Growth Of Human Lung Fibroblasts Research Articles

The effect of PM10 on human lung fibroblasts.

Asthma diagnoses are increasing nationally with the highest rates in the New England states. Epidemiological studies have suggested a relationship between airborne particulate matter (PM) and severity of an asthma attack. However, because particulate matter, PM, is such a complex mixture, it is difficult to isolate the exacerbating factors. In this paper we investigate the effect of NIST (National Institute of Standards and Technology) and Maine PM and the soluble metals released from the PM on the growth of human lung fibroblasts. While the NIST PM itself had the most pronounced effect on cell survival rates, solutions of metals extracted from the PM also affected cell survival. Treatment of cells with 10, 50, 100 and 200 ug/cm(2) resulted in 84 +/- 13%, 69 +/- 15%, 58 +/- 14% and 58 +/- 16% survival, respectively. Appropriate concentrations of eight acid soluble metals from NIST PM were determined and tested on cells giving 91 +/- 11%, 87 +/- 10%, 72 +/- 18% and 66 +/- 20% survival, respectively. Soluble metals from Maine PM were extracted and mixtures of appropriate concentrations of these metals were used to treat cells, resulting in 88 +/- 5%, 81 +/- 5%, 79 +/- 3% and 57 +/- 9% survival rate. To determine which, if any, of the metals individually affected the cells, Mn, Cu, V and As were used to treat the cells. At the metal concentrations tested, only As and V affected cell survival.

Dermatophagoides extract-treated confluent type II epithelial cells (cA549) and human lung mesenchymal cell growth

Chronic severe persistent asthma is associated with damaged epithelial cells with discontinuous tight junctions that contribute to dysregulated fibroblast and endothelial cell (mesenchymal) growth. Dermatophagoides species-derived proteases have been shown to cause damage to epithelial cell tight junctions. To determine whether Dermatophagoides species can stimulate confluent A549 (cA549), a cell type with discontinuous tight junctions that approximate differentiated type II cells, to undergo altered growth and secrete putative soluble factors that affect the growth of human lung fibroblasts and microvascular endothelial cells. Dialyzed Dermatophagoides pteronyssinus or Dermatophagoides farinae extracts (0, 300, 600, and 1000 AU/mL) were cultured with and without cA549 in serum-free media for 24 hours. After changes in cA549 growth were recorded, conditioned media from extracts with cA549 (CM) and without cA549 (control media [CTLM]) were transferred to fibroblasts and endothelial cells for 24 hours. Fibroblast and endothelial cell growth responses to CM and CTLM were observed and measured. All conditions showed greater than 95% cell viability. Confluent A549 showed dose-dependent growth changes characterized by increased aggregation when incubated with 300, 600, and 1000 AU/mL of D pteronyssinus in serum-free media relative to control. The CM, but not the CTLM, induced dose-dependent aggregation by fibroblasts and endothelial cells. Fibroblasts also showed decreased adhesion when incubated with CM. Dermatophagoides farinae-treated cA549 showed similar but weaker results. The use of serum, boiled CM, or boiled extract inhibited these findings. Dialyzed Dermatophagoides species extracts altered cA549 growth and stimulated the secretion of factors that dysregulate mesenchymal cell growth in vitro.

IL-13 and IL-1beta promote lung fibroblast growth through coordinated up-regulation of PDGF-AA and PDGF-Ralpha.

Peribronchiolar fibrosis is a prominent feature of airway remodeling in asthma and involves fibroblast growth and collagen deposition. Interleukin-13 (IL-13), a T-helper 2 cytokine, is a key mediator of airway remodeling in asthma, yet the mechanism through which IL-13 promotes fibroblast growth has not been investigated. In this study, we show that IL-13 stimulates the mitogenesis of mouse, rat, and human lung fibroblasts through release of a soluble mitogen that we identified as PDGF-AA. The IL-13-induced growth of human lung fibroblasts was attenuated by an anti-PDGF-AA neutralizing antibody, and IL-13 stimulated human lung fibroblasts to secrete PDGF-AA. Fibroblasts derived from mouse embryos possessing the lethal Patch mutation, which lack the PDGF-Ralpha, showed no mitogenic response to IL-13. However, Patch cells did exhibit IL-13-induced STAT-6 phosphorylation. Stable transfection of the PDGF-Ralpha into Patch cells restored the growth response to PDGF-AA and IL-13. Through the use of lung fibroblasts from STAT-6-deficient mice, we showed that IL-13-induced PDGF-AA release is STAT-6 dependent, but PDGF-AA-induced growth is STAT-6 independent. Finally, we showed that IL-1beta enhanced IL-13-induced mitogenesis of rat lung fibroblasts through up-regulation of the PDGF-Ralpha. Our findings indicate that IL-13 acts in synergy with IL-1beta to stimulate growth by coordinately up-regulating PDGF-AA and the PDGF-Ralpha, respectively.

