Biomedical subjects

R F Ertl

Publications and source records attributed to R F Ertl.

30 records · Page 2Linked to original sources

PubMed1990

Fractional processing of sequential bronchoalveolar lavage to separate bronchial and alveolar samples.

Bronchoalveolar lavage has been widely used to sample the lower respiratory tract. Most of the material recovered with this technique represents alveolar contents. A number of modifications have been suggested in order to obtain samples relatively enriched for bronchial material. In order to be able to use a standard technique for bronchoalveolar lavage to sample both airways and "routine" alveolar material, a simple modification of the technique as described by Reynolds and Newball was used: five sequential 20-ml aliquots were infused into the lower respiratory tract, and each aliquot was immediately aspirated. The return from the first aliquot was processed separately from the return from the subsequent four aliquots. These last four aliquots were pooled. Analysis of the first aliquot revealed it to be enriched for ciliated epithelial cells when compared with the subsequent aliquots. There were also differences in inflammatory cell composition with the bronchial sample containing relatively more neutrophils and relatively less lymphocytes. Aspiration during transoral bronchoscopy was documented by quantifying salivary amylase in the bronchial and alveolar lavage fluids. It was estimated, however, that the aspiration was not of quantitative significance in the vast majority of subjects studied. Finally, with the technique of fractional processing of bronchoalveolar lavage samples, it was possible to compare the protein concentrations in bronchial and alveolar lavages. Most prominent among the differences was a marked relative enrichment in the bronchial samples for immunoglobulin A. The technique of fractional processing of bronchoalveolar lavage samples provides a simple means to obtain samples enriched for bronchial and alveolar components. This should facilitate analysis of lower respiratory tract specimens in airway disease.

Adult↗

PubMed1990

Bronchial epithelial cells produce chemotactic activity for bronchial epithelial cells. Possible role for fibronectin in airway repair.

Bronchial mucosal injury initiates a complex series of repair mechanisms, one of which is reepithelialization of a denuded lumenal surface. This suggests the hypothesis that bronchial epithelial cells, the cells initially affected by bronchial injury, might be able to initiate repair of an injured area by producing a chemotactic activity for intact bronchial epithelial cells. To evaluate this, bronchial epithelial cells were prepared from bovine lung by protease digestion and cultured in medium 199 (M199) with 10% fetal calf serum (FCS) until confluence, after which the cells were rinsed with Hanks' balanced salt solution, and serum-free fresh M199 was added. This conditioned medium was then collected and used to test the chemotactic response of bronchial epithelial cells using a blindwell chamber technique. Target cells for this assay were isolated from airways by protease digestion, grown to confluence in M199 with 10% FCS, and then harvested with trypsin. Bronchial epithelial cell-conditioned medium harvested after 3 days attracted more cells (197.0 +/- 5.7 cells/10 high power fields) than did M199 without FCS alone (4.3 +/- 0.9) (p less than 0.01). Checkerboard analysis showed that the migration was chemotactic. The chemotactic activity was nondialyzable, pepsin-labile, acid-stable, heat-labile, and lipid-inextractable. The chemotactic activity accumulated in the culture medium with time. The addition of 25 micrograms/ml of cycloheximide inhibited this accumulation. Column chromatography with Sephadex G-150 revealed a single peak of chemotactic activity in the high molecular weight range. The chemotactic activity was bound to gelatin-Sepharose 4B and was eluted with 6 M urea.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Lung fibroblasts produce growth stimulatory activity for bronchial epithelial cells.

Interaction between lung fibroblasts and airway epithelial cells may play an important role in lung morphogenesis and airway repair after injury. Even though many reports have suggested that mesenchymal fibroblasts have a growth stimulatory effect on surrounding epithelial cells, very few have attempted to confirm and characterize the growth stimulatory activity in isolated cell systems. We cultured human fetal lung (HFL-1) fibroblasts, harvested supernatant-conditioned media under serum-free conditions, and examined their growth stimulatory effect on bovine bronchial epithelial cells by a variety of techniques, including direct enumeration, thymidine uptake, and metaphase arrest. We also partially characterized the growth stimulatory activity and examined the effect of an inhibitor of DNA synthesis on its release. The results demonstrate that HLF-1 fibroblasts release growth stimulatory activity for bovine bronchial epithelial cells. This activity is nondialyzable, protease-sensitive, acid-stable, and lipid-inextractable and thus appears to have the property of protein; it may be produced by de novo synthesis. Molecular sieve chromatography on Sephadex G-75 indicates an approximate molecular mass of 6,000 D. This growth stimulatory activity is likely to play roles in lung morphogenesis and airway repair after injury.

