Interleukin-1beta decreases expression of the epithelial sodium channel alpha-subunit in alveolar epithelial cells via a p38 MAPK-dependent signaling pathway

Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with protein-rich edema fluid. Interleukin-1beta (IL-1beta) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1beta has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1beta would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1beta on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1beta significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1beta decreased basal and dexamethasone-induced epithelial sodium channel alpha-subunit (alpha ENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1beta on alpha ENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of alpha v beta6 integrin and transforming growth factor-beta. These results indicate that IL-1beta may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in alpha ENaC expression through p38 MAPK-dependent inhibition of alpha ENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.     

Keratinocyte growth factor promotes cell motility during alveolar epithelial repair in vitro

Epithelia play a key role as protective barriers, and mechanisms of repair are crucial for restoring epithelial barrier integrity, especially in the lung. Cell spreading and migration are the first steps of reepithelialization. Keratinocyte growth factor (KGF) plays a key role in lung epithelial repair and protects against various injuries. We hypothesized that KGF may protect the lung not only by inducing proliferation but also by promoting epithelial repair via enhanced epithelial cell migration. In an in vitro wound-healing model, we found that KGF enhanced wound closure by 33%. KGF acted primarily by inducing lamellipodia emission (73.2 +/- 3.9% of KGF-treated cells had lamellipodia vs 61.3 +/- 3.4% of control cells) and increasing their relative surface area (59 +/- 2.7% with KGF vs 48 +/- 2.0% in controls). KGF reduced cytoskeleton stiffness as measured by magnetic twisting cytometry and increased cell motility (5.8 +/- 0.42 microm/h with KGF vs 3.7 +/- 0.41 microm/h in controls). KGF-increased cell motility was associated with increased fibronectin deposition during wound closure and with fibronectin reorganization into fibrils at the rear of the cells. Taken together, our findings strongly suggest that KGF may promote epithelial repair through several mechanisms involved in cell migration.     

Telomerase in alveolar epithelial development and repair

Telomerase expression and activity were examined in the developing lung and in the adult lung during repair after injury. Both whole lung tissue and primary cultures of type 2 alveolar epithelial cells (AEC2) isolated from fetal and adult rodents were analyzed for 1) telomerase expression by immunohistochemistry and 2) telomerase activity with a telomerase repeat amplification protocol. We found that telomerase was expressed in a temporally regulated manner in fetal lung through the late stages of gestation, with peak expression just before birth. Expression persisted for a brief period in neonates, then decreased to nearly undetectable levels by postnatal day 9. Telomerase expression and activity were reinduced in normally quiescent adult lung by in vivo treatment with hyperoxia. In populations of AEC2 isolated from both developing and repairing lungs, telomerase expression and activity showed a strong correlation with the proliferation marker proliferating cell nuclear antigen. It has been suggested that telomerase expression and activity are hallmarks of stem or progenitor cells. Our observations suggest that a telomerase-positive subpopulation is present within the general AEC2 population. Telomerase may act as a marker for the proliferative status of this subpopulation.     

TGF-beta1 induces alveolar epithelial to mesenchymal transition in vitro

The aim of this study was to investigate whether transforming growth factor-beta1 (TGF-beta1) could induce alveolar epithelial to mesenchymal transition (EMT) in vitro. Alveolar epithelial cells (AECs) from SD rats were isolated by elastase cell dispersion and IgG panning. Expression of alpha-smooth muscle actin (alpha-SMA) was assayed using Western blotting and immunostaining analysis. Morphological changes, the markers of epithelial cell (E-cadherin), and stress fiber by actin reorganization were detected by an indirect immunostaining. The contents of collagen I were determined by spectrophotometry. The levels of endogenous TGF-beta1 were measured with ELISA. Incubation of AECs with TGF-beta1 (0.1 approximately 10 ng/mL) induced abundant expression of alpha-SMA protein, and alpha-SMA expression in AECs reached a plateau when TGF-beta1 was > 3 ng/mL. Furthermore, we found that TGF-beta1 (3 ng/mL) exposure of AECs induced an authentic EMT characterized by abundant expression of alpha-smooth muscle actin, transformation of myofibroblastic morphology, increased formation of stress fiber by actin reorganization, and loss of epithelial marker E-cadherin. Meanwhile, significant increase in the levels of collagen I from 32.0 +/- 6.6 mg/g in control to 98 +/- 10.8 mg/g in TGF-beta1-treated group was found over a 72 h incubation period. Moreover, following stimulated by TGF-beta1 (3 ng/mL), a marked and time-dependent increase in endogenous TGF-beta1 released from AECs was observed. At time points 72 h, TGF-beta1 release mounted to 3451 pg/ml, which was much enough to induce EMT in vitro. These results demonstrated that AECs, under stimulation of TGF-beta1, underwent a conversion process into myofibroblasts in vitro.     

