Elevation of nitrotyrosine and nitrate concentrations in cystic fibrosis sputum.

Nitric oxide (NO) is increased in the exhaled air of some patients with inflammatory lung disorders, but not in others. NO may combine with superoxide to form peroxynitrite, which lowers NO gas concentrations, increases formation of nitrate, and increases nitration of tyrosine residues on proteins. We hypothesized that superoxide released from neutrophils in the lower respiratory tract of cystic fibrosis (CF) results in increased nitrate and nitrotyrosine levels in sputum. In order to test this hypothesis, exhaled NO was collected from 5 stable adult CF subjects and from 5 nonsmoking normal controls. Consistent with previous reports, exhaled NO concentrations were not increased in CF exhaled air (22.6 +/- 1.5 ppb vs. 28.6 +/- 1.5 ppb in normals, P > 0.05). Sputum was collected from 9 adult CF subjects and the same 5 normal controls and evaluated for nitrite, nitrate, and nitrotyrosine. Nitrate and nitrotyrosine levels, but not nitrite, were significantly elevated in CF. Recently, myeloperoxidase has also been implicated as a mechanism of nitrotyrosine formation. Therefore, myeloperoxidase was measured and found to be elevated in the CF sputum (64.2 +/- 35.9 vs. 0.73 +/- 0.16 U/mL, P < 0.001), and was found to correlate with concentrations of nitrotyrosine (r = 0.87, P < 0.05). However, in vitro studies with myeloperoxidase and murine lung epithelial cells did not demonstrate a reduction of NO gas with nitrotyrosine or an increase in nitrate formation. These data demonstrate that nitrate and nitrotyrosine are elevated in the sputa of CF subjects and suggest increased production of NO in the lower respiratory tract of CF patients, despite the relatively low exhaled NO levels. Pediatr Pulmonol. 2000; 30:79-85. Published 2000 Wiley-Liss, Inc.

The pathogenesis of chronic obstructive pulmonary disease.

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD). An accelerated rate of lung function decline that causes clinically significant COPD, however, is present in only a minority of smokers. In addition to the cumulative amount of cigarettes smoked, other environmental and genetic properties contribute to this variable physiological response. This article reviews the role of airway hyperresponsiveness, mucus hypersecretion, infection, and proteases in the development of COPD.

Outpatient management of chronic obstructive pulmonary disease.

The outpatient management of chronic obstructive pulmonary disease (COPD) is designed to limit the decline in respiratory function over time, to relieve the symptoms and improve the patient's functional status, and to manage complications when they arise. Factors that predispose to airway inflammation, including cigarette smoking and respiratory infections, are prevented by behavioral modification programs, measures such as exercise and nutrition to improve general health, and regular vaccination. Symptoms are relieved by bronchodilator and anti-inflammatory therapy, based upon the specific needs of the patient. Hypoxemia and acute infections are treated with oxygen administration and the use of antibiotics when necessary. The management of acute exacerbations of COPD is addressed elsewhere in this symposium (ie, choice of antibiotics is not discussed here). Also, certain aspects of management, such as surgical procedures, chest physical therapy, and other aspects of pulmonary rehabilitation, are also subjects of subsequent articles in this series. Although none of these modalities, except for smoking cessation and oxygen administration, have been shown to alter the course of COPD, the careful choice of the therapeutic measures discussed here can lead to significant relief of symptoms in the patient with chronic airway obstruction.

Surgical treatment of chronic obstructive pulmonary disease.

Over the past several decades, a number of surgical techniques have been developed for the treatment of chronic obstructive pulmonary disease. Many of these procedures have been abandoned because of lack of efficacy and/or high morbidity and mortality. At the present time, lung transplantation, reduction pneumoplasty for giant bullous emphysema, and lung volume reduction surgery are being performed in a number of centers. Data concerning the effectiveness of these procedures is accumulating and will ultimately need careful analysis to determine long-term outcomes in this group of patients.

Superoxide released from neutrophils causes a reduction in nitric oxide gas.

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide (O-2) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing O-2 could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce O-2 were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that O-2 released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Differential regulation of extracellular signal-regulated kinase and nuclear factor-kappa B signal transduction pathways by hydrogen peroxide and tumor necrosis factor.

