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Moore, Susan A.; Strieter, Robert M.; Rolfe, Mark W.; Standiford, Theodore J.; Burdick, Marie D.; Kunkel, Steven L.
doi: 10.1165/ajrcmb/6.6.569pmid: 1534243
The alveolar macrophage (AM) is the sentinel immune cell of the distal airspace of the lung. These mononuclear phagocytic cells represent the major host defense against inhaled environmental agents. When activated, the AM has the capacity to release reactive oxygen and arachidonic acid metabolites and produce a number of cytokines, such as interleukin-1 (IL-1). This latter cytokine has pleiotropic effects on a variety of cells and has been implicated as one of the preeminent mediators of acute inflammation. Recently, an IL-1 receptor antagonist (IRAP) has been isolated, purified, and cloned from peripheral blood monocytes (PBM) stimulated with either adherent IgG (adhIgG), lipopolysaccharide (LPS), or phorbol myristate acetate. IRAP acts as a true receptor antagonist without agonist activity. We postulated that the AM would be a significant cellular source of IRAP from the lung. To test this hypothesis, normal human AM were immediately isolated or stimulated in a dose-dependent fashion with either LPS or adhIgG. For comparison, PBM were also isolated and treated in a similar manner. PBM expressed steady-state IRAP mRNA by Northern blot analysis only in response to LPS or adhIgG. In contrast, AM were found to express significant levels of antigenic IRAP by Western blot analysis, immunostaining, and specific ELISA, and express steady-state levels of IRAP mRNA under unstimulated culture conditions. Moreover, LPS or adhIgG failed to induce AM-derived IRAP antigen generation over unstimulated control. In further studies, we demonstrated that interleukin-4 (IL-4), a T lymphocyte-derived cytokine known to block the production of monocyte-derived IL-1, was a potent stimulus for the production of both PBM- and AM-derived steady-state IRAP mRNA and antigen. These results suggested that the AM may be a significant cellular source of IRAP in the lung, and the production of this cytokine antagonist may modulate IL-1-dependent pulmonary inflammation. Furthermore, by modulating the expression of IRAP and IL-1, IL-4 may play an important anti-inflammatory role in the interstitium and airspace of the lung.
Rolfe, Mark W.; Kunkel, Steven L.; Rowens, Brad; Standiford, Theodore J.; Cragoe, Edward J.; Strieter, Robert M.
doi: 10.1165/ajrcmb/6.6.576pmid: 1591007
Various human alveolar macrophage (AM)-derived cytokines in the lungs have been shown to be present under conditions of normal homeostasis as well as during the pathogenesis of inflammation. Although extensive investigation has demonstrated the induction of cytokines from AM, relatively little is known regarding endogenous and exogenous regulation of their production. Several pharmacologic agents, including corticosteroids, cyclooxygenase inhibitors, prostaglandins, and methyl-xanthines have been examined for their role in the modulation of mononuclear phagocyte-derived cytokines. In this study, we examine the role of amiloride for the regulation of AM-derived interleukin (IL)-8, tumor necrosis factor (TNF), IL-6, and IL-1β. Amiloride in concentrations of 10−4 to 10−6 M, concentrations capable of being achieved in the distal airways via nebulization, were shown to inhibit lipopolysaccharide-stimulated, AM-derived IL-8 and TNF in both a time- and dose-dependent fashion. In addition, 5-(N,N-hexamethylene) amiloride hydrochloride, an amiloride analogue with specific sodium channel antiport inhibition, resulted in a similar dose-dependent suppression of lipopolysaccharide-stimulated, AM-derived IL-8 production. Furthermore, the suppressive effect of amiloride appeared to be at the level of mRNA for IL-8, TNF, IL-1β, and IL-6, whereas steady-state levels of β-actin mRNA remained unaltered. These findings would suggest that amiloride has a potentially important modulating influence for the regulation of AM-derived cytokines.
