No abstract

Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR 2) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR 2 or the use of PAR 2 ؊/؊ mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its ␣2-and ␤-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immunemediated diseases that can be targeted for therapeutic interventions.autoimmune diseases ͉ epidermal growth factor receptor ͉ gut permeability ͉ proteinase-activated receptor 2 ͉ celiac disease

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time-and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization.In IEC6 cells ZOT induced a peak increment of the PKCct isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function. (J. Clin. Invest. 1995. 96:710-720.)

Gram-negative bacterial sepsis remains a common, life-threatening event. The prognosis for patients who develop sepsis-related complications, including the development of acute respiratory distress syndrome (ARDS), remains poor. A common finding among patients and experimental animals with sepsis and ARDS is endothelial injury and/or dysfunction. A component of the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, has been implicated in the pathogenesis of much of the endothelial cell injury and/or dysfunction associated with these disease states. LPS is a highly proinflammatory molecule that elicits a wide array of endothelial responses, including the upregulation of cytokines, adhesion molecules, and tissue factor. In addition to activation, LPS induces endothelial cell death that is apoptotic in nature. This review summarizes the evidence for LPS-induced vascular endothelial injury and examines the molecular signaling pathways that activate and inhibit LPS-induced endothelial apoptosis. Furthermore, the role of apoptotic signaling molecules in mediating LPS-induced activation of endothelial cells will be considered.

Pulmonary leukostasis can be associated with acute lung injury. We studied lung peroxidase activity using myeloperoxidase (MPO) as a granulocyte marker to quantitate pulmonary leukostasis in rabbits. Lungs were homogenized in detergent, freeze-thawed, sonified, and centrifuged, and supernatants were assayed for MPO. Seven extractions were performed, and greater than 80% of cumulative MPO was found in the first three extractions. By use of a three-extraction procedure, the mean lung MPO (delta A X min-1 X g tissue-1) was determined in normal [20.9 +/- 5.2 (SE)], granulocyte-depleted (6.5 +/- 2.0), saline-injected (22.2 +/- 5.6), and pneumococcus (PNC)-challenged (69.7 +/- 10.6) animals. Lung MPO was significantly decreased in granulocyte-depleted compared with normal animals (P less than 0.005) and significantly increased in PNC-challenged compared with saline-injected animals (P less than 0.001). MPO extracted from granulocytes and lungs from normal as well as PNC-challenged animals were all biochemically identical. Lung extract did not inhibit MPO, and no MPO was detected in bronchoalveolar lavage fluid obtained from leukostatic lungs. Lung MPO significantly (P less than 0.01) correlated with intravascular intrapulmonary granulocytes. Determination of lung MPO is a relatively simple quantitative method that can be used to detect pulmonary leukostasis.