Reactive oxygen metabolites are increasingly recognized for their ability to stimulate signal transduction pathways. This is important because these oxidants are frequently generated at sites of inflammation. However, little is known about the manner in which reactive oxygen species may selectively stimulate distinct signaling pathways. We have examined this question by stimulating mesothelial cells with hydrogen peroxide (H2O2) as a model oxidant stimulus. The response to H2O2 was examined by measuring the activation of the extracellular signal-regulated kinase (ERK1/2) and the nuclear factor-kappa B (NF-kappa B) signal transduction pathways. We found that H2O2 stimulated activity of the ERK1/2 pathway in a dose- and time-dependent manner. The ability of H2O2 to activate ERK1/2 was similar to that found with tumor necrosis factor (TNF) stimulation. The oxidant effect was inhibited by various reactive oxygen scavengers. An inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase, the upstream kinase that activates ERK1/2, inhibited the oxidant effect. The superoxide anion (O2-) also stimulated ERK1/2 activity. In contrast, H2O2 did not stimulate proteolysis of I kappa B-alpha and induced only a small degree of NF-kappa B nuclear translocation. Stimulation of the cells with O2- also induced a minimal degree of NF-kappa B activation. TNF was a potent stimulus for I kappa B-alpha proteolysis and NF-kappa B activation, demonstrating that the cells did have a functional NF-kappa B pathway. These results suggest that oxidants may selectively stimulate certain pathways, thereby preserving some specificity of the signaling process. Furthermore, different cell types and distinct signaling pathways within cells may demonstrate unique profiles in the manner in which they respond to oxidant stimulation.

Tuberculosis in Louisiana: an update.

The incidence of tuberculosis in the United States declined steadily until 1985 when increases were seen, in part due to the AIDS epidemic. Although the decline resumed in 1992, tuberculosis remains a public health problem in Louisiana and nationally. In Louisiana in 1997, HIV infection was present in 14% of persons with tuberculosis whose HIV status was known. In that year there were 16 cases of tuberculosis that were resistant to at least one first-line anti-tuberculous drug. Infection with Mycobacterium tuberculosis is diagnosed with the tuberculin skin test; the size of induration considered positive varies with the risk status of the person tested. TB infection in immunocompetent persons under age 35 is treated with 6 months of isoniazid. TB disease is generally treated with 4 drugs until antimicrobial sensitivities are known. Directly observed therapy is an inexpensive way to ensure compliance and is routinely used for patients in Louisiana.

Effects of reactive metabolites of oxygen and nitrogen on gelatinase A activity.

The regulation of matrix metalloproteinase activity is crucial for maintaining the proper balance of tissue remodeling vs. injury. Metalloproteinase proenzymes are activated when the active site zinc is exposed via a cysteine switch mechanism. Peroxynitrite, the product generated from the interaction between nitric oxide and superoxide, has been shown to release zinc from zinc-thiolate groups, suggesting that it might alter metalloproteinase activity. This study examined the effects of nitric oxide and superoxide generators on gelatinase A activity. Results showed that nitric oxide alone had no effect on gelatinase A activity relative to control, whereas superoxide-derived metabolites increased activity. The simultaneous generation of both nitric oxide and superoxide caused an inhibition of gelatinase A activity. This inhibition was reversed by the addition of hemoglobin, superoxide dismutase, or sodium urate, suggesting that peroxynitrite and/or peroxynitrous acid caused the inhibition. Authentic peroxynitrite also inhibited gelatinase A activity. We postulate that the relative fluxes of nitric oxide and superoxide at sites of inflammation may modulate metalloproteinase activity and thus affect matrix protein metabolism.

Inhibition of rat pleural mesothelial cell nitric oxide synthesis by transforming growth factor-beta 1.

Pleuritis is a common initial clinical manifestation of tuberculosis. It is associated with an accumulation of a variety of cytokines in the pleura and pleural fluid. We have recently shown that these proinflammatory cytokines induce the pleural mesothelial cell to produce large amounts of nitric oxide, a nitrogen intermediate that has been shown to have a tuberculocidal effect. TGF-beta has also been found in situ in tuberculous effusions and pleural tissues and is thought to suppress the immune response and promote tissue repair. This study examined the effects of TGF-beta on cytokine-induced NO synthesis by rat pleural mesothelial cells in vitro. Results demonstrated that TGF-beta significantly inhibited NO synthesis and that this inhibition was associated with a proportionate decrease in iNOS mRNA and iNOS protein. Suppression of pleural mesothelial cell NO synthesis by TGF-beta may be important in the pathogenesis of tuberculous pleuritis.

Inhibition of IkappaB-alpha and IkappaB-beta proteolysis by calpain inhibitor I blocks nitric oxide synthesis.