Winter, Michael C.; Carson, Mark R.; Sheldon, Rebecca A.; Shasby, D. Michael
doi: 10.1165/ajrcmb/6.6.583pmid: 1317191
Mastoparan is a tetradecapeptide. Mastoparan added to the apical surface of monolayers of Madin-Darby canine kidney (MDCK) epithelial cells, cultured on micropore filters, activated ion transport and increased the permeability of the paracellular pathway across the monolayers. In monolayers of similar MDCK cells in which the basolateral membrane was permeabilized with Staphylococcus aureus α toxin (Staph. α toxin), the effects of mastoparan on apical membrane ion conductances were dependent on the presence of guanosine triphosphate (GTP). Mastoparan and GTP increased apical membrane chloride conductance more than potassium conductance, with very little change in sodium conductance. In intact monolayers, addition of barium to the apical bath prevented mastoparan activation of ion transport and the increase in paracellular permeability. Increasing bath potassium to 130 mM also reduced ion transport and prevented the increase in paracellular permeability. We hypothesized that these observations could be linked by mastoparan activation of apical chloride and potassium conductances, with consequent decreases in cell volume and resultant increases in paracellular permeability. Addition of 270 mM mannitol to isosmotic media to decrease cell volume decreased MDCK monolayer transepithelial resistance. Addition of mastoparan to monolayers of MDCK cells grown on micropore filters decreased cell volume to the same extent as addition of 270 mM mannitol to isosmotic media. Addition of the potassium channel inhibitor, barium, prevented the decrease in cell volume in response to mastoparan. Mastoparan activates apical membrane chloride and potassium conductances in MDCK cells. The loss of these ions from the cells decreases cell volume, and the decrease in cell volume increases the permeability of the paracellular pathway.
Kalina, Moshe; Mason, Robert J.; Shannon, John M.
doi: 10.1165/ajrcmb/6.6.594pmid: 1591008
The expression of the surfactant-associated proteins in bronchiolar cells remains to be defined. We used in situ hybridization to identify sites of message expression of the surfactant-associated proteins A, B, and C (SP-A, SP-B, and SP-C) in adult and fetal rat lung. The expression of these messages by in situ hybridization was also compared with the localization of SP-A by immunocytochemistry. The localization of SP-A was used to identify type II cells and nonciliated bronchiolar epithelial (Clara) cells in these sections. The cRNA antisense probes for SP-A, SP-B, and SP-C appeared to hybridize over type II cells. Sense probes showed no localization or apparent specific hybridization. Messages for both SP-A and SP-B were also found in nonciliated bronchiolar epithelial (Clara) cells. However, no message for SP-C was observed in these cells. Clara cells from terminal to large bronchioles lacked detectable mRNA for SP-C. Expression of surfactant protein mRNAs was not detectable in type I cells, alveolar macrophages, interstitial cells, or vascular cells. Similarly, in fetal rat lung the messages for SP-A and SP-B but not SP-C were detected in bronchiolar cells. We conclude that rat Clara cells do not express SP-C mRNA, and thus SP-C can be regarded as a specific marker for rat type II cells.
Keller, Andreas; Steinhilber, Wolfram; Schäfer, Klaus P.; Voss, Tilman
doi: 10.1165/ajrcmb/6.6.601pmid: 1591009
We have studied the intracellular transport of the pulmonary surfactant SP-C precursor in vitro and in vivo. In order to monitor the route of the SP-C precursor, we constructed various C-terminally truncated forms of SP-C, which were tagged with a sequence derived from the C-terminus of the human c-myc gene (aa 409-419). Expression of these constructs under the control of the SV40 enhancer and the huMT-II promoter in stably transformed Chinese hamster ovary (CHO) cells revealed that the complete precursor molecule is localized mostly in vesicular structures, probably of lysosomal origin. The truncated precursor lacking the last 22 amino acids at the C-terminus (SP-C/Ctag), however, was restricted to the endoplasmic reticulum as shown by immunofluorescence, using antibodies directed against the tag-sequence, the lysosomal enzyme cathepsin D, the enzyme disulfide isomerase, and the Golgi zone. The intracellular localization was substantiated by subcellular fractionation analysis, suggesting that the last 22 amino acids are necessary for intracellular targeting. Furthermore, Triton X-114 extractions from CHO cells revealed a modification of the SP-C precursor. In vitro translation and pulse-chase experiments in the absence or presence of microsomes showed that the modification occurs post-translationally and in a time-dependent manner. Membrane association studies using an SP-C precursor lacking the mature peptide indicated that the modification is of hydrophobic nature but not a thioester-linked fatty acid.