Cytostatic properties of some angiotensin I converting enzyme inhibitors and of angiotensin II type I receptor antagonists.

Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) type 1 receptor antagonists have strong cytostatic properties on in vitro cultures of many normal and neoplastic cells. They are effective, in particular, in reducing the growth of human lung fibroblasts, renal canine epithelial cells, bovine adrenal endothelial cells, simian T lymphocytes, and of neoplastic cell lines derived from human neuroblastomas, a ductal pancreatic carcinoma of the Syrian hamsters, human salivary glands adenocarcinomas, and two lines of human breast adenocarcinomas. ACE inhibitors are also effective in protecting lungs, kidneys and bladders from the development of nephropathy, pneumopathy, cystitis, and eventually fibrosis in different models of organ-induced damage such as exposure to radiation, chronic hypoxia, administration of the alkaloid monocrotaline or bladder ligation. ACE inhibitors and AII type 1 receptor antagonists are also effective in reducing excessive vascular neoformation in a model of injury to the cornea of rats and rabbits, and in controlling the excessive angiogenesis observed in the Solt-Farber model of experimentally induced hepatoma, in methylcholantrene or radiation-induced fibrosarcomas, in radiation-induced squamous cell carcinomas and in the MA-16 viral-induced mammary carcinoma of the mouse. Captopril was, in addition, effective in controlling tumor growth in a case of Kaposi's sarcoma in humans. The inhibition of AII synthesis and/or its blockade by AII receptors is likely to be an important mechanism for this cytostatic action. The mitogenic effect of AII is well established and a reduction of AII synthesis may well explain cell and neoplasm delayed growth. Moreover, AII regulates and enhances the activity of several growth factors including transforming growth factor B (TGFB) and smooth muscle actin (SMA); and many of these factors are reduced in tissues of animals treated with ACE inhibitors and AII type 1 receptor antagonists. These processes seem to be particularly relevant in the control of fibroblast growth and in the control of the ensuing fibrosis. The ACE inhibitors containing a sulphydril (SH) or thiol radical in their moiety (Captopril and CL242817) seemed to be more effective in controlling fibrosis and the growth of some neoplastic cells than those ACE inhibitors without this thiol radical in their structure, even if the second group of these drugs show in vitro a stronger inhibitory effect on converting enzyme activity. Pharmacologically it is known that ACE inhibitors containing a thiol radical also have antioxidant properties and they are efficient in controlling metalloproteinase action. However, although these additional properties are pharmacologically relevant, the blockade of AII synthesis plays an essential role in the cytostatic activity of these two categories of drugs. These observations underline that in addition to the beneficial effect of these drugs on the cardiovascular system, new potential applications are opening for their wider deployment.

Effect of toluene diisocyanate and its corresponding amines on viability and growth of human lung fibroblasts in culture.