Lipopolysaccharide (LPS) stimulates the induction of the inducible isoform of nitric oxide synthase (iNOS) in part by inducing the nuclear translocation of the transcription factor nuclear factor-kappa B (NF-kappaB). LPS induces ubiquination and phosphorylation of the IkappaB inhibitory subunit of NF-kappaB. Subsequently, the ubiquitin-proteasome multicatalytic enzyme complex catalyzes the proteolytic degradation of IkappaB with resultant nuclear translocation of NF-kappaB. Our results demonstrate that the proteasome inhibitor calpain inhibitor I dose-dependently inhibited LPS-induced nitric oxide synthesis in RAW macrophages. The inhibitor was found to block iNOS transcription and protein translation as noted by Northern analysis and Western blotting, respectively. LPS stimulated rapid proteolytic degradation of IkappaB-alpha which was inhibited by approximately 50% in the presence of calpain inhibitor I. In contrast, LPS induced the delayed proteolytic degradation of IkappaB-beta which was almost totally inhibited by calpain inhibitor I. Calpain inhibitor I also decreased the LPS-induced nuclear translocation of NF-kappaB. These results demonstrate that the ubiquitin-proteasome complex has an important role in induction of iNOS in response to stimuli which act via the NF-kappaB/IkappaB signal transduction pathway. Furthermore, the results suggest that the ubiquitin-proteasome complex is important in the degradation of IkappaB-beta as well as IkappaB-alpha. Finally, we have demonstrated that there is a marked difference in the extent of proteolysis of IkappaB-alpha and IkappaB-beta when the ubiquitin-proteasome complex is inhibited with calpain inhibitor I.

Early albumin leakage in pulmonary endothelial monolayers exposed to varying levels of hyperoxia.

We assessed the effect of varying levels of hyperoxia on 14C-albumin flux across bovine pulmonary artery endothelial cell (BPAEC) monolayers. Endothelialized nitrocellulose filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts of 14C-labeled and unlabeled albumin were added to the "hot" and "cold" chambers, respectively, and the monolayers were exposed to 3 hours of varying levels of oxygen (16%, 30%, 40%, 60%, and 95%). When compared to 16% O2, exposure to hyperoxic gas mixtures of 40% or greater progressively increased albumin permeability across endothelial monolayers within 3 hours to a value 2.5 times higher at 95% O2 compared to 16% O2 (p < 0.001). Hyperoxia-induced permeability increases were prevented by catalase, superoxide dismutase, desferrioxamine, and allopurinol. Our data indicate that hyperoxia induces endothelial permeability changes more rapidly than previously reported even at O2 concentrations as low as 40%.

Augmentation of cytokine-induced nitric oxide synthesis by hydrogen peroxide.

The inducible isoform of nitric oxide synthase (iNOS) is induced upon stimulation of cells with cytokines and lipopolysaccharide (LPS). Stimulation of rat pleural mesothelial cells with combinations of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite (NO2-) and nitrate (NO3-). Addition of 25-50 microM H2O2 to the cytokines significantly augmented the synthesis of NO2- and NO3-. Stimulation with IL-1 beta and TNF-alpha plus H2O2 or IL-1 beta and LPS plus H2O2 increased the synthesis of NO2- and NO3- by 3.8- and 3.5-fold, respectively. These effects were inhibited by NG-nitro-L-arginine methyl ester and cycloheximide as well as by catalase. Immunoblotting demonstrated that H2O2 augmented cytokine-induced synthesis of iNOS protein. These effects were inhibited by certain antioxidants and metal chelators, suggesting that the hydroxyl radical may mediate the oxidant-induced effect. Northern blotting demonstrated that H2O2 greatly augmented steady-state levels of iNOS mRNA, suggesting that H2O2 acted in part at the transcriptional level.

Inhibition of pleural mesothelial cell collagen synthesis by nitric oxide.

