Background Epithelial barrier dysfunction and increased permeability may contribute to antigen sensitization and disease progression in asthma. Claudin-18.1 is the only known lung-specific tight junction protein, but its contribution to airway barrier function or asthma is unclear. Objectives To test the hypotheses that claudin-18 is a determinant of airway epithelial barrier function that is down regulated by IL-13, and that claudin-18 deficiency results in increased aeroantigen sensitization and airway hyperresponsiveness. Methods Claudin-18.1 mRNA levels were measured in airway epithelial brushings from healthy controls and asthmatics. In the asthmatics, claudin-18 levels were compared with a three-gene-mean marker of TH2 inflammation. Airway epithelial permeability changes due to claudin-18 deficiency were measured in 16HBE cells and claudin-18 null mice. The effect of IL-13 on claudin expression was determined in primary human airway epithelial cells and in mice. Airway hyperresponsiveness and serum IgE levels were compared in claudin-18 null and wild type mice following aspergillus sensitization. Results Epithelial brushings from asthmatic subjects (n=67) had significantly lower claudin-18 mRNA levels than healthy controls (n=42). Claudin-18 levels were lowest among TH2-high asthmatics. Loss of claudin-18 was sufficient to impair epithelial barrier function in 16HBE cells and in mouse airways. IL-13 decreased claudin-18 expression in primary human cells and in mice. Claudin-18 null mice had significantly higher serum IgE levels and increased airway responsiveness following intranasal aspergillus sensitization. Conclusions These data support the hypothesis that claudin-18 is an essential contributor to the airway epithelial barrier to aeroantigens. Furthermore, TH2 inflammation suppresses claudin-18 expression, potentially promoting sensitization and airway hyperresponsiveness.

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-β-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Keratinocyte growth factor (KGF) has efficacy in several experimental models of lung injury; however, the mechanisms underlying KGF's protective effect remain incompletely understood. This study was undertaken to determine whether KGF augments barrier function in primary rat alveolar epithelial cells grown in culture, specifically whether KGF alters tight junction function via claudin expression. KGF significantly increased alveolar epithelial barrier function in culture as assessed by transepithelial electrical resistance (TER) and paracellular permeability. Fluorescence-activated cell sorting of freshly isolated type 1 (AT1) and type 2 (AT2) cells followed by quantitative real-time RT-PCR revealed that more than 97% of claudin mRNA transcripts in these cells were for claudins-3, -4, and -18. Using cultured AT2 cells, we then examined the effect of KGF on the protein levels of the claudins with the highest mRNA levels: -3, -4, -5, -7, -12, -15, and -18. KGF did not alter the levels of any of the claudins tested, nor of zona occludens-1 (ZO-1) or occludin. Moreover, localization of claudins-3, -4, -18, and ZO-1 was unchanged. KGF did induce a marked increase in the apical perijunctional F-actin ring. Actin depolymerization with cytochalasin D blocked the KGF-mediated increase in TER without significantly changing TER in control cells. Together, these data support a novel mechanism by which KGF enhances alveolar barrier function, modulation of the actin cytoskeleton. In addition, these data demonstrate the complete claudin expression profile for AT1 and AT2 cells and indicate that claudins-3, -4, and -18 are the primary claudins expressed in these cell types.

Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.

ABSTRACT:The neonatal brain responds differently to hypoxicischemic injury and may be more vulnerable than the mature brain due to a greater susceptibility to oxidative stress. As a measure of oxidative stress, the immature brain should accumulate more hydrogen peroxide (H 2 O 2 ) than the mature brain after a similar hypoxicischemic insult. To test this hypothesis, H 2 O 2 accumulation was measured in postnatal day 7 (P7, neonatal) and P42 (adult) CD1 mouse brain regionally after inducing HI by carotid ligation followed by systemic hypoxia. H 2 O 2 accumulation was quantified at 2, 12, 24, and 120 h after HI using the aminotriazole (AT)-mediated inhibition of catalase spectrophotometric method. Histologic injury was determined by an established scoring system, and infarction volume was determined. P7 and P42 animals were subjected to different durations of hypoxia to create a similar degree of brain injury. Despite similar injury, significantly less H 2 O 2 accumulated in P42 mouse cortex compared with P7 at 2, 12, and 24 h after HI. In addition, less H 2 O 2 accumulated in P42 mouse hippocampus compared with P7 hippocampus at 2 h. Since immature neurons are more vulnerable to the toxic effects of H 2 O 2 than mature neurons, this increased accumulation in the immature brain may explain why the neonatal brain may be more devastated, even after a milder degree of acute hypoxicischemic injury. (Pediatr Res 59: 680-683, 2006)

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Acute respiratory distress syndrome (ARDS) is a devastating disorder characterized by increased alveolar permeability with no effective treatment beyond supportive care. Current mechanisms underlying ARDS focus on alveolar endothelial and epithelial injury caused by products of innate immune cells and platelets. However, the role of adaptive immune cells in ARDS remains largely unknown. Here we report that expansion of antigen-specific αβT helper 17 (αβTH17) cells contribute to ARDS by local secretion of IL-17A, which in turn directly increases alveolar epithelial permeability. Mice with a highly restrictive defect in antigen-specific αβTH17 cells were protected from experimental ARDS induced by a single dose of endotracheal lipopolysaccharide (LPS). Loss of IL-17 receptor C or antibody blockade of IL-17A was similarly protective, further suggesting that IL-17A released by these cells was responsible for this effect. LPS induced a rapid and specific clonal expansion of αβTH17 cells in the lung, as determined by deep sequencing of the hypervariable CD3RβVJ region of the T cell receptor. Our findings could be relevant to ARDS in humans, since we found significant elevation of IL-17A in bronchoalveolar lavage (BAL) fluid from patients with ARDS and recombinant IL-17A directly increased permeability across cultured human alveolar epithelial monolayers. These results reveal a previously unexpected role for adaptive immune responses that increase alveolar permeability in ARDS and suggest that αβTH17 cells and IL-17A could be novel therapeutic targets for this currently untreatable disease.