Toluene diisocyanate (TDI) is a highly volatile chemical known to cause occupational asthma in exposed workers. TDI-induced asthma is associated with airway epithelium injury and repair, and subepithelial fibrosis. We investigated the effect of TDI and its hydrolysis products, the 2,4- and 2,6-toluenediamines (TDA), on viability and growth of human lung fibroblasts (HLFs) in culture, using a tetrazolium-based cell viability assay. The effects of increasing concentrations of each of these chemicals were evaluated on quiescent cells seeded at two densities (2500 and 5000 cells/well) and treated for 24 or 48 h. TDI (10(-4)-10(-5) mol/L, as a mixture of 80% 2,4-TDI and 20% 2,6-TDI) exhibited a partial but significant cytotoxic effect (10-24%, p<0.05) on HLFs. This effect was observed at both cell densities, and was time- and concentration-dependent. 2,4-TDA, at lower concentrations (10(-8)-10(-6) mol/L) applied for 48 h, also partially reduced HLF viability (10-15%, p<0.05), whereas it tended to trigger cell growth at concentrations higher than 10(-5) mol/L. 2,6-TDA exhibited both a cytotoxic and a proliferative effect on HLFs that depended on concentration, time of exposure and cell culture density. Significant cytotoxicity was only observed after 24 h of treatment with 10(-7)-10(-6) mol/L 2,6-TDA, and reached greater intensity in cells cultured at the highest density. In contrast, 2,6-TDA stimulated HLF growth only after 48 h of incubation at 10(-4) mol/L on cells cultured at the lowest density. Taken together, our results showed that TDI and 2,4-TDA somewhat decreased HLF viability, whereas 2,6-TDA appeared to exhibit both a cytotoxic and a growth stimulatory effect on these cells. TDI and 2,4-TDA are thus suggested to contribute to airway epithelium damage associated with TDI-induced asthma, whereas 2,6-TDA might either trigger epithelial damage or induce cell proliferation that could contribute to epithelium repair or subepithelial fibrosis.

Proliferation of cultured fibroblasts is inhibited byL‐Iduronate—containing glycosaminoglycans

We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth-promoting effects in densely seeded cultures.

Monocyte inhibition of lung fibroblast growth: relationship to fibroblast prostaglandin production and density-defined monocyte subpopulations.

Mononuclear cell fibroblast interactions in the normal human lung are poorly understood. Mononuclear cells can regulate fibroblast function and blood monocytes are known to migrate to the lung and participate in pulmonary inflammation. Thus, to clarify mononuclear cell-fibroblast interactions in the normal lung, we obtained supernatants from adherent monocytes and characterized their effect on the log phase growth of human lung fibroblasts. Monocyte supernatants inhibited fibroblast growth in a dose-dependent fashion. The inhibition was the result of an approximately 16,000 MW soluble factor(s) that was heat stable, trypsin sensitive, and chymotrypsin resistant. Elaboration of the factor(s) required monocyte protein synthesis and was not restricted to a density-defined monocyte subpopulation. The inhibitory capacity of a monocyte supernatant was directly related to its ability to stimulate fibroblast prostaglandin production. Blocking fibroblast prostaglandin production reversed the inhibition of fibroblast growth caused by monocyte supernatants. Thus, monocyte inhibition of fibroblast growth may be mediated by fibroblast prostaglandin production. Recruitment of monocytes to the lung and subsequent monocyte inhibition of fibroblast growth may be important in regulating pulmonary fibrosis.

The effect of beryllium on the growth of human lung fibroblasts

The effect of beryllium on the proliferative capacity of human fetal lung fibroblasts (IMR-90) was investigated. The addition of BeCl 2 to cultures, at concentrations ranging from 0.02 to 0.50 μg Be/ml, increased the population doubling time and decreased the maximal growth level in a dose-dependent manner. Upon achieving maximal growth, cultures exposed to beryllium concentrations of 0.05 μg/ml and above began to decline in cell number, presumably because of cell death. Studies with 7BeCl 2 showed that fibroblasts began to remove beryllium from the growth medium within 24 hr after dosing and continued to accumulate beryllium for 120 hr; after that time, the amount of beryllium per cell appeared to level off. Cells grown in 0.05 μg Be/ml accumulated 2.5 times more beryllium than cells grown in 0.02 μg Be/ml. Exposure of fibroblasts to a concentration of 0.005 μg Be/ml had no detectable effect on the number of population doublings after a single subculture, but reduced the population doublings by 42% after the second subculture. Comparative studies using different beryllium compounds showed that beryllium citrate had a greater inhibitory effect than BeCl 2, which was, in turn, more inhibitory than BeSO 4; BeO, even at a concentration of 2.0 μg Be/ml, had no effect. The degree to which fibroblast growth is inhibited by a particular beryllium compound may be related to the ease with which the cell can accumulate the beryllium complex present.