Exposure to febrile temperature modifies endothelial cell response to tumor necrosis factor-alpha.

Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.

Human zonulin, a potential modulator of intestinal tight junctions.

Intercellular tight junctions are dynamic structures involved in vectorial transport of water and electrolytes across the intestinal epithelium. Zonula occludens toxin derived from Vibrio cholerae interacts with a specific intestinal epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tight junction permeability. We postulated that this toxin may mimic the effect of a functionally and immunologically related endogenous modulator of intestinal tight junctions. Affinity-purified anti-zonula occludens toxin antibodies and the Ussing chamber assay were used to screen for one or more mammalian zonula occludens toxin analogues in both fetal and adult human intestine. A novel protein, zonulin, was identified that induces tight junction disassembly in non-human primate intestinal epithelia mounted in Ussing chambers. Comparison of amino acids in the active zonula occludens toxin fragment and zonulin permitted the identification of the putative receptor binding domain within the N-terminal region of the two proteins. Zonulin likely plays a pivotal role in tight junction regulation during developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory/autoimmune disorders.

Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease.

We identified zonulin, a novel human protein analogue to the Vibrio cholerae derived Zonula occludens toxin, which induces tight junction disassembly and a subsequent increase in intestinal permeability in non-human primate intestinal epithelia. Zonulin expression was raised in intestinal tissues during the acute phase of coeliac disease, a clinical condition in which tight junctions are opened and permeability is increased.

Direct effects of endotoxin on the endothelium: barrier function and injury.

LPS directly disrupts EC barrier function in vitro and in vivo. This barrier dysfunction has been reported to occur in EC derived from both the macro- and microvasculature of varying species, including humans. Unlike other EC responses, LPS-induced loss of endothelial barrier function is protein-synthesis independent. In fact, protein synthesis inhibition enhances the LPS effect. The lipid A moiety is responsible for LPS-induced activation of the non-CD14-bearing EC, and agents that bind to and neutralize this highly conserved portion of the LPS molecule can crossprotect against EC barrier dysfunction elicited by LPS derived from diverse species of Gram-negative bacteria. Although the presentation of LPS to CD14-bearing cells such as macrophages and monocytes has been well characterized, far less is known about the interactions of LPS with the non-CD14-bearing EC. An EC receptor involved in LPS binding and cellular activation has yet to be identified. The presence of the accessory molecules, LBP and sCD14, are prerequisite to LPS-induced activation of EC at clinically relevant LPS concentrations. As with monocytes and macrophages, the CD14 dependence of LPS-induced endothelial barrier dysfunction can be overcome with high concentrations of LPS. In the absence of LBP and sCD14, a 200,000-fold increase in LPS concentration is required to elicit the same increments in EC monolayer permeability relative to when these accessory molecules are present. Within 30 minutes after LPS exposure, PTK activation is observed. PTK inhibition blocks LPS-induced EC actin depolymerization and endothelial barrier dysfunction which are seen only after a > or = 2-hour stimulus-to-response lag time. Furthermore this LPS-induced actin depolymerization is a prerequisite to opening up the paracellular pathway and loss of monolayer integrity. Interestingly LPS-induced increments in transendothelial 14C-BSA flux and EC detachment parallel caspase-mediated cleavage of ZA and FA proteins that participate in cell-cell and cell-matrix adhesion. The cleavage of the ZA components, beta- and gamma-catenin, does not affect their ability to bind the transmembrane protein, cadherin, or the actin-binding protein, alpha-catenin, suggesting that the linkage of the ZA to the actin cytoskeleton remains intact. LPS-induced cleavage of the FA protein, FAK, leads to dissociation of its catalytic domain from paxillin substrate and decreased paxillin phosphotyrosine content. Caspase inhibition protects against LPS-provoked apoptosis, cleavage of adherens junction proteins, paxillin dephosphorylation, cell-shape changes, and EC detachment. In contrast it fails to block LPS-induced increments in transendothelial 14C-BSA flux. PTK inhibition, which does protect against increased transendothelial 14C-BSA flux, does not block LPS-induced proteolytic cleavage events and only partially inhibits EC detachment. These findings suggest that the EC detachment and endothelial barrier dysfunction elicited by LPS are mediated through distinct pathways (Fig. 6). Much of the work to date has focused on LPS interactions with mCD14-bearing cells, such as monocytes and macrophages, which are central to the inflammatory response elicited by endotoxin. EC, which line the vasculature, are one of the first host tissue barriers to encounter circulating LPS. Because damage to the endothelium is known to contribute to the development of multiorgan failure, including ARDS, understanding LPS-induced EC dysfunction in the setting of Gram-negative septicemia has clear pathophysiologic implications. (ABSTRACT TRUNCATED)