Animals↗

PubMed1990

Pulmonary macrophages can stimulate cell growth of bovine bronchial epithelial cells.

Macrophages are thought to participate in tissue repair following injury by releasing growth factors into the local environment. To evaluate whether pulmonary macrophages can mediate airway epithelial repair, we attempted to determine if pulmonary macrophages can stimulate growth of bovine bronchial epithelial cells in vitro. Bronchial epithelial cells isolated by protease digestion of the bovine bronchi were plated into tissue culture dishes with and without macrophage-conditioned medium. Bronchial epithelial cells cultured with macrophage-conditioned medium showed a significantly greater cell growth than those without macrophage-conditioned medium when assessed by direct enumeration of the cell numbers and by clonal growth assay. Stimulation of proliferation was confirmed by autoradiography using [3H]thymidine uptake into cell nuclei. Co-culture of pulmonary macrophages with bronchial epithelial cells also led to an increase in cell number. Immunohistochemical staining of the proliferating cells showed that these cells were positively stained by anti-keratin antibodies confirming that they were bronchial epithelial cells. Partial characterization of the activity in macrophage-conditioned medium showed that it was nondialyzable, pepsin- and acid-labile, and lipid-inextractable. Sephadex G-75 column fractionation indicated this activity existed in a high molecular fraction, thus suggesting a peptide. DEAE ion exchange chromatography revealed 3 peaks of stimulating activity. One peak resulted in a decrease in cell number, suggesting a possible inhibitory activity. The DEAE results thus suggest that macrophages may release several factors that can affect bronchial epithelial cell proliferation. In conclusion, pulmonary macrophages stimulate cell proliferation of bronchial epithelial cells in vitro. The stimulatory activity that may be heterogeneous appears to have the properties of a peptide.

Animals↗

PubMed1990

Bronchial epithelial cells respond to insulin and insulin-like growth factor-I as a chemoattractant.

Migration of epithelial cells to cover areas of injury is thought to be important in the repair process following airway insult. Insulin is reported to be a growth factor for bronchial epithelial cells, and growth factors have been known to be chemotactic for many types of cells. Thus, we hypothesized that insulin may be a chemoattractant for bronchial epithelial cells. To evaluate this, we prepared bronchial epithelial cells and measured their chemotactic activity toward insulin. Bronchial epithelial cells were isolated by overnight digestion with bacterial protease, filtered through 100-microns nitex mesh, and then cultured at 1 x 10(6) cells/ml in tissue culture dishes in medium 199 supplemented with transferrin, insulin, epidermal growth factor, hydrocortisone, antibiotics, and 10% FCS for 3 d. The cultured cells were rinsed twice to remove supplements, trypsinized and resuspended at 1 x 10(6) cells/ml in medium 199 without supplements, and used as the cell source for chemotaxis. Chemotactic activity of bronchial epithelial cells was measured by the blindwell chamber technique using 8-microns Nuclepore filter membranes coated with 0.1% gelatin. The cells were added to the top wells in a 48-multiwell chamber with insulin in the bottom wells and incubated for 6 h at 37 degrees C, 5% CO2. Bronchial epithelial cells migrated in response to insulin in a dose-dependent manner up to an optimal dose of insulin, 100 micrograms/ml, and decreased at higher concentrations. The number of migrated cells per 10 high power fields was 33.7 +/- 1.9 at the optimum and 3.7 +/- 0.7 without insulin (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Separation of bovine bronchial epithelial cell subpopulations by density centrifugation: a method to isolate ciliated and nonciliated cell fractions.

Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95% ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 +/- 3.88% nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2% fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 +/- 5.28 ng/10(6) cells/h) than fraction A cells (73.4 +/- 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.

Animals↗

PubMed1989

Lung fibroblasts produce chemotactic factors for bronchial epithelial cells.

The interaction between the epithelial cells and the subjacent mesenchymal cells in the airway is thought to play a major role during tissue repair and morphogenesis. To evaluate this interaction, we cultured human lung fibroblasts and bovine bronchial epithelial cells and determined that fibroblast-conditioned medium has chemotactic activity for bronchial epithelial cells. This activity was nondialyzable, heat labile, pepsin labile, acid stable, lipid inextractable, and eluted from Sephadex G-150 column chromatography in the high-molecular-weight range. DEAE-Sephacyl ion exchange and gelatin-Sepharose affinity chromatography revealed two peaks containing chemotactic activity, one of which may be fibronectin, since it binds to gelatin, reacts in a specific immunoassay, and is inhibited of chemotactic activity by anti-fibronectin antiserum, and another of which does not appear to be fibronectin, since it does not bind to gelatin nor react in the immunoassay. Thus lung fibroblasts can produce at least two chemotactic factors for bronchial epithelial cells that may play a role during lung tissue repair and morphogenesis by modulating bronchial epithelial cell migration.

Animals↗

PubMed1989

Bronchial epithelial cells produce lung fibroblast chemotactic factor: fibronectin.

The interaction between the epithelial cells and the subjacent mesenchymal cells in the airway is thought to play a major role during tissue repair after airway injury and lung morphogenesis. To evaluate this interaction, we cultured human lung fibroblasts, and bovine and human bronchial epithelial cells, and determined that bronchial epithelial cell-conditioned medium has a chemotactic activity for lung fibroblasts. This activity had the characteristics of protein: it was nondialyzable, heat-labile, pepsin-labile, acid-stable, and lipid-inextractable. Molecular sieve chromatography on Sephadex G-150 and affinity chromatography on gelatin-Sepharose revealed that there was one peak of chemotactic activity in high molecular weight range, which bound to gelatin, thus suggesting that the chemotactic factor might be fibronectin. Production and secretion of fibronectin into the culture media were demonstrated by biosynthetic incorporation of radioactive amino acid into fibronectin followed by immunoprecipitation on SDS-PAGE and autoradiography. Release into the culture medium was confirmed by ELISA. The identity of fibronectin as the chemotactic activity was confirmed by the addition of antifibronectin antibody to the conditioned medium, which inhibited chemotaxis in dose-dependent manner. Thus, bronchial epithelial cells produce fibronectin which can function as a chemotactic factor for lung fibroblasts. This production of fibronectin by bronchial epithelial cells may play an important role in regulating interaction between the bronchial epithelial cells that line the lumenal surface of the bronchial epithelial wall and the mesenchymal fibroblasts that underlie the bronchial epithelial basement membrane.

Antibodies↗

PubMed1988

Mechanisms of neutrophil recruitment to the lung by grain dust exposure.

Inhalation of grain dusts can cause symptoms of both acute and chronic bronchitis, which has been associated with a neutrophilic inflammatory response in the lung. Since this influx of neutrophils may potentially result in bronchial injury, mechanisms of neutrophil recruitment to grain dust were examined. Sterile, aqueous grain dust extracts were prepared from settled dusts of grain sorghum, corn, oats, and soybeans. The extracts were evaluated for their ability to directly attract human neutrophils using a blindwell neutrophil chemotaxis assay. Each extract was found to possess significant chemotactic activity compared to control (p less than 0.01). To evaluate if the dusts could attract neutrophils indirectly by activating humoral inflammatory mechanisms, the grain dust extracts were evaluated for their ability to activate the complement system. When incubated with normal human serum, each of the grain dusts caused cleavage of the complement proteins C3 and properdin factor B (PFB). Importantly, the grain dust extracts lead to the generation of C5a, a potent neutrophil chemoattractant generated by complement activation. Since activation of the alveolar macrophage to release chemotactic activity represents an additional indirect mechanism of neutrophil recruitment, an extract from grain sorghum dust was evaluated for its ability to stimulate guinea pig and human alveolar macrophages to release neutrophil chemotactic activity. The results demonstrate that supernatants obtained from alveolar macrophages cultured in the presence of grain sorghum dust extract possessed increased chemotactic activity compared to controls (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomechanical Phenomena↗

PubMedDate not supplied

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix.