McCray, Paul B.; Bettencourt, Jeffrey D.; Bastacky, Jacob
doi: 10.1165/ajrcmb/6.6.609pmid: 1317192
We studied lung expiants in submersion organ culture to examine the role of the developing fetal alveolar epithelium in the production of lung fluid. Fourteen-day-gestation fetal rat lungs were grown in a collagen gel matrix supplemented with F-12 media and 10% fetal calf serum. In this model, the lung continues to grow, secrete fluid, and become progressively cystic in morphology. There is gradual thinning of the distal epithelial layer, which is lined by alveolar type II cells and their precursors. After 6 to 8 days in culture, we impaled the cyst walls with a microelectrode and continuously recorded the transepithelial potential (ψt). Stable, baseline transepithelial potentials of −1.1 to −6.2 mV (mean ± SEM = −3.3 ± 0.11 mV, lumen negative, n = 34) were measured in bicarbonate-buffered Ringer's solution, suggesting active electrolyte transport. When bumetanide, an inhibitor of chloride secretion in other systems, was added to the bathing solution, ψt decreased from a baseline of −3.5 ± 0.07 mV (mean ± SEM) to a value of −2.2 ± 0.07 mV, suggesting chloride transport contributes to the voltage (n = 18, P < 0.0005). Isoproterenol hyperpolarized ψt from a baseline of −4.3 ± 1.0 mV to −6.5 ± 1.0 mV (n = 7, P < 0.005). 8-(4-Chlorophenylthio) adenosine 3′:5′cyclic monophosphate (CPT-cAMP) plus isobutylmethylxanthine (IBMX) similarly hyperpolarized ψt from a baseline of −4.6 ± 0.4 mV to −7.3 ± 0.7 mV (n = 11, P < 0.005). Addition of bumetanide after stimulation with isoproterenol or CPT-cAMP/IBMX depolarized ψt. Bath chloride was replaced with gluconate to further test the hypothesis that the change in ψt in response to isoproterenol was due to chloride transport. In Cl− free solution, baseline ψt decreased and there was no significant change in ψt in response to isoproterenol. These findings suggest: (1) the developing fetal alveolar epithelium actively produces lung fluid by a process that is chloride dependent, (2) chloride secretion can be stimulated by a β-adrenergic agonist, and (3) chloride secretion is mediated in part through cAMP-dependent pathways.
Hassoun, P. M.; Shedd, A. L.; Lanzillo, J. J.; Thappa, V.; Landman, M. J.; Fanburg, B. L.
doi: 10.1165/ajrcmb/6.6.617pmid: 1591010
We have previously reported that medium conditioned by hypoxic pulmonary artery endothelial cells (ECCM) contains a factor of small molecular weight that inhibits the growth of pulmonary artery smooth muscle cells (SMC). We postulated that this factor might be a breakdown product of ATP and, therefore, measured the levels of hypoxanthine/xanthine (HX/X) and uric acid (UA) in ECCM and cell lysates from endothelial cells (EC) exposed to hypoxia and normoxia. Although hypoxic and normoxic cell lysates contained no UA and an equal amount of HX/X (2.9 ± 0.3 and 2.9 ± 0.5 µM, respectively), there was a 5-fold increase in the amount of HX/X present in hypoxic compared with normoxic ECCM (3.4 ±0.3 versus 0.6 ± 0.4 µM, respectively; P < 0.001) but no difference in UA levels (5 ± 2 versus 5 ± 1 µM, respectively). In separate experiments, we examined the effects of exogenous HX, X, and UA (doses ranging from 0.1 to 100 µM) on the proliferation of pulmonary and aortic SMC and pulmonary artery EC. Our results indicate that HX, X, and UA inhibit the proliferation of SMC in a dose-dependent manner without causing injury to the cells. The proliferation of EC, on the other hand, was not affected by UA and was significantly inhibited by HX and X only at doses of 100 µM. In conclusion, we have found that significant amounts of HX/X accumulate in hypoxic ECCM and that HX, X, and UA inhibit the proliferation of SMC. The relevance of these findings to conditions where hypoxia prevails is discussed.
