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.)

Tumor necrosis factor-alpha (TNF-alpha) influences pulmonary vascular endothelial barrier function in vitro. We studied whether recombinant TNF-alpha (rTNF-alpha) regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to human rTNF-alpha (1,000 U/ml) and evaluated for 1) transendothelial [14C]albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the deoxyribonuclease I inhibition assay. rTNF-alpha induced increments in [14C]albumin flux (P < 0.04) and intercellular gap formation at > or = 2-6 h. During this same time, the endothelial F-actin pool decreased (P = 0.0064), with reciprocal increases in the G-actin pool (P < 0.0001). Prior F-actin stabilization with phallicidin protected against the rTNF-alpha-induced increments in G-actin (P < 0.002) as well as changes in barrier function (P < 0.01). Prior protein synthesis inhibition enhanced the rTNF-alpha-induced decrement in F-actin (P < 0.0001), blunted the G-actin increment (P < 0.002), and increased rTNF-alpha-induced changes in endothelial barrier function (P < 0.003). Therefore, rTNF-alpha induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. rTNF-alpha also increased total actin (P < 0.02) and new actin synthesis (P < 0.002), which may be a compensatory endothelial cell response to rTNF-alpha-induced F-actin depolymerization.

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: 1) transendothelial 14C-albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C-albumin flux (P < 0.001) and intercellular gap formation at > or = 2-6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (+/- SE) G-actin (micrograms/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization.

SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of iqjury and induces rounding in adherent endothelial cells in vitro. To determine whether SPARC might influence endothelial permeability, we studied the effect of exogenous SPARC on the movement of 14C-labeled bovine serum albumin across postconfluent bovine pulmonary artery endothelial cells. SPARC increased (P < 0.02) transendothelial albumin flux in a dose-dependent manner at concentrations -0.5 jug/ml. At a fixed dose (15 pg/mi), exposure times > 1 h augmented (P < 0.005) albumin flux by 1.3-to 3.6-fold; this increase was blocked by anti-SPARC antibodies but not by inhibition of protein synthesis. Barrier dysfunction was not associated with loss of cell viability. Monolayers exposed to SPARC exhibited a rounded morphology and intercellular gaps. Prior stabilization ofF-actin with phallicidin protected against the changes in barrier function (P = 0.0001) that were otherwise induced by SPARC. Bovine aortic and retinal microvascular endothelia also responded to SPARC. We propose that SPARC regulates endothelial barrier function through F-actin-dependent changes in cell shape, coincident with the appearance of intercellular gaps, that provide a paracellular pathway for extravasation of macromolecules.The vascular endothelium presents a selective barrier that actively regulates movement of circulating macromolecules and cells into extravascular tissues and compartments (1, 2). Although mechanisms regulating this endothelial barrier are not well understood, a structure-function relationship appears to exist between endothelial cell (EC) shape and barrier function. Specific agonists can induce changes in EC shape coincident with the formation of intercellular gaps, which in turn provide a paracellular pathway for the flux of macromolecules (3-5). Actin organization is postulated to regulate EC shape as well as barrier function (5)(6)(7)(8)(9)(10)(11)(12). Actin filamentdisrupting agents increase endothelial permeability (6), prior stabilization of actin protects against increases in permeability (7-9), and mediators of permeability have been shown to induce cytoskeletal rearrangement (8-12 (20).Assay of Albumin Flux. EC were grown to confluence in 0.5 ml of medium on gelatin-impregnated polycarbonate filters (13-mm diameter, 0.4-,im pore size; Nucleopore) mounted in chemotactic chambers (ADAPS, Dedham, MA) as described (4). These chambers, which served as the upper compartment for the assay chambers, were inserted into wells of 24-well plates, each well containing 1.5 ml of medium and serving as the lower compartment of the assay chamber. 14C-BSA (Sigma; 30.1 pCi/mg of protein; 1 uCi = 37 kBq), the tracer molecule, was prepared in serum-free medium supplemented with BSA (34 mg/ml) to produce a final protein concentration equivalent to medium enriched with 10%o FBS. The baseline barrier function of each monolayer was established by application of an equivalent amount of 14C-BS...

Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 011 1:B4 LPS on movement of '4C-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 2 0.5 ng/ml, and LPS (10 ng/ml) exposures of 2 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 Ag/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 . 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. (J. Clin. Invest. 1994. 93:692-702.)

We studied whether Staphylococcal enterotoxin B (SEB) has direct effects on endothelial cells (EC) in the absence of effector cells or their products. Bovine or human pulmonary artery EC were grown to confluence on filters mounted in chemotaxis chambers. Barrier function was assessed by placing [14C]bovine serum albumin in the chamber and sampling the lower well for 14C activity. SEB exposures induced a significant (P < 0.001) dose- and time-dependent increase in albumin flux across both bovine and human EC monolayers. Albumin flux was temperature dependent, and cycloheximide pretreatment of the monolayers did not block the SEB-induced increase in permeability. Preincubation of SEB with trypsin or anti-SEB antibody significantly (P < 0.0001) reduced the effect, whereas pretreatment with polymyxin B did not. SEB at > or = 10 micrograms/ml significantly (P < 0.03) increased EC injury as measured by 51Cr release in a dose- and time-dependent manner. Herbimycin and genistein, inhibitors of protein tyrosine kinases, each protected against SEB-induced cytotoxicity, barrier dysfunction, and intercellular gap formation. We conclude that SEB perturbs endothelial barrier function and viability in the absence of effector cells or their mediators.

Human recombinant interleukin-1 alpha (rIL-1 alpha) and -beta were studied to determine whether either could alter the permeability of bovine pulmonary artery endothelial cell monolayers. Endothelial cells were grown to confluence on filters mounted in chemotaxis chambers placed in wells. Barrier function of the monolayers was assessed by placing 14C-labeled bovine serum albumin ([14C]BSA) in the upper chamber and sampling the lower well for [14C]BSA. rIL-1 alpha induced a significant (P less than 0.01) dose- and time-dependent increase in transendothelial [14C]BSA flux. rIL-1 alpha exposures as brief as 30 min increased permeability, but the increased albumin transfer could not be demonstrated before 4 h after exposure. Exposures up to 6 h were reversible at 24 h. rIL-1 alpha induced significantly (P less than 0.01) greater increments in [14C]BSA flux than did equivalent exposures to rIL-1 beta. No important differences between bovine and human rIL-1 beta were demonstrated. Increased transendothelial flux could not be ascribed to either endothelial cytotoxicity or growth inhibition. There was no additive or synergistic relationship between rIL-1 alpha and human recombinant tumor necrosis factor-alpha. Our studies suggest that IL-1 alpha and -beta may play a role in the pathogenesis of pulmonary vascular leak.