After the initial infection with HIV, there is evidence of immune dysfunction despite an apparent normal clinical state. In the context that the lung is a major site affected by opportunistic ...infection during the progression of this immune dysfunction, and that some components of the immune system are activated during early HIV infection, we hypothesized that there may be activation of alveolar macrophages (AM), a key component of the pulmonary host defense system, during the asymptomatic phase of HIV infection. Compared to normals, in HIV-infected individuals the class II MHC molecules DR, DQ, and DP were all expressed more frequently and in greater cell surface density on AM (p < 0.03, all comparisons), and there was increased spontaneous release of superoxide anion (O2-.) by AM (p < 0.002). To gain insight into whether the activation of the AM was an inherent property of the cells or dependent on the in vivo milieu, AM were evaluated after 24 h in culture for O2-. release. In contrast to the findings in fresh AM, after 24 h in culture, O2-. release by HIV AM was not different from normals (p > 0.7), suggesting that these AM had been activated in vivo. To assess whether IFN-gamma could be mediating these effects, mRNA levels of the IP-10 gene (a gene specifically induced by increased concentrations of IFN-gamma) were quantified in AM. Strikingly, the IP-10 gene was expressed only in AM of HIV-seropositive individuals, suggesting the AM had been exposed to IFN-gamma in vivo. Overall, these observations are consistent with the concept that the HIV-seropositive state is associated with activation of AM, in part due to local exposure to IFN-gamma.
Prostaglandin E (PGE), a cyclooxygenase metabolite normally present in high concentrations in respiratory epithelial lining fluid (ELF), is capable of suppressing mesenchymal cell proliferation ...mediated by polypeptide-derived growth factors. Although PGE is normally abundant in respiratory ELF, PGE levels in ELF of individuals with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disorder characterized by intraalveolar mesenchymal cell accumulation and fibrosis, were found to be 50% lower than normal (p less than 0.01): that is, a relative PGE "deficiency" in ELF may enhance intraalveolar mesenchymal cell proliferation in IPF. With this background, it is rational to consider augmenting PGE levels in ELF as a future therapy for IPF. Since systemic administration of PGE is associated with significant adverse effects, in vitro and experimental animal studies were carried out to evaluate whether aerosol PGE administration could augment ELF PGE levels. Greater than 50% of a solution of PGE1 could be placed in droplets less than 3 microns mass median aerodynamic diameter without loss of function. Aerosolization of PGE1 to sheep (n = 14) resulted in a marked augmentation of ELF PGE1 levels (preaerosol 20 +/- 7 nM, 30 min postaerosol 1,150 +/- 210 nM; p less than 0.0 to 0.1). ELF PGE1 levels remained elevated for up to 2 h (p less than 0.05 compared with baseline) and returned to baseline by 3 h (p greater than 0.2). Lung interstitial fluid (lymph) PGE1 levels increased slightly, but to levels far less than ELF levels (preaerosol 7 +/- 1 nM, 30 min postaerosol 13 +/- 2 nM; p less than 0.01), and plasma PGE1 levels did not change (p greater than 0.1).
The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar ...epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be glycosylated differently from kidney and other tissues. These differences appear to be due to organ- or cell-specific posttranslational processing of the beta 1-subunit and may result in altered regulation of sodium pumps in lung compared with other epithelia.
T1alpha is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of ...modulating alveolar epithelial cell (AEC) phenotype in vitro on T1alpha expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1alpha expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1alpha expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1 (DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1alpha expression was quantified by Northern and Western blotting, respectively. Expression of T1alpha progressively increases in AEC grown in MDSF +/- BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1alpha expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1alpha on days 4 and 8. T1alpha expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1alpha expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.
In April 2010, a NIH workshop was convened to discuss the current state of understanding of lung cell plasticity, including the responses of epithelial cells to injury, with the objectives of ...summarizing what is known, what the field needs to know, and how to get there. The proximal stimulus for this workshop is the body of recent evidence suggesting that plasticity is a prominent but incompletely characterized property of lung epithelial cells, and that a focus on understanding this aspect of epithelial cell biology in particular, may be an important window into disease pathobiology and pathogenesis. In addition to their many vital functions in maintaining tissue homeostasis, epithelial cells have emerged as both a central target of disease initiation and an active contributor to disease progression, making a workshop to investigate the role of cell plasticity in lung injury and repair timely. The workshop was organized around four major themes: lung epithelial cell plasticity, signaling control of plasticity, fibroblast plasticity and crosstalk, and translation to human disease. Although this breakdown was recognized to be somewhat artificial, it was felt that this approach would promote cross-fertilization among groups that ordinarily do not communicate and lend itself to the generation of new approaches. The summary reports of individual group discussions below are followed by consensus priorities and recommendations of the workshop participants.
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