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Levy, Paul C.; Utell, Mark J.; Fleit, Howard B.; Roberts, Norbert J.; Ryan, Daniel H.; Looney, R. John
doi: 10.1165/ajrcmb/5.4.307pmid: 1654955
Three classes of Fcγ receptors (FcR) have been identified on blood leukocytes: FcRI, FcRII, and FcRIII. Two forms of FcRIII have recently been characterized; a phosphatidylinositol linked form is found on neutrophils, whereas a transmembrane form of the molecule is found on a subset of peripheral blood lymphocytes. Peripheral blood monocytes express low levels of FcRIII on their surface, whereas FcRIII is readily expressed by tissue macrophages. The purpose of this investigation was to characterize the form of FcRIII expressed by normal human alveolar macrophages (AM) obtained from normal subjects by bronchoalveolar lavage. We found FcRIII expressed by AM has a molecular mass of 50 to 60 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis and migrates as a single band with a molecular mass of 35 kD after digestion with endoglycosidase F. Macrophage FcRIII was resistant to cleavage by phosphatidylinositol-specific phospholipase C. These results demonstrate that FcRIII expressed by AM is a transmembrane glycoprotein similar to the molecule found on peripheral blood lymphocytes. Scatchard binding analysis using 125I-labeled mAb 3G8 showed that AM express similar numbers of FcRIII as found on neutrophils (73,300 ± 16,300 versus 69,300 ± 8,500 receptor sites/cell, respectively; P = 0.73), whereas fewer binding sites were found on FcRIII-positive peripheral blood lymphocytes (35,300 ± 13,900; P = 0.04). Of note, we found expression of FcRIII by AM was selectively and dramatically reduced during short term in vitro incubation at 37° C. Receptor shedding as a result of proteolytic cleavage is probably responsible for the reduced expression that occurs during short-term in vitro culture.
Rahmoune, Hassan; Lamblin, Geneviève; Lafitte, Jean-Jacques; Galabert, Claude; Filliat, Monique; Roussel, Philippe
doi: 10.1165/ajrcmb/5.4.315pmid: 1910815
In order to ascertain whether or not the presence of glycosaminoglycans in sputa of patients suffering from chronic bronchial disorders was related to tracheobronchial infection, an electrophoretic procedure was set up. The different acidic macromolecular components of sputum, namely nucleic acids, glycosaminoglycans, and bronchial glycopeptides could be identified in proteolyzed sputum using agarose electrophoresis before and after the action of different enzymes: nucleases, chondroitinases, hyaluronidase and heparinase. This procedure was used to analyze 13 sputum samples from patients suffering from cystic fibrosis (CF) and 12 sputum samples from patients suffering from chronic bronchitis. Chondroitin sulfate was identified in 11 infected sputum samples from patients with CF and also in the noninfected sputum from a patient with chronic bronchitis. These data suggest a relationship between the presence of chondroitin sulfate proteoglycans in sputum and severe tracheobronchial infection in CF.
Patel, J. M.; Sekharam, K. Madhavi; Block, Edward R.
doi: 10.1165/ajrcmb/5.4.321pmid: 1910816
The stimulatory effects of angiotensin (Ang) I, Ang II, and Ang III on production of diacylglycerol (DAG), a second messenger, were examined in porcine pulmonary artery endothelial cells. Ang I, Ang II, and Ang III provoked rapid increases in [3H]glycerol labeling of DAG. The stimulatory effect on DAG production was maximal after 1 and 5 min. Pretreatment of cells with angiotensin-converting enzyme activity inhibitors prevented the stimulatory effect of Ang I on DAG production, indicating that Ang II but not Ang I is responsible for increased DAG production. The stimulatory effects of Ang II and Ang III on DAG production were concentration dependent and were maximal at a 10-nM concentration of both Ang II and Ang III. Data from further experiments revealed that the Ang II- and Ang III-elicited formation of DAG is derived from the coordinated hydrolysis of membrane phosphatidylinositol and phosphatidylcholine by phospholipase C- and phospholipase D-catalyzed pathways. The angiotensin analogue [Sar1 Ile8] Ang II, an Ang II receptor antagonist, blocked the hydrolysis of phosphatidylinositol and phosphatidylcholine and thus the increased production of DAG by Ang II and Ang III. These results indicate that Ang II- and Ang III-induced stimulation of DAG production in pulmonary artery endothelial cells involves multiple pathways of phospholipid hydrolysis and is mediated by angiotensin receptors.