Fibroblasts in vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but it also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E2 (PGE2), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE2 inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.

Cell Count↗

PubMedDate not supplied

Glucocorticoids augment fibroblast-mediated contraction of collagen gels by inhibition of endogenous PGE production.

Glucocorticoids are currently regarded as the drug of choice in the treatment of inflammatory airway and lung diseases, however, they are not routinely effective in fibrotic phases of inflammation. In the current study, glucocorticoids were investigated for their ability to affect fibroblast mediated contraction of a three dimensional collagen gel, a measure of one aspect of tissue remodeling. Dexamethasone, budesonide, hydrocortisone and fluticasone propionate were all able to significantly augment fibroblast contractility in a concentration dependent manner. Glucocorticoids also had an augmentative effect on collagen gel contraction mediated by fibroblasts from bronchi, skin and bone marrow. The increased contractility was not due to cell proliferation or to collagen degradation, since the glucocorticoids did not alter the amounts of DNA and hydroxyproline in the gels. The concentration of prostaglandin E2 (PGE2) in supernatant media was lower from glucocorticoid-treated gels compared to control gels. Consistent with this, addition of exogenous PGE2 to the culture system restored the contractile properties and indomethacin augmented contraction similar to the glucocorticoids suggesting that inhibition of prostaglandins or related eicosanoids may be the mechanism by which the increased contractility occurs. DBcAMP, forskolin and the long lasting beta2-agonist formoterol were able to reverse the effect of the glucocorticoids on fibroblast mediated collagen gel contraction suggesting that enhancers of cAMP can counteract the effect of glucocorticoids. Thus, we provide evidence that glucocorticoids have the ability to directly augment fibroblast contractility by inhibiting fibroblast endogenous PGE synthesis. The findings could be one possible mechanism to explain the poor therapeutic response to glucocorticoids on the later stages of fibrotic diseases.

Animals↗

PubMedDate not supplied

Antiproteases attenuate the release of neutrophil chemotactic activity from bronchial epithelial cells induced by smoke.

The released neutrophil chemotactic activity (NCA) from bronchial epithelial cells (BECs) in response to smoke extract was evaluated by reverse-phase, high-performance liquid chromatography (RP-HPLC) and the involvement of proteolytic activity was assessed for the release of NCA from BECs. Smoke extract stimulated the release of NCA (55.3 +/- 5.2 vs. 17.3 +/- 4.1 cells per high-power field [HPF], p < .001). The released activity determined by RP-HPLC analysis was 15-hydroxyeicosatetraenoic acid and leukotriene B4. Several structurally and functionally different serine protease inhibitors, including alpha-1-protease inhibitor (alpha-1-PI), chloromethyl ketone (CK) derivatives, N-tosyl-L-lysine CK (TLCK), methoxysuccinyl-Ala-Ala-Pro-Val CK (SPCK), N-alpha-tosyl-L-phenylalanine CK (TPCK), and N-alpha-p-tosyl-L-arginine methyl ester hydrochloride (TAME), attenuated the release of NCA (P < .01) in a dose-dependent fashion. Leupeptin, a cysteine protease inhibitor, has only a small effect on the release of NCA (p < .05), and phosphoramidon, a neutral endopeptidase inhibitor, had no effect. The measurement of proteolytic enzyme activity using synthetic substrate S-2288 revealed that smoke extract significantly (p < .05) augmented the serine protease activity in BEC layers. Culture supernatant fluids and cell lysates of BECs in response to smoke extract solubilized 14C-labeled casein. These results suggest that BECs may release lipoxygenase-derived NCA in response to smoke extract and that the release of NCA may involve the activation of proteolytic activity of BECs which was inhibited by serine protease inhibitors.

Albumins↗