American Journal of Respiratory Cell and Molecular Biology

Zimmerman, G. A.; Lorant, D. E.; McIntyre, T. M.; Prescott, S. M.

doi: 10.1165/ajrcmb/9.6.573pmid: 7504925

Intercellular interactions in which one cell sends a signal to another cell, inducing a change in function of the second cell, are common in morphogenesis, development, inflammation, and repair of the lung and other organs. In juxtacrine intercellular signaling, the molecule that induces the functional changes in the target cell remains associated with the plasma membrane of the signaling cell, rather than acting in the fluid phase. This feature distinguishes juxtacrine signaling from endocrine and paracrine stimulation and provides a mechanism for strict spatial control of activation of one cell by another. Juxtacrine signaling is likely to be common in physiologic events that require tight regulation, and disruption of juxtacrine signaling may lead to pathologic outcomes. In this minireview, general principles as well as several specific examples of juxtacrine signaling are discussed.

McCray, Paul B.; Bettencourt, Jeffrey D.; Bastacky, Jacob; Denning, Gerene M.; Welsh, Michael J.

doi: 10.1165/ajrcmb/9.6.578pmid: 7504926

During development the fetal lung secretes fluid that is osmotically linked to chloride (Cl−) transport. One possible pathway for Cl− secretion across the fetal pulmonary epithelium is through the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in epithelia and functions as a Cl− channel regulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and intracellular ATP. Previous studies have shown that CFTR mRNA is expressed throughout the human fetal pulmonary epithelium and CFTR protein can be immunoprecipitated from human fetal lung homogenates. In cultured fetal lung tissue explants, CFTR mRNA was localized to alveolar epithelial cells. To test the hypothesis that fetal alveolar epithelial cells express functional CFTR, we immunolocalized CFTR in human fetal lung and looked for evidence of Cl− secretion in cultured alveolar epithelial cell monolayers. Monoclonal anti-CFTR antibodies localized CFTR in cultured lung explants to the epithelial cells, predominantly at the apical surface. Bioelectric properties of cultured monolayers of midgestation fetal alveolar epithelial cells were measured in modified Ussing chambers. In unstimulated monolayers, transepithelial electrical potential difference (Ψt) = −1.1 ± 0.1 mV, transepithelial resistance (Rt) = 768 ± 58 Ω·cm2, and short-circuit current (Isc) = 1.9 ± 0.2 µA/cm2 (mean ± SE, n = 17). Addition of amiloride to the apical surface significantly decreased basal Isc. Apical diphenylamine-2-carboxylate (DPC), a Cl− channel inhibitor, caused no significant change in basal Isc. In the presence of apical amiloride, isoproterenol significantly increased Cl−, a response that was inhibited by apical DPC and submucosal bumetanide. The cAMP agonists forskolin and IBMX also stimulated Cl−. These results suggest that human fetal alveolar epithelial cells express CFTR, which mediates cAMP-stimulated transepithelial Cl− secretion.

Bloemen, Pauline G. M.; van den Tweel, Marja C.; Henricks, Paul A. J.; Engels, Ferdi; Wagenaar, Sjoerd S.; Rutten, Alfons A. J. J. L.; Nijkamp, Frans P.

doi: 10.1165/ajrcmb/9.6.586pmid: 7504927

Epithelial damage in the airways is a feature often observed in patients with asthma and is probably caused by the interaction of epithelial cells with leukocytes. As adhesion molecules are thought to be important in this interaction, we analyzed the expression and modulation of adhesion molecules on primary cultured human bronchial epithelial cells and the bronchial epithelial cell lines BEAS-2B and NCI-H292. E-selectin, P-selectin, and VCAM-1 were absent under basal and stimulated conditions. The adhesion molecules ICAM-1 (CD54), LFA-3 (CD58), and CD44 (H-CAM) were expressed basally on primary cultured human bronchial epithelial cells and the BEAS-2B and NCI-H292 cell lines. CD44 and LFA-3 expression did not change after stimulation with IFN-γ or TNF-α. In contrast, ICAM-1 expression on human bronchial epithelial cells and BEAS-2B cells could be increased by incubation with PMA, IFN-γ, TNF-α, and especially with the combination of IFN-γ and TNF-α. The maximal ICAM-1 expression on both epithelial cell types was obtained with the combination of TNF-α and IFN-γ after 48 h of incubation. The NCI-H292 cell line was different in that it only showed increased ICAM-1 expression after stimulation with PMA and IFN-γ and not by the combination of IFN-γ and TNF-α or with TNF-α alone. In conclusion, the bronchial epithelial cells tested express several adhesion molecules, but only ICAM-1 expression was influenced by inflammatory cytokines.

