ORIGINAL CONTRIBUTIONSOxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton
Introduction
The intestinal mucosa is an essential barrier between body and the environment. However, pathological events may diminish barrier integrity causing an increase in the absorption of toxic luminal antigens (e.g., endotoxin) and leading to intestinal inflammation and oxidative tissue damage [1], [2]. Indeed, an abnormal intestinal barrier has been linked to several gastrointestinal (GI) disorders including inflammatory bowel disease (IBD) [1], [2], [3], [4], [5], [6], [7], [8]. The pathophysiology of mucosal barrier dysfunction in IBD is not completely established but it appears to involve leukocyte infiltration and oxidant-induced disruption of the mucosa. For example, we and others have shown that reactive oxygen metabolites (ROM) such as hydrogen peroxide and hypochlorous acid derived from epithelial cells and leukocytes are prominent in IBD mucosa [9], [10]. Nevertheless, the underlying difficulty in managing this disorder is due to a lack of preventive strategies, which is in turn due to our lack of understanding of the specific mechanisms responsible for its onset and for the perpetuation of inflammation. In particular, the precise mechanism underlying the oxidant-induced loss of intestinal mucosal barrier function in IBD and an effective means of preventing such an abnormality remain elusive.
Rapid resealing of the barrier after oxidant disruption is essential to the preservation of normal mucosal homeostasis. Protective factors should restore normal barrier function that will limit penetration of dangerous substances (e.g., toxic antigens such as endotoxin) into mucosal tissues following oxidant insult, and subsequently limit or control mucosal inflammatory events and tissue damage. Increasing our knowledge of the underlying mechanism of mucosal protection may provide new insights into more effective treatment regimes for IBD.
Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) are the prototypic members of a family of at least seven different peptides that appear to be key constituents in the maintenance, growth, repair, and barrier integrity of GI mucosa [11], [12], [13], [14], [15], [16], [17]. These two polypeptides share 35% homology, bind to the same receptor (epidermal growth factor receptor [EGFR]), and have qualitatively similar spectrums of biological activities [18]. GI mucosal disruption induced by a wide spectrum of damaging insults is rapidly prevented by these growth factors, independent of their known antisecretory properties. Despite the accumulation of a substantial body of knowledge over the past 20 years concerning the protective actions of EGF and TGF-∞ in the GI tract, many issues about their mechanism of action remain unresolved.
We hypothesized that oxidant-induced disruption of mucosal barrier integrity and its prevention by growth factors are mediated by alterations in the cytoskeleton. In recent years, the importance of the microtubule cytoskeleton to the maintenance of normal cellular homeostasis and physiology has been demonstrated. This cytoskeletal component plays an essential role in cellular polarity, structure, and transport functions [19], [20], [21]. Moreover, we have previously shown that the microtubules may be involved in mucosal healing under in vivo as well as under in vitro conditions [22], [23], [24]. Nevertheless, the role of this structural component in the maintenance of mucosal barrier function and in the loss of mucosal barrier induced by oxidants, such as occurs under the pathophysiological conditions of IBD, remains largely unexplored. Furthermore, the role that microtubules might play in the protective actions of EGF or TGF-α on mucosal barrier integrity also remains unknown. A human colonic cell line (Caco-2) was chosen for our studies because these cells resemble intestinal cells in that they have defined apical brush borders, form tight junctions, and more importantly exhibit a highly organized microtubule network [25], [26]. The objective of the current study was to investigate the interrelationships among microtubule/tubulin stability, mucosal barrier integrity, and EGF or TGF-α protection after oxidant-induced insult under in vitro conditions.
Section snippets
Cell culture
Caco-2 cells (a human colonic cell line) were obtained from the ATCC (Rockville, MD, USA) at passage 15. Cells were maintained at 37°C in complete Dulbecco’s minimum essential medium (DMEM). They were split at a ratio of 1:6 upon confluency every 6 d, and set up in either 6 or 24 well plates for experiments, or T-175 asks for propagation. Cells grown for barrier integrity work were split at a ratio of 1:2 and seeded at a density of 200,000 cells/cm2 into 0.4 μm Biocoat Collagen I Cell Culture
Results
Monolayers of Caco-2 cells incubated on the apical side with a range of concentrations (0.01 to 5 mM) of H2O2 or HOCl demonstrated graded disruption of barrier integrity as shown by increased clearance of FSA (Fig 1, H2O2 data shown), which first became statistically significant at a concentration of 0.1 mM (both oxidants). Exposure of monolayers on the basolateral side to these same oxidants caused a similar loss of barrier function Fig. 1, H2O2 shown) Apical oxidant insult was a
Discussion
Oxidant-induced intestinal barrier dysfunction may be involved in the pathophysiology of several disorders, in particular of IBD [9], [10]. The intestinal mucosa constitutes the key barrier against mucosal penetration of a wide array of toxic antigens and molecules such as bacteria and bacterial by-products, food compounds, and other proinflammatory agents [1], [2], [3]. Specifically, the epithelial layer of mucosa acts as a highly selective barrier against the penetration of normally excluded
Acknowledgements
This work was supported in part by a grant from Otsuka America Pharmaceutical Company.
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