The pleural mesothelial cell has a critical role in repairing the mesothelium after injury via its ability to produce connective tissue macromolecules. We have recently shown that proinflammatory cytokines and lipopolysaccharide induce pleural mesothelial cells to produce nitric oxide. The present study examined the effect of nitric oxide on pleural mesothelial cell protein synthesis. Rat pleural mesothelial cells were exposed to various combinations of tumor necrosis factor, interleukin-1, interferon-gamma, and lipopolysaccharide or to the nitric oxide donors: 6-morpholino-sydnonimine, S-nitroso-N-acetyl-D,L-penicillamine, sodium nitroprusside, and spermine-NO adduct for 24-48 h. Nitrate and nitrite (an index of nitric oxide production) and not collagen and noncollagen protein production (uptake of 3H-proline into collagenase-sensitive protein) were then determined. Net collagen production was significantly inhibited by the cytokine-lipopolysaccharide combinations tested. Collagen inhibition paralleled the time course of increased nitric oxide production. The inhibition of collagen production was also significantly reversed by the addition of NG-nitro-L-arginine methyl ester, and was reproduced by the addition of a 5:1 molar excess of L-arginine to NG-nitro-L-arginine methyl ester. Additionally, nitric oxide-generating compounds significantly inhibited collagen production in a dose-dependent manner compared to unexposed control cells. Net collagen production was inhibited to a greater degree than noncollagen protein synthesis. These results suggest that nitric oxide may be a significant mediator of PMC collagen production during conditions of significant pleural inflammation.

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes.

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG-monomethyl-L-arginine (L-NMMA)-inhibitable conversion of 14C-labeled L-arginine to L-[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L-NMMA-inhibitable nitrite (NO2-) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 x 10(6) cells/well) were unable to generate significant amounts of NO2- when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4- and 6-h elicited rat PMNs increased 21- and 42-fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4- and 6-h elicited rat PMNs, respectively. Approximately 20-30% of the total iNOS activity was Ca(2+)-dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 microM/10(6) cells for the 4- and 6-h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation-induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time-dependent fashion and that 20-30% of the total inducible NOS is Ca(2+)-dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Nitric oxide-dependent N-nitrosating activity of rat pleural mesothelial cells.

Recent studies have demonstrated that nitric oxide (NO)-derived N-nitrosating agents may promote mutagenesis and carcinogenesis from the nitrosative deamination of DNA bases via the formation of nitrosamine intermediates. The objective of this study was to determine if pleural mesothelial cells (PMC) stimulated with proinflammatory cytokines could promote the N-nitrosation of a primary aromatic amine via the L-arginine-dependent formation of NO-derived N-nitrosation agents. N-nitrosating activity was determined by measuring the N-nitrosation of a model amine, 2,3-diaminonapthalene, to yield its fluorescent triazole (1-naptho-2,3-triazole) derivative. Results show that specific combinations of TNF, IL-1, interferon gamma, and LPS significantly increased N-nitrosating activity. There was a significant positive correlation between nitrite plus nitrate and triazole production. Triazole formation was inhibited by NG-nitro-L-arginine methyl ester, suggesting that triazole was derived from L-arginine-dependent formation of NO. These data indicate that PMC have the capacity to promote the N-nitrosation of primary aromatic amines via the formation of NO.

Nitric oxide synthesis by rat pleural mesothelial cells: induction by growth factors and lipopolysaccharide.

The purpose of this study was to determine if certain growth factors and bacterial products induce pleural mesothelial cells (PMC) to produce nitric oxide (NO). Confluent monolayers of rat PMC were exposed to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or lipopolysaccharide (LPS) individually and in various combinations for 24-72 h. Concentrations of nitrite and nitrate were quantified and used as an indirect measure of NO production. LPS stimulation resulted in a significant increase in nitrite/nitrate concentration, but neither EGF nor PDGF alone or combined had any significant effect relative to control. However, LPS combined with either EGF or PDGF caused a significant increase in nitrite/nitrate concentration relative to LPS alone and growth factor alone. The highest level level of nitrite/nitrate concentration was observed with the triple combination of LPS, EGF, and PDGF. Nitrite/nitrate accumulation was significantly increased at 24 h by all combinations, and continued to increase, with the highest concentration observed after 72 h of exposure. Nitrite/nitrate production was significantly inhibited by NG-nitro-L-arginine methyl ester and this inhibition was reversed by the addition of L-arginine, suggesting that nitrite and nitrate were derived from the L-arginine-dependent formation of NO. These data indicate that PMC can be induced to produce relatively large amounts of NO in response to growth factors combined with LPS.

Pleuritis and pleural effusions.

A variety of diseases either directly or indirectly affect the pleura, resulting in the accumulation of pleural fluid. A pleural effusion develops whenever the influx of fluid into the pleural space is greater than the efflux. It is now clear that the parietal pleura has the primary role in the reabsorption of pleural fluid normally and during pathologic conditions. Recently, models of experimental pleuritis have demonstrated the importance of inflammatory cytokines in the pathogenesis of both asbestos- and endotoxin-induced pleural effusions.

