Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4µM [~300 µg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 µM) and submicromolar (0.8 µM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells.

As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

Laminin-332 alters connexin profile, dye coupling and intercellular Ca2+ waves in ciliated tracheal epithelial cells.

BACKGROUND: Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin alpha3beta3gamma2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC). METHODS: Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca2+ waves. Expression of connexin (Cx) mRNA and proteins were assayed by reverse transcriptase - polymerase chain reaction and immunocytochemistry, respectively. RESULTS: When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular Ca2+ waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca2+waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins. CONCLUSION: Alterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function.