Search

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 PKC-alpha 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.

Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.

Abnormal granulocyte chemotaxis has been described in chronic hemodialysis patients. In this study, sera from 53 hemodialysis patients were tested for chemotactic inhibitory activity by a modified Boyden technique. Chemotactic inhibitory activity, defined as >20% inhibition of normal granulocyte chemotaxis, was found in 45% of patients. Only sera from patients having undergone >3 mo hemodialysis displayed chemotactic inhibitory activity and retained this inhibitory activity when retested 9 mo later. Four of five patients who had initially undergone <3 mo hemodialysis and lacked serum chemotactic inhibitory activity developed inhibitory activity when tested 9 mo later. Clinical evaluation of patients with serum chemotactic inhibitory activity showed that these patients did not have a significantly increased incidence of infection, although a trend toward decreased mortality during the time of study was observed (P = 0.0721). Serum chemotactic inhibitory activity was heat stable at 56 degrees C for 30 min and concentration dependent. The major inhibitory component was found to have a sedimentation coefficient of 4S by sucrose density gradient centrifugation. The chemotactic inhibitory activity was not precipitated by 30% ammonium sulfate, but was partially precipitated by 50% ammonium sulfate. Inhibitory sera effectively suppressed neutrophil migration in response to chemotactic C5 fragment and Escherichia coli derived chemotactic factor but was least effective in a system mediated by casein. Furthermore, normal neutrophils preincubated in hemodialysis patient sera displayed normal chemotactic responsiveness indicating a lack of cell-directed inhibition. Serum fractions that contained the inhibitor were found to directly act on the chemotactic C5 fragment, reducing its chemotactic activity. This study indicates that a circulating 4S, heat-stable, factor-directed inhibitor of granulocyte chemotaxis is present in the sera of many hemodialysis patients and probably results from the hemodialysis procedure.