Characterization of proteoglycans produced by rat pleural mesothelial cells in vitro.

The mesothelial cell envelopes the surface of the parietal and visceral pleura. These cells are known to synthesize most of the protein constituents of the pleural basement membrane and interstitium. This study examined the ability of a rat pleural mesothelial cell line to synthesize proteoglycans in vitro. Cells were labeled with inorganic 35SO4 to label the glycosaminoglycan moiety of proteoglycans. The medium and combined cell membrane/extracellular matrix fractions contained 73 and 25% of the proteoglycan radioactivity, respectively. The medium contained a single chondroitin/dermatan sulfate proteoglycan of approximately 190 kDa, consistent with biglycan. As determined by Northern analysis of steady-state levels of messenger RNA, the cells contained message for biglycan. Stimulation of the cells with epidermal growth factor resulted in the appearance of a second chondroitin/dermatan sulfate proteoglycan of approximately 97 kDa, characteristic of decorin. The cell membrane/matrix contained a biglycan-like chondroitin/dermatan proteoglycan and several heparan sulfate proteoglycans. Pleural mesothelial cells in vitro are capable of synthesizing a variety of interstitial and basement membrane proteoglycans.

Modulation of endothelial cell permeability by lung carcinoma cells: a potential mechanism of malignant pleural effusion formation.

This study examined the hypothesis that tumor cells metastatic to the pleura secrete a soluble factor(s) that directly increases endothelial cell permeability. Nitrocellulose filters were endothelialized with bovine pulmonary artery endothelial cells and exposed to conditioned media from either human lung adenocarcinoma (Calu-3), human lung squamous cell carcinoma (SK-MES-1), or control media for 16 h. The diffusional permeability (Pd x 10(-5) cm/sec) to [14C]albumin was then determined for each monolayer with Ussing-type chambers. Both adenocarcinoma conditioned media (ACCM) and squamous cell carcinoma conditioned media (SCCM) caused a two- to threefold increase in endothelial monolayer permeability. The addition of indomethacin (10 micrograms/ml) blocked the observed permeability increase in ACCM but not in SCCM, suggesting that the increase in permeability by ACCM was secondary to the production of prostaglandins. To confirm this, a variety of prostanoids previously shown to be produced by the Calu-3 cell line were added directly to the endothelial monolayer. Prostaglandin F2 alpha (PGF2 alpha) in both low (10 ng/ml) and high (100 ng/ml) concentrations for 16 h resulted in a three- to fourfold increase in permeability. Prostaglandin E2 (PGE2) resulted in a small increase in [14C]albumin permeability but only at high concentrations (100 ng/ml). PGF2 alpha production by the two tumor cell lines was measured using radioimmunoassay. Baseline adenocarcinoma production of PGF2 alpha was 117.5 pmol/10(6) cells and fell to 24.2 pmol/10(6) cells hours following incubation with indomethacin. The decrease in PGF2 alpha occurred in parallel with the changes in permeability. Concomitant, reversible changes in cell shape and F-actin distribution were detected in endothelial cells exposed to ACCM. No significant production of PGF2 alpha by the squamous cell carcinoma cell line was detected. These results suggest that both adenocarcinoma and squamous cell carcinoma secrete a soluble factor(s) that directly increases endothelial cell permeability to albumin and that in the case of adenocarcinoma this soluble factor may be a prostanoid such as PGF2 alpha.

Growth factor modulation of rat pleural mesothelial cell mitogenesis and collagen synthesis. Effects of epidermal growth factor and platelet-derived factor.

This study examined the effects of an epithelial and a mesenchymal growth factor on pleural mesothelial cell proliferation and collagen synthesis, functions that may be important in the response of the pleura to injury. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) added singly caused significant increases relative to control in both the uptake of [3H]thymidine into the cellular DNA of subconfluent monolayers and of [3H]proline into collagenase-sensitive protein. Combinations of EGF and PDGF resulted in more than additive increases in proliferation and additive increases in collagen production relative to each factor alone. Media from control and growth factor-stimulated PMC demonstrated no gelatinase or collagenase activity, suggesting that the increase in net collagen production was secondary to enhanced synthesis. These data demonstrate that both epithelial and mesenchymal growth factors can stimulate PMC proliferation and collagen synthesis and that these growth factors have even greater effects when combined, particularly in regard to cellular proliferation. Increases in PMC proliferation and collagen synthesis in response to these growth factors may be important in healing the pleura after injury by a variety of disease processes.