Leikauf, George D.; Zhao, Qiyu; Zhou, Shaoying; Santrock, Jeffrey

doi: 10.1165/ajrcmb/9.6.594pmid: 8257591

When inhaled, ozone reacts at the airway luminal surface with unsaturated fatty acids contained in the extracellular fluid and plasma membrane to form an aldehyde and hydroxyhydroperoxide. The resulting hydroxyhydroperoxide degrades in aqueous systems to yield a second aldehyde and hydrogen peroxide (H2O2). Previously, we demonstrated that ozone can augment eicosanoid metabolism in bovine airway epithelial cells. To examine structure-activity relationships of ozone-fatty acid degradation products on eicosanoid metabolism in human airway epithelial cells, 3-, 6-, and 9-carbon saturated aldehydes and hydroxyhydroperoxides were synthesized and purified. Eicosanoid metabolism was evaluated by determination of total 3H-activity release from confluent cells previously incubated with [3H]arachidonic acid and by identification of specific metabolites with high performance liquid chromatography and radioimmunoassay. The major metabolites detected were prostaglandin E2, prostaglandin F2α, and 15-hydroxyeicosatetraenoic acid. The 9-carbon aldehyde, nonanal, in contrast to 3- or 6-carbon aldehydes, stimulated release at concentrations ≥ 100 µM, suggesting that the stimulatory effect increases with increasing chain length. When tested under identical conditions, the 3-, 6-, and 9-carbon hydroxyhydroperoxides were more potent than the corresponding aldehydes. Again, a greater effect was noted when the chain length was increased. One possible explanation for the increased potency of the hydroxyhydroperoxides over the aldehydes could be due to degradation of the hydroxyhydroperoxide into H2O2 and aldehyde. We consider this an unlikely explanation because responses varied with chain length (although each hydroxyhydroperoxide would produce an equivalent amount of H2O2) and because exposure to H2O2 alone or H2O2 plus hexanal produced a response dissimilar to 1-hydroxy-l-hexanehydroperoxide. Inasmuch as these compounds are ozonolysis products of unsaturated fetty acids of the membrane phospholipids, this study ascribes pathophysiologic significance to this chemical mechanism of ozone toxicity.

Kinnula, Vuokko L.; Mirza, Zermeena; Crapo, James D.; Whorton, A. Richard

doi: 10.1165/ajrcmb/9.6.603pmid: 8257592

We have investigated factors that regulate hydrogen peroxide (H2O2) release from vascular endothelial cells. Endothelial cells produce H2O2 at an intracellular site in the vicinity of peroxisomes and at a second site near the cell surface that is inaccessible to intracellular catalase or glutathione peroxidase. Regulation of H2O2 generation at the intracellular site was studied using aminotriazole, which inactivates catalase in the presence of H2O2. Regulation of H2O2 generation at the second site was studied by measuring H2O2 release into the medium. The rate of H2O2 release was constant over 2 h when cells were incubated in room air. Changing O2 levels in the atmosphere from 0% to 10% O2 resulted in a threefold increase in the rate of H2O2 release. Elevation of O2 levels from 10% to 95% O2 produced no further enhancement in the rate of release. Preincubation of cells under hypoxic conditions did not lead to an exaggerated rate of H2O2 release when cells were returned to room air. Pretreatment of cells with exogenous H2O2 inhibited subsequent H2O2 release while pretreatment with catalase enhanced H2O2 release. Although arachidonic acid transiently enhanced the rate of H2O2 release through a mechanism dependent on PGH synthase, basal H2O2 release was independent of this enzyme. Neither hypoxia, hyperoxia, or hypoxia followed by reoxygenation altered H2O2 generation at the intracellular site accessible to peroxisomal catalase. These data demonstrate that H2O2 release from endothelial cells is responsive to changes in O2 concentrations over a narrow range. The mechanisms involved are subject to product inhibition and appear to be saturated at 10% O2 in the atmosphere.

Russell, William J.; Jackson, Robert M.