Nelson, Jeanne M.; Duane, Peter G.; Rice, Kathryn L.; Niewoehner, Dennis E.
doi: 10.1165/ajrcmb/5.4.328pmid: 1910817
Cadmium exposure is capable of causing acute and chronic lung injuries, but the specific pathogenetic mechanisms are uncertain. The effects of cadmium ion (Cd2+) on phospholipid metabolism were examined in cultured bovine pulmonary artery endothelial cells (BPAEC), as endothelial cells appear to be particularly vulnerable to the toxic effects of this metallic ion. Exposure of radiolabeled BPAEC to millimolar concentrations of Cd2+ causes liberation of substantial amounts of [3H]arachidonic acid ([3H]AA), but only small amounts of [14C]stearic acid, from each of the major phospholipid subclasses. Analyses of hydrolytic products in BPAEC radiolabeled with [3H]myo-inositol and exposed to Cd2+ indicate that degradation of complex phospholipids is mediated by phospholipase A2. The ability of BPAEC to incorporate fatty acids or lysophosphatides into complex phospholipids is similarly impaired after exposure to Cd2+, suggesting that the liberation of [3H]AA might be due to impairment of reacylation mechanisms and not to increased hydrolytic activity of phospholipase A2. Of the two enzymes involved in reacylation reactions, Cd2+ is found to inhibit the activity of arachidonyl-specific acyl coenzyme A synthetase but not the activity of acyltransferase. Cd2+ also causes a profound time- and dose-dependent depletion of adenosine triphosphate levels in BPAEC, and these changes closely correlate with the liberation of [3H]AA. We suggest that impairment of reacylation mechanisms, and the consequent accumulation of arachidonic acid, may be important in the development of the acute inflammatory reaction that is characteristic of Cd2+-induced lung injury.
doi: 10.1165/ajrcmb/5.4.337pmid: 1910818
Epidermal growth factor (EGF) increases the rate of choline incorporation into disaturated phosphatidylcholine in cultured fetal rat type II cells via an indirect mechanism. Whereas EGF has no effect on the rate of disaturated phosphatidylcholine synthesis when added directly to type II pneumocytes, the growth factor is effective if it is present during preliminary conditioning of the media by lung fibroblasts. This effect is concentration dependent with a maximal effect at 20 ng/ml. When lung fibroblasts are incubated with both glucocorticoids and EGF, there is no significant effect of the growth factor over and above that seen with the steroid alone. This suggests that the two agents might act via a similar mechanism. This is supported by the observation that each inducer leads to the production by lung fibroblasts of a stimulatory factor that has a similar, if not identical, chromatographic elution profile. We conclude that EGF may contribute significantly to the normal onset of lung maturation by elaborating a fibroblast-derived factor that stimulates phosphatidylcholine synthesis in type II pneumocytes.
doi: 10.1165/ajrcmb/5.4.344pmid: 1910819
Alveolarization of the immature lung is thought to be influenced by the presence of elastic fibers that could provide structural support for developing septa. Although morphometric studies have established that alveolar septal development occurs from days 4 to 13 in the neonatal rat, the precise time period over which elastin synthesis occurs has proved difficult to determine. We have evaluated the usefulness of in situ hybridization techniques to follow tropoelastin message expression in parenchymal tissue, small vessels, and bronchioles in the developing rat lung from days 4 through 18. This method proved to be sufficiently sensitive to detect differences in rates of tropoelastin message expression from days 4 through 18 (P < 0.0001). Peak tropoelastin message expression was observed in the small vessels on day 4 and in parenchymal tissue on days 9 through 11. Because the time course of tropoelastin message expression in small vessels differs from that in parenchymal tissue, the use of lung extracts to analyze rates of tropoelastin synthesis in the developing lung may be in error.
Rannels, D. Eugene; Stockstill, Barbara; Mercer, Robert R.; Crapo, James D.