Pennington, Daniel W.; Ruoss, Stephen J.; Gold, Warren M.
doi: 10.1165/ajrcmb/6.6.625pmid: 1591011
It is suspected that mast cells play a part in the pathogenesis of fibrotic diseases, but the mediators that might be involved in induction of fibrosis have not been identified. We asked whether cultured dog mast cell lines produced growth factor(s) for fibroblasts. Three mastocytoma cell lines were found to secrete proliferative activity for human, hamster, and rabbit fibroblasts. Both mastocytoma cell-conditioned medium and cell extract served as competence factors for induction of DNA synthesis in confluent mouse Swiss 3T3 fibroblasts. The mitogenic activity in the conditioned medium was stable to heat, acid, and high concentrations of chaotropic agents or organic solvents but was decreased by treatment with proteases or reducing agents. The activity had an apparent molecular mass of 10 kD and did not bind to heparin. Activity eluted in a single peak from reverse-phase HPLC, and retention time differed from that of typical mesenchymal mitogens. We offer the hypothesis that mast cells produce growth factors for fibroblasts, possibly including a novel growth factor, and that this may contribute to pathologic fibrosis.
Bhat, Ganesh B.; Block, Edward R.
doi: 10.1165/ajrcmb/6.6.633pmid: 1591012
To determine whether changes in the lipid dynamics of the plasma membrane bilayer are responsible for hypoxic stimulation of serotonin (5-hydroxytryptamine [5-HT]) transport in pulmonary artery endothelial cells, we solubilized and isolated phospholipid and protein fractions from plasma membrane vesicles derived from endothelial cells exposed to 20% O2 (normoxia) or 0% O2 (hypoxia) for 24 h. Four different combinations of proteoliposomes were prepared by reconstituting (1) normoxic protein and normoxic phospholipid, (2) normoxic protein and hypoxic phospholipid, (3) hypoxic protein and normoxic phospholipid, and (4) hypoxic protein and hypoxic phospholipid. Fluorescence anisotropy of diphenylhexatriene (DPH), a measure of fluidity, and 5-HT transport were evaluated in each of the four groups of reconstituted proteoliposomes. 5-HT transport by the reconstituted proteoliposomes was saturable, linear with protein (5 to 25 µg) and time (15 to 60 s), and optimal with a phospholipid-to-protein ratio of 3:1. There were no significant differences in intravesicular volume, phospholipid-to-protein ratio, and size distribution among the four different groups of proteoliposomes. 5-HT transport was significantly higher and fluorescence anisotropy of DPH was significantly lower in proteoliposomes made from hypoxic phospholipids irrespective of the source of protein. Hypoxia also had a direct effect on the 5-HT transporter since uptake was increased slightly in proteoliposomes from group 3. These results indicate that changes in the plasma membrane phospholipids, and to a much lesser extent changes in the 5-HT transporter, are responsible for increases in the transmembrane transport of 5-HT by hypoxic endothelial cells. Increased fluidity of the plasma membrane lipid bilayer, leading to freer movement of the 5-HT transporter and increased accessibility to substrate, is likely to be the primary mechanism responsible for increased 5-HT transport in hypoxic endothelial cells.
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