Lipopolysaccharide induces functional ICAM-1 expression in rat alveolar epithelial cells in vitro

Lipopolysaccharide (LPS)-induced lung inflammation is known to increase pulmonary intercellular adhesion molecule-1 (ICAM-1) expression. In the present study, L2 cells, a cell line of alveolar epithelial cells, were stimulated with LPS, and ICAM-1 expression was studied. ICAM-1 protein on L2 cells peaked at 6 (38% increase; P < 0.01) and 10 (48% increase; P < 0.001) h after stimulation with Escherichia coli and Pseudomonas aeruginosa LPS, respectively. ICAM-1 mRNA expression was markedly increased, with a peak at 2-4 (E. coli) and 4-6 (P. aeruginosa) h. Adherence assays of neutrophils to LPS-stimulated L2 cells showed a threefold increase in adherence (P < 0.001). Pretreatment of the neutrophils with anti-lymphocyte function-associated antigen-1 and anti-Mac-1 antibodies reduced adherence by 54% (P < 0.001). Analysis of immunofluorescence staining for ICAM-1 showed an exclusive apical expression of ICAM-1. These results indicate that LPS upregulates functional active ICAM-1 on the apical part of the membrane in rat pneumocytes.     

Interleukin-1beta (IL-1beta) induces a crosstalk between cAMP and ceramide signaling pathways in thyroid epithelial cells

Interleukin-1 beta (IL-1beta) is an important regulator of the thyroid cell function. This cytokine has been largely described to trigger an important biological signaling cascade: the sphingomyelin/ceramide pathway. In this report, we show that IL-1beta induces the transient activation of a neutral sphingomyelinase in porcine thyroid cells. Moreover, IL-1beta and ceramides are demonstrated to inhibit the TSH-induced cAMP production via the implication of alphaGi subunit of the adenylyl cyclase system. This crosstalk between cAMP and ceramide pathways constitutes a preponderant process in the TSH-controlled differentiation state of thyrocytes. All these results argue for the involvement of ceramides and IL-1beta in the thyroid function regulation, leading to a cell dedifferentiated state.     

Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms

Background

Mesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. Furthermore, MSC can ameliorate pulmonary fibrosis in animal models although mechanisms of action remain unclear. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration.

Methods

To investigate the paracrine role of human MSC (hMSC) on pulmonary epithelial repair, hMSC-conditioned media (CM) and a selected cohort of hMSC-secretory proteins (identified by LC-MS/MS mass spectrometry) were tested on human type II alveolar epithelial cell line A549 cells (AEC) and primary human small airway epithelial cells (SAEC) using an in vitro scratch wound repair model. A 3D direct-contact wound repair model was further developed to assess the migratory properties of hMSC.

Results

We demonstrate that MSC-CM facilitates AEC and SAEC wound repair in serum-dependent and –independent manners respectively via stimulation of cell migration. We also show that the hMSC secretome contains an array of proteins including Fibronectin, Lumican, Periostin, and IGFBP-7; each capable of influencing AEC and SAEC migration and wound repair stimulation. In addition, hMSC also show a strong migratory response to AEC injury as, supported by the observation of rapid and effective AEC wound gap closure by hMSC in the 3D model.

Conclusion

These findings support the notion for clinical application of hMSCs and/or their secretory factors as a pharmacoregenerative modality for the treatment of idiopathic pulmonary fibrosis (IPF) and other fibrotic lung disorders.     

Rapid expression of IL-1beta by intestinal epithelial cells in vitro

Intestinal epithelial cells have been shown to produce IL-1beta in vivo. This gene expression is rapid and precedes most determinants of inflammation, suggesting a pivotal role for IL-1beta in the early events leading to inflammation. To better understand the mechanisms leading to this IL-1beta production, we have developed an in vitro model system employing a nontransformed intestinal epithelial cell line that does not constitutively express IL-1beta. Following detachment, these cells rapidly expressed IL-1beta mRNA. This expression was enhanced, but not induced, by LPS. IL-1beta protein was detected by immunoprecipitation in the culture medium from passaged IEC-18 but not intracellularly, suggesting an efficient secretion of the molecule following induction. Interestingly, culture supernatants from passaged cells were without IL-1 bioactivity, suggesting the presence of an inhibitor as well. RT-PCR and Western blot analysis showed expression of IL-1RII by IEC-18 following detachment, possibly explaining the observed lack of bioactivity. These results indicate a novel pathway for IL-1beta production and suggest that proinflammatory effects of IEC-derived IL-1 may be modulated by the simultaneous production of IL-1 antagonists.     