doi: 10.1165/ajrcmb/9.6.610pmid: 8257593

Previous studies using an in vivo rabbit model in which lung tissue hypoxia/hypoperfusion was created by unilateral lung collapse for 7 days demonstrated a decrease in MnSOD activity in previously hypoxic/hypoperfused lungs. In the present study, we determined whether tissue hypoxia/hypoperfusion decreased MnSOD protein concentration or mRNA expression in the lung as well, changes that would suggest pretranslational regulation of enzyme activity. Expression of MnSOD may be critical in determining the degree of tissue injury during re-oxygenation because the mitochondrial electron transport system produces reactive oxygen species (ROS) both during hypoxia and re-oxygenation. We purified MnSOD protein from rabbit livers to a specific activity of ∼3,500 U/mg protein and found the amino terminal sequence nearly identical to those of the rat and human MnSOD proteins. Lung MnSOD protein content was quantitated by immunoassay, and MnSOD mRNA content was determined by slot blotting. Results from five control and six experimental rabbits, the right lungs of which had been hypoxic/hypoperfused because of collapse for 7 days, demonstrated a 32% decrease (P < 0.03) in MnSOD protein content (42 ± 8 µg/mg DNA in hypoxic lungs compared with 61 ± 3 µg/mg DNA in contralateral lungs) that was not due to decreased numbers of mitochondria. Lung succinate dehydrogenase activity, a mitochondrial marker, did not change in hypoxic/hypoperfused lungs. The mRNA for MnSOD did not change relative to B-actin mRNA in lungs that had been hypoxic and hypoperfused for 7 days. Lung tissue hypoxia/hypoperfusion, resulting from unilateral lung collapse, was associated with parallel decreases in both MnSOD activity and protein content. MnSOD activity in hypoxic lung tissue appears related to changes in enzyme protein content rather than mRNA, suggesting that in this model of lung tissue hypoxia MnSOD is regulated at the post-transcriptional level.

Lovchik, J. A.; Lipscomb, M. F.

doi: 10.1165/ajrcmb/9.6.617pmid: 8257594

Although C5a-induced intravascular pulmonary sequestration of neutrophils has been investigated with regards to lung injury, relatively few studies have addressed the possible role for this mechanism in the intravascular clearance of circulating microorganisms. A murine model was used in which the complement-fixing, encapsulated yeast Cryptococcus neoformans (Cne) was inoculated intravenously (IV), and lung clearance of the organism was measured 24 h later. In normal mice, clearance was remarkably effective, but in leukocyte-depleted or C5-deficient (C5−) animals, clearance was significantly decreased. In vitro assays indicated that C5 was necessary for neutrophils to kill encapsulated Cne and evidence was obtained that C5a was involved. In vivo studies using light and electron microscopy demonstrated that 30 min after an IV inoculation of encapsulated yeast into C5-sufficient (C5+) mice, neutrophils accumulated in pulmonary vessels and engulfed Cne. However, in C5− mice, neutrophils failed to accumulate in pulmonary vessels and there was no endocytosis of encapsulated yeasts. These studies suggested that following Cne interaction with complement in the blood, release of C5a activated circulating neutrophils to adhere to Cne, and perhaps to adjacent endothelium, which facilitated rapid phagocytosis and killing of the organism. In contrast to the IV infection model, when Cne was inoculated into the tracheas of C5+ and C5− mice, no evidence was obtained for an early PMN-C5-dependent clearance mechanism. C5a-dependent neutrophil killing in the lung vasculature may provide important host protection against Cne during vascular dissemination and perhaps against other disseminating microorganisms that activate complement.

Karmiol, Soverin; Remick, Daniel G.; Kunkel, Steven L.; Phan, Sem H.

doi: 10.1165/ajrcmb/9.6.628pmid: 7504928

Previous studies have shown upregulation of lung cell interleukin-6 (IL-6) production in bleomycin-induced pulmonary fibrosis. To further elucidate the regulatory mechanisms governing this disease, the effects of bleomycin on the production of the pleiotropic cytokine, IL-6, were investigated in lung endothelial cells. Rat pulmonary artery endothelial cells were treated with bleomycin at doses previously shown to be effective in upregulating cytokine production in these cells, and the conditioned media was collected and assayed for IL-6 activity. The results show that these endothelial cells constitutively produced IL-6 and that bleomycin increased the production in a time- and dose-dependent manner. Feeding rats diets deficient in n-6 fatty acids is known to ameliorate bleomycin-induced lung fibrosis. In order to examine if fatty acids could modulate IL-6 production in vitro, cells were lipid depleted and then supplemented with 18:1n-9, 18:2n-6, or 18:3n-3 fatty acids, and the effects of bleomycin on IL-6 production reexamined. This regimen resulted in significant depletion of arachidonate in the 18:1n-9 and 18:3n-3 supplemented cells, which was associated with significantly reduced IL-6 production relative to the 18:2n-6-supplemented cells, both constitutively and when stimulated with bleomycin. Preincubation with indomethacin did not significantly inhibit the production of IL-6 by all three groups of cells, nor did supplementation with a stable prostacyclin analog increase IL-6 production. These results suggest that endothelial cell IL-6 production is not directly dependent on prostacyclin or other cyclooxygenase metabolites but may require or be upregulated by 18:2n-6 and/or metabolites derived from it. This conclusion would be consistent with the previous in vivo observation that diets low in 18:2n-6 have a protective effect on bleomycin-induced pulmonary fibrosis.