doi: 10.1165/ajrcmb/5.4.351pmid: 1910820
The time course and nature of the cellular response to left pneumonectomy, with or without prior adrenalectomy, were evaluated in the right lungs of male Sprague-Dawley rats using morphometric techniques. Animals were studied at days 2, 5, and 14 following pneumonectomy, intervals prior to, during the course of, and following significant compensatory changes in right lung mass. The postoperative increase in right lung mass and volume in pneumonectomized animals involved minimal changes in the ratios of most tissue components, when compared to the lungs of sham-operated controls. A transient disproportionate increase in type II cell volume and epithelial thickness was evident on day 14. Postpneumonectomy changes in the type II epithelium were accentuated in the lungs of adrenalectomized-pneumonectomized animals. Adrenalectomy 5 days prior to pneumonectomy resulted in a substantial increase in the volume of all right lung tissue components, associated with thickening of the alveolar wall and with increases in the volume of both cellular and noncellular interstitium. Effects of adrenalectomy on the endothelium also were evident. In both adrenal-intact and adrenalectomized animals, pneumonectomy increased alveolar number by day 14 but had no effect on the volume of individual alveoli. These results confirm a coordinated pattern of compensatory growth following pneumonectomy in the adrenal-intact rat. The data further suggest that in adrenalectomized animals compensatory lung growth is more poorly synchronized, with pronounced postoperative elevations in volume of the interstitial and type II epithelial compartments leading to increased thickness of the alveolar wall. Adrenal hormones thus appear to be required for coordination and control of compensatory lung growth and for rapid restoration of normal tissue structure.
Caesar, Petula A.; McElroy, Mary C.; Kelly, Frank J.; Normand, I. Colin S.; Postle, Anthony D.
doi: 10.1165/ajrcmb/5.4.363pmid: 1910821
The molecular specificity of phosphatidylcholine (PC) synthesis by the de novo pathway in postmortem samples of human fetal lung (15 to 20 wk of gestation) was determined from the incorporation pattern in isolated microsomal preparations of CDP:[14C]choline into individual molecular species of PC. These analyses are based on the assumption that the molecular species composition of the pool of endogenous diacylglycerol used for PC synthesis by isolated microsomes reflects that of the authentic pool of diacylglycerol converted to PC by intact cells. Comparison of this microsomal incorporation pattern of radiolabel into PC with tissue PC composition suggested that even at this early stage of gestation 50% of lung dipalmitoyl PC was derived from synthesis de novo, with the remainder coming from acyl remodeling mechanisms. Analysis of PC synthesis de novo by organ cultures of human fetal lung showed that these acyl remodeling mechanisms were lost in culture. Despite evidence for differentiation of type II alveolar epithelial cells in culture, equilibrium labeling of PC with [14C]choline over 18 h resulted in a progressive decline in fractional incorporation into dipalmitoyl PC with time in culture. By 4 days in culture, this value was no different from the fractional incorporation of CDP:[14C]choline into microsomal PC in vitro over 3 h. The pattern of PC synthesized was not altered when total PC synthesis was stimulated by exposure of cultures to dexamethasone and tri-iodothyronine but was readily manipulated by exposure to exogenous fatty acids. These results demonstrate for the first time the activity of PC acyl remodeling mechanisms in human fetal lung, well before the initiation of surfactant production. They support the conclusion that conditions that permit accelerated differentiation of type II epithelial cells from human fetal lung in organ culture do not necessarily support the spectrum of PC species synthesis characteristic of the differentiated state.
Buch, Shilpa; Jones, Colin; Sweezey, Neil; Tanswell, Keith; Post, Martin
doi: 10.1165/ajrcmb/5.4.371pmid: 1910822
The autocrine, paracrine, or systemic growth factors responsible for fetal lung cell growth are not completely defined. The progression-type insulin-like growth factors and epidermal growth factor, or transforming growth factor-α acting through the epidermal growth factor receptor, appear to act on the developing lung epithelium. The competence factors that facilitate the actions of progression factors during lung growth are unknown. Fetal rat lung cells in vitro synthesize a platelet-derived growth factor (PDGF)-like polypeptide, which we have hypothesized may play a paracrine role in normal lung development. Slot blot and Northern blot analyses of fetal rat lung mRNA have been used to determine if there is a relationship between expression of message for PDGF-A or PDGF-B chains, or their cognate receptors, and periods of maximal growth during late fetal rat lung development. Whole lung mRNA was extracted on 18, 19, 20, 21, and 22 days of gestation (term = 22 days). The peak of DNA synthesis, as assessed by expression of message for DNA polymerase α, hist one 3, and the proto-oncogenes c-fos and c-myc, which are stimulated by binding of growth factors including PDGF, occurred during the canalicular stage of lung development on days 19 and 20 of gestation. Expression of message for PDGF-A and PDGF-B chains was low during the pseudoglandular stage on day 18, peaked during the canalicular stage on days 19 and 20, then fell again during the saccular stage at days 21 and 22 of gestation. Message for PDGF receptors was not apparently related to the stage of lung development but did decline dramatically immediately prior to delivery.
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