Interleukin-1beta and depressive states

Administration of Interleukin-1 beta (IL-1 beta) in pyrogenic and subpyrogenic doses induced a depression of social and exploratory behaviour in rats. A reduction in locomotor activity only occurred with pyrogenic doses of the IL-1 beta. The low dose induced the reduction whereas the high dose the increase of anxiety in elevated plus maze. The opposite effects of two doses of IL-1 beta were observed also in a test with saccharine.     

Transforming growth factor-beta1 regulates basement membrane formation by alveolar epithelial cells in vitro

Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-beta1. Corresponding to lamina densa formation, immunohistochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells. Synthesis of these basement membrane constituents was significantly enhanced by TGF-beta1 in a dose-dependent manner. On the other hand, TGF-beta1 did not affect the synthesis of extracellular matrix-regulatory enzymes and their inhibitors, such as type II transglutaminase, matrix metalloproteinase-2, plasminogen activator inhibitor-1, or tissue inhibitor of matrix metalloproteinase-1. These results indicate that basement membrane formation in the presence of 1.0 ng/ml TGF-beta1 is attributable to enhanced synthesis of basement membrane constituents. However, formation of a continuous basement membrane was inhibited at a TGF-beta1 concentration of 5.0 ng/ml. Synthesis of the basement membrane constituents was further enhanced at this concentration and the extracellular matrix-regulatory enzymes remained unchanged. The deposits of cellular fibronectin and type I collagen beneath SV40-T2 cells were significantly augmented. Thus excessive production of interstitial extracellular matrix components appears to obstruct the integration of basement membrane constituents into a continuous architecture. These results indicate that the basement membrane formation by SV40-T2 cells is achieved at the optimal TGF-beta1 concentration.     

Interleukin-1beta induces macrophage inflammatory protein-1beta expression in human hepatocytes

The investigation of factors that regulate expression of CC-chemokines, the important mediators in immune responses and inflammation processes, has an important significance in understanding the immunopathogenesis of liver diseases. We examined the role of interleukin-1beta (IL-1beta), a multifunctional cytokine, in regulating the expression of macrophage inflammatory protein (MIP)-1beta in human hepatocytes (Huh7 and HepG2). IL-1beta significantly enhanced MIP-1beta expression in these cells at both the mRNA and protein levels. Cytokine-enriched supernatants from monocyte-derived macrophage (MDM) cultures also induced MIP-1beta expression. IL-1beta is responsible for MDM supernatant-mediated up-regulation of MIP-1beta since the antibody to IL-1beta abolished MDM supernatant action. Investigation of the mechanism involved in MIP-1beta induction by IL-1beta showed that IL-1beta activated the nuclear factor kappa B (NF-kappaB) promoter in Huh7 cells. In addition, caffeic acid phenethyl ester (CAPE), a specific inhibitor of the activation of NF-kappaB, not only abolished IL-1beta-mediated NF-kappaB promoter activation, but also blocked IL-1beta-induced MIP-1beta expression. These observations suggest that IL-1beta-mediated up-regulation of MIP-1beta production in the hepatic cells may contribute a critical mechanism for continuous recruitment of inflammatory cell to liver and maintenance of inflammation.     

Involvement of KATP and KvLQT1 K+ channels in EGF-stimulated alveolar epithelial cell repair processes

Several respiratory diseases are associated with extensive damage of lung epithelia, and the regulatory mechanisms involved in their regeneration are not clearly defined. Growth factors released by epithelial cells or fibroblasts from injured lungs are important regulators of alveolar repair by stimulating cell motility, proliferation, and differentiation. In addition, K(+) channels regulate cell proliferation/migration and are coupled with growth factor signaling in several tissues. We decided to explore the hypothesis, never investigated before, that K(+) could play a prominent role in alveolar repair. We employed a model of mechanical wounding of rat alveolar type II epithelia, in primary culture, to study their response to injury. Wound healing was suppressed by one-half upon epidermal growth factor (EGF) titration with EGF-antibody (Ab) or erbB1/erbB2 tyrosine-kinase inhibition with AG-1478/AG-825. The addition of exogenous EGF slightly stimulated the alveolar wound healing and enhanced, by up to five times, alveolar cell migration measured in a Boyden-type chamber. Conditioned medium collected from injured alveolar monolayers also stimulated cell migration; this effect was abolished in the presence of EGF-Ab. The impact of K(+) channel modulators was examined in basal and EGF-stimulated conditions. Wound healing was stimulated by pinacidil, an ATP-dependent K(+) channel (K(ATP)) activator, which also increased cell migration, by twofold, in basal conditions and potentiated the stimulatory effect of EGF. K(ATP) or KvLQT1 inhibitors (glibenclamide, clofilium) reduced EGF-stimulated wound healing, cell migration, and proliferation. Finally, EGF stimulated K(ATP) and KvLQT1 currents and channel expression. In summary, stimulation of K(+) channels through autocrine activation of EGF receptors could play a crucial role in lung epithelia repair processes.     