Thrombospondin-1 induces tyrosine phosphorylation of adherens junction proteins and regulates an endothelial paracellular pathway.

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC-EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations >/=1 microg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 microg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, gamma-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.

Bacterial lipopolysaccharide disrupts endothelial monolayer integrity and survival signaling events through caspase cleavage of adherens junction proteins.

Bacterial lipopolysaccharide or endotoxin induces actin reorganization, increased paracellular permeability, and endothelial cell detachment from the underlying extracellular matrix in vitro. We studied the effect of endotoxin on transendothelial albumin flux and detachment of endothelial cells cultured on gelatin-impregnated filters. The endotoxin-induced changes in endothelial barrier function and detachment occurred at doses and times that were compatible with endotoxin-induced apoptosis. Since the actin cytoskeleton and cell-cell and cell-matrix adhesion all participate in the regulation of the paracellular pathway and cell-matrix interactions, we studied whether protein components of the actin-linked adherens junctions were modified in response to endotoxin. Components of cell-cell (beta- and gamma-catenin) and cell-matrix (focal adhesion kinase and p130(Cas)) adherens junctions were cleaved by caspases activated during apoptosis with dose and time requirements that paralleled those seen for barrier dysfunction and detachment. Cleavage of focal adhesion kinase led to its dissociation from the focal adhesion-associated signaling protein, paxillin, resulting in reduced paxillin tyrosine phosphorylation. Inhibition of caspase-mediated cleavage of these proteins protected against detachment but not opening of the paracellular pathway. Therefore, endotoxin-induced disruption of endothelial monolayer integrity and survival signaling events is mediated, in part, through caspase cleavage of adherens junction proteins.

The counteradhesive protein SPARC regulates an endothelial paracellular pathway through protein tyrosine phosphorylation.

SPARC (Secreted Protein Acidic and Rich in Cysteine) regulates the transendothelial flux of macromolecules through a paracellular pathway. We now have demonstrated that SPARC-induced increments in albumin flux across postconfluent endothelial cell (EC) monolayers are mediated, in part, through protein tyrosine phosphorylation. SPARC increased tyrosine phosphorylation of EC proteins up to 12-fold within 1 h. The phosphotyrosine-containing proteins were immunolocalized to the intercellular boundaries. Two substrates for SPARC-induced tyrosine phosphorylation were identified as beta-catenin and paxillin. Inhibition of tyrosine kinases with herbimycin A or genistein reversed the barrier dysfunction induced by SPARC by 71% and 49%, respectively. Herbimycin A also protected against SPARC-induced intercellular gap formation. In contrast, inhibition of tyrosine phosphatases with sodium orthovanadate or phenylarsine oxide enhanced the loss of barrier function associated with SPARC treatment by 120% and 88%, respectively. These data indicate that SPARC influences EC-EC interactions through a tyrosine phosphorylation-dependent signaling pathway.

Endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction.

Bacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived from Limulus antilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of 14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial 14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (> or =10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of > or =10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.

Concepts of fever: recent advances and lingering dogma.

