo. To accomplish this, human fetal lung fibroblasts were cultured and, after achieving confluence, were incubated for 60 min with and without indomethacin (2 ⫻ 10⫺6 mol/L) to inhibit cyclooxygenase. Fibroblasts were then trypsinized and placed in the upper portion of a Boyden blindwell chemotaxis chamber. Fibronectin, 20 ␮g/mL, was placed in the lower portion of the chamber as the chemoattractant. Indomethacin (2 ⫻ 10⫺6 mol/L) was added to the upper side of the chemotaxis chamber. Indomethacin alone resulted in a slight but variable stimulation of chemotaxis (159 ⫾ 33% compared to control p ⬍ 0.05). Ariflo added to control fibroblasts inhibited chemotaxis in a concentration-dependent manner. In three separate experiments, the concentration of Ariflo required to inhibit chemotaxis by 50% was 4.9 ⫾ 2.5 ␮g/mL. In the presence of indomethacin, Ariflo inhibited chemotaxis only at the highest concentration tested, 10 ␮g/mL, reducing the response to 58.7 ⫾ 21.0% of control (p ⬍ 0.005) compared to indomethacin. The current study, therefore, demonstrates that the PDE4 inhibitor Ariflo is dependent, at least in part, on endogenous cyclooxygenase activity and, presumably, prostaglandin production in order to exert its inhibitory effect on fibroblast chemotaxis. That Ariflo is active even in the presence of indomethacin suggests alternate mechanisms for stimulating cyclic adenosine monophosphate may also play a role. Since cyclooxygenase activity and prostaglan*From the University of Nebraska Medical Center, Omaha, NE. Correspondence to: Tadashi Kohyama, MD, Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 600 South 42nd St, Omaha, NE 68198-5300

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Thomas L. Petty 43rd Annual Aspen Lung Conference: Mechanisms of Pulmonary Fibrosis

din production can be modulated by a variety of mediators present in an inflammatory milieu, fibroblasts in such a setting may be particularly sensitive to the inhibitory effects of Ariflo. Such an effect may have therapeutic potential in fibrotic disorders.

Interleukin-13, a Mediator of Subepithelial Fibrosis, Enhances Growth Factor Production and Proliferation in Human Airway Epithelial Cells* Brian Booth, BS; James Bonner, PhD; Nancy Akley, MS; Mariangela Macchione, PhD; Kenneth Adler, PhD; and Linda D. Martin, PhD Abbreviations: IL ⫽ interleukin; TGF ⫽ transforming growth factor

(CHEST 2001; 120:15S) fibrosis is a prominent feature of the S ubepithelial remodeled asthmatic airway. The cytokine interleukin

(IL)-13, implicated as a mediator in the development of asthma, induces a significant degree of subepithelial fibrosis in the lungs of transgenic mice. Since IL-13 has been shown to exert effects on the airway epithelium, including the development of a mucous phenotype, we have begun to determine whether IL-13 provokes production of factors from the epithelium that could elicit the observed subepithelial fibrotic response. In the studies reported herein, injured airways with regions of regenerating/differentiating cells and regions of normal fully differentiated cells have been mimicked by examining the effects of IL-13 on normal human bronchial epithelial cells during mucociliary differentiation in air/liquid interface culture. Exposure of normal human bronchial epithelial cells to IL-13 resulted in increased production of soluble transforming growth factor (TGF)-␣, with the growth factor interacting in an autocrine manner with the epidermal growth factor receptor. Production of soluble TGF-␣ was very rapid, with a threefold increase observed in response to IL-13 (10 ng/mL) by 1 h of exposure. Continuous exposure to IL-13 throughout the course of mucociliary differentiation (a total of 10 days) resulted in a twofold increase in cell number by day 7 when cells are differentiated. Exposure to IL-13 (10 ng/mL; 24 h) provoked a threefold increase in proliferation once the cells were differentiated, an effect that could be duplicated in differ-

*From the North Carolina State University (Mr. Booth, Ms. Akley, and Drs. Macchione, Adler, and Martin), Raleigh, NC; and National Institute of Environmental Health Sciences (Dr. Bonner), Research Triangle Park, NC. Supported by National Institutes of Health grants HL36982 and HL09869, and a grant from the state of North Carolina. Correspondence to: Linda D. Martin, PhD, Research Assistant Professor of Cell Biology, North Carolina State University, College of Veterinary Medicine, 4700 Hillsborough St, Raleigh, NC 27606

entiated, but not undifferentiated cells, by the direct addition of TGF-␣ (5 ng/mL or 25 ng/mL; 24 h). Proliferation of differentiated cells in response to continuous IL-13 treatment was followed 2 days later by a decrease in proliferation compared to control mice. Soluble TGF-␣, however, continued to be produced from these nonproliferating cultures. Thus, an increase in soluble TGF-␣ in response to IL-13 may serve to promote proliferation of injured epithelial cells in an autocrine manner. Once this proliferative effect is no longer necessary, the soluble TGF-␣ may promote proliferation of other cells. These data suggest that any injury to the airway epithelium resulting in the production of IL-13 from infiltrating inflammatory cells may provoke the release of soluble TGF-␣ from the airway epithelium. The availability of this growth factor may contribute to the subepithelial fibrosis observed in chronic asthma.

Monocytes May Regulate Tissue Fibrosis* Role of Reactive Oxygen Species in Monocyte Survival and in the Activation of Latent Transforming Growth Factor-␤ Clay B. Marsh, MD; Todd W. Kelley, MS; Mandy M. Graham; Chunming Dong, PhD; and Pascal J. Goldschmidt-Clermont, MD Abbreviations: DPI ⫽ diphenyleneiodonium; IPF ⫽ idiopathic pulmonary fibrosis; M-CSF ⫽ macrophage colony-stimulating factor; PI 3-K ⫽ phosphatidylinositol 3-kinase; ROS ⫽ reactive oxygen species; TGF ⫽ transforming growth factor; UPA ⫽ urokinase plasminogen activator

(CHEST 2001; 120:15S–16S) samples from patients with idiopathic pulP athologic monary fibrosis (IPF) show accumulations of mono-

cytes and macrophages in the lung. Monocytes and macrophages recovered from the lungs of patients with IPF show enhanced survival of recently recruited, young monocyte/macrophages that spontaneously produce oxidants.1– 4 Moreover, antioxidant strategies have been used successfully to treat patients with IPF.5–7 We previously demonstrated that the macrophage colony-stimulating factor (M-CSF) promoted monocyte survival through the activation of phosphatidylinositol 3-kinase (PI 3-K) and the serine threonine kinase Akt.8 M-CSF also promoted the production of reactive oxygen species (ROS) in human monocytes. ROS produced in M-CSF–stimulated monocytes were inhibited by PI 3-K inhibitors, flavoprotein inhibitors (diphenyleneiodonium [DPI]), or the intracellular superoxide dismutase mimetic Mn(III)tetrakis(4*From the Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH. Correspondence to: Clay B. Marsh, MD, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, The Ohio State University, N325 Means Hall, 1654 Upham Dr, Columbus, OH 43210 CHEST / 120 / 1 / JULY, 2001 SUPPLEMENT

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