H(2)O(2) inhibits alveolar epithelial wound repair in vitro by induction of apoptosis

Reactive oxygen species (ROS) are released into the alveolar space and contribute to alveolar epithelial damage in patients with acute lung injury. However, the role of ROS in alveolar repair is not known. We studied the effect of ROS in our in vitro wound healing model using either human A549 alveolar epithelial cells or primary distal lung epithelial cells. We found that H(2)O(2) inhibited alveolar epithelial repair in a concentration-dependent manner. At similar concentrations, H(2)O(2) also induced apoptosis, an effect seen particularly at the edge of the wound, leading us to hypothesize that apoptosis contributes to H(2)O(2)-induced inhibition of wound repair. To learn the role of apoptosis, we blocked caspases with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). In the presence of H(2)O(2), zVAD inhibited apoptosis, particularly at the wound edge and, most importantly, maintained alveolar epithelial wound repair. In H(2)O(2)-exposed cells, zVAD also maintained cell viability as judged by improved cell spreading and/or migration at the wound edge and by a more normal mitochondrial potential difference compared with cells not treated with zVAD. In conclusion, H(2)O(2) inhibits alveolar epithelial wound repair in large part by induction of apoptosis. Inhibition of apoptosis can maintain wound repair and cell viability in the face of ROS. Inhibiting apoptosis may be a promising new approach to improve repair of the alveolar epithelium in patients with acute lung injury.     

Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1

Background

Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor β1 (TGFβ1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFβ1 may become activated in the alveolar space of the alcoholic lung.

Methods

Primary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague–Dawley rats. Expression of TGFβ1 and the epithelial integrin αvβ6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran.

Results

TGFβ1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFβ1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFβ1 antibody treatment, indicating the presence of bioactive TGFβ1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFβ1 and anti-αvβ6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFβ1 in vitro, increased the expression of αvβ6 integrin, which is known to activate TGFβ1, in alveolar epithelial cells.

Conclusions

Taken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFβ1 at points of cell-cell contact.     

Neopterin inhibits ATP-induced calcium release in alveolar epithelial cells in vitro

BACKGROUND: Serum neopterin concentrations rise during activation of the cellular immune system. It is suggested that neopterin interacts with cellular redox mechanisms. This induces oxidative stress, which inhibits intracellular Ca2+ transients in various cell types. In type II alveolar epithelial cells, Ca2+ increase is considered involved in the exocytosis of surfactants. This exocytosis is disturbed during inflammation. AIMS: To clarify whether neopterin affects adenosine triphosphate (ATP)-induced Ca2+ transients in an alveolar epithelial cell line (L2). METHODS: Ca2+ transients were detected as fura-2 fluorescence by an image analysis system. RESULTS: Cells were exposed for 100 sec to ATP (1 microM, repeated four times). The first application of ATP induced an increase of the fluorescence ratio by approximately 100%, while the following stimulations resulted in smaller transients. In a second set of experiments, L2 cells were exposed to ATP or ATP + neopterin (100 nM), alternately. Simultaneous application of neopterin inhibited Ca2+ transients almost completely. CONCLUSIONS: Inhibition of Ca2+ transients by neopterin may lead to suppressed exocytosis of surfactant proteins in alveolar epithelial cells. This might contribute to the deterioration of pulmonary functions in the course of inflammatory processes.     

Keratinocyte growth factor can enhance alveolar epithelial repair by nonmitogenic mechanisms

Pretreatment with keratinocyte growth factor (KGF) ameliorates experimentally induced acute lung injury in rats. Although alveolar epithelial type II cell hyperplasia probably contributes, the mechanisms underlying KGF's protective effect remain incompletely described. Therefore, we tested the hypothesis that KGF given to rats in vivo would enhance alveolar epithelial repair in vitro by nonproliferative mechanisms. After intratracheal instillation (48 h) of KGF (5 mg/kg), alveolar epithelial type II cells were isolated for in vitro alveolar epithelial repair studies. KGF-treated cells had markedly increased epithelial repair (96 +/- 22%) compared with control cells (P < 0.001). KGF-treated cells had increased cell spreading and migration at the wound edge but no increase in in vitro proliferation compared with control cells. KGF-treated cells were more adherent to extracellular matrix proteins and polystyrene. Inhibition of the epidermal growth factor (EGF) receptor with tyrosine kinase inhibitors abolished the KGF effect on epithelial repair. In conclusion, in vivo administration of KGF augments the epithelial repair rate of alveolar epithelial cells by altering cell adherence, spreading, and migration and through stimulation of the EGF receptor.