Walsh, James; Absher, Marlene; Kelley, Jason

doi: 10.1165/ajrcmb/9.6.637pmid: 7504929

During acute lung injury, there is an outpouring of growth factors into the alveolar space that drive local repair and fibrosis. During the remodeling that follows the instillation of bleomycin via the trachea into the adult rat, at least two platelet-derived growth factor (PDGF)-like peptides are released sequentially into lung lining fluid. Groups of four to five animals were killed at 3, 6, 15, and 26 days after exposure to bleomycin and lungs lavaged with isotonic saline. PDGF-like peptides in epithelial lining fluid (ELF) were partially purified by cation exchange chromatography and concentrated. Isolated peptides were analyzed by immunoblotting to determine their molecular weight and immunologic identity. Western blots were probed with polyclonal antibodies to PDGF-BB and PDGF-AA. PDGF-like peptides of two distinct size classes (38–40 kD and 29 kD) were present in alveolar fluid from all rats with lung injury induced by bleomycin. No PDGF-like peptides were found in comparably prepared ELF from control animals. The 38–40 kD peptide was detected only with anti-PDGF-BB antibody; the 29 kD peptide was detected only with anti-PDGF-AA antibody The presence of these two peptides varied independently with time after exposure to bleomycin. The 38–40 kD peptide was at peak levels at 3 to 6 days. In contrast, the 29 kD peptide was present at all times following injury but with far less variation over time. In parallel with these immunoassays for PDGF-like molecules, there was abundant growth-promoting activity for fibroblasts present in concentrated ELF during the course of injury. Bioassays of processed lavage fluid were used to determine the mitogenic potency of ELF for lung fibroblasts. The amount of growth-promoting activity paralleled over time the amount of the 38–40 kD peptide detected on immunoblots. At day 3, the mitogenic activity of chromatographically purified PDGF-like peptides was equivalent in potency to 12.1 ± 1.6 ng/ml of authentic PDGF. Anti-PDGF antibodies blocked the activity of ELF: 64% of the activity was inhibited by anti-PDGF-BB antibodies; 15% of the activity was inhibited by anti-PDGF-AA antibodies. The presence of several PDGF-like peptides in ELF during the evolution of tissue repair in this model supports the notion that these important mitogens for mesenchymal cells are central to the remodeling process in lung.

De, Swades; Zelazny, E. T.; Souhrada, J. F.; Souhrada, Magdalena

doi: 10.1165/ajrcmb/9.6.645pmid: 8257595

Bronchoalveolar lavage fluid from symptomatic asthmatics contains elevated levels of several proinflammatory interleukins including interleukin-1β (IL-1β). Biologic activities of IL-1β are considered to be critical in the inflammatory process. Since these characteristics include mitogenic properties, we investigated the effect of IL-1β on the proliferation of airway smooth muscle (ASM) cells isolated from guinea pig tracheas. Primary tissue culture of ASM cells was maintained in media containing 0%, 1%, or 10% fetal bovine serum (FBS). Cultures were exposed up to 6 days to a human recombinant IL-1β (20, 40, or 100 pg/ml) in the presence or absence of indomethacin. The proliferation of ASM cells was assessed with two techniques: a direct counting of cells with a hemocytometer and/or with a [3H]-thymidine incorporation, an established marker of DNA synthesis. The evaluation was done daily, up to the sixth day after exposure of cells to different doses of IL-1β. We found that the exposure of ASM cells to human recombinant IL-1β significantly (P < 0.01) increased the number of tracheal myocytes as well as the [3H]-thymidine incorporation into ASM cells. These changes were dependent upon the dose of IL-1β and the concentration of FBS in the cultured medium. The most active proliferation of ASM cells was observed in medium containing 1% FBS, indomethacin (1 µg/ml), and IL-1β (100 pg/ml). The presence of indomethacin in the culture medium was essential to demonstrate the proliferative effect of IL-1β. Pretreatment with specific polyclonal antibodies against platelet-derived growth factor (PDGF-BB homodimer) completely inhibited the IL-1β-induced increase in [3H]-thymidine incorporation by ASM cells. In contrast, the presence of human recombinant PDGF-BB (1.5 ng/ml) in the incubation medium further potentiated the ASM proliferation induced by IL-1β. This potentiation of proliferation was also dose-dependent: PDGF-BB (1.5 and 2.5 ng/ml) alone potentiated (P < 0.01) proliferation of ASM cells. It can be concluded that proinflammatory cytokine, such as IL-1β which is produced in asthma, stimulates proliferation of ASM cells through the PDGF-dependent mechanism. This effect may be critically important in an increase of ASM cell mass observed in asthma.