Fever has been a preoccupation of clinicians since medicine's beginning. One might therefore expect that basic concepts relating to this physiological response would be well delineated and that such concepts would be widely known. In fact, only in the past several decades has the febrile response been subjected to scientific scrutiny. As a result of recent scientific investigation, modern concepts have evolved from a perception of fever as nothing more than a rise in core temperature to one in which fever is recognized as a complex physiological response characterized by a cytokine-mediated rise in temperature, as well as by generation of acute-phase reactants and activation of a panoply of physiological, endocrinologic, and immunologic systems. The average clinician appears to have little more than a regrettably rudimentary knowledge of these modern concepts of fever. This symposium summary considers many such concepts that have immediate relevance to the practice of medicine.

SEB is cytotoxic and alters EC barrier function through protein tyrosine phosphorylation in vitro.

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.

Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation.

Bacterial lipopolysaccharide (LPS) induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in vitro. We studied whether LPS-induced increments in 14C-labeled bovine serum albumin (BSA) flux across bovine pulmonary artery endothelial cell (EC) monolayers and actin depolymerization are mediated through protein tyrosine phosphorylation. Lysates from EC exposed to LPS derived from Escherichia coli 0111:B4 (100 ng/ml, 1 h) demonstrated increased tyrosine phosphorylation of the cytoskeletal protein paxillin. Protein tyrosine kinase inhibition, with either herbimycin A (1 microM) or genistein (50 micrograms/ml), protected against LPS-induced actin depolymerization, intercellular gap formation, and increments in [14C]BSA flux. In contrast, inhibition of tyrosine phosphatases with sodium orthovanadate (2.5 microM) or phenylarsine oxide (0.1 microM) enhanced the LPS-induced increments in the G-actin pool and the transendothelial flux of [14C]BSA compared with that seen after exposure to LPS alone. Our data indicate that the influence of LPS on EC actin organization and barrier function is mediated, in part, through a signaling pathway that is dependent on tyrosine phosphorylation.

Bronchoalveolar macrophage CD14 expression: shift between membrane-associated and soluble pools.

The bacterial endotoxin [lipopolysaccharide (LPS)]-binding protein CD14 modulates the host response to LPS, but membrane-associated and soluble forms of the molecule exert different biological effects. CD14 anchored to the mononuclear phagocyte membrane (mCD14) enhances response to LPS. Soluble CD14 (sCD14) may block LPS stimulation of CD14-bearing cells while supporting LPS presentation to non-CD14-bearing cells. We analyzed cell mCD14 and sCD14 expression in simultaneously collected human bronchoalveolar macrophages (BAM) and peripheral blood monocytes (PBM). Expression of mCD14 in freshly isolated BAM was only 9% as high as in PBM. Levels of sCD14 in 48 h in BAM culture supernatants were 19% as high as in PBM cultures. Interleukin (IL)-6 increased CD14 expression in both BAM and PBM but exerted different effects on CD14 distribution in these cell types. IL-6 increased only sCD14 release (2.5-fold) in BAM while increasing only mCD14 expression (2.5-fold) in PBM. IL-4 reduced both mCD14 (> 40%) and sCD14 (> 60%) expression in both cell types. We speculate that the balance between sCD14 and mCD14 expression influences the response to aspirated or inhaled LPS in the bronchoalveolar compartment. Cytokine expression and monocyte recruitment may influence this process by modulating CD14 expression.

Asthma and endotoxin: lipopolysaccharide-binding protein and soluble CD14 in bronchoalveolar compartment.

In allergic asthma, inhalation of antigen provokes an early increase in microvascular permeability with protein extravasation and a delayed recruitment of inflammatory cells. We showed that similar concentrations of lipopolysaccharide (LPS) are present in bronchoalveolar lavage fluid (BALF) in 12 subjects without asthma (86.5 +/- 53.8 pg/ml) and 12 subjects with mild asthma (111 +/- 37.0 pg/ml). These LPS levels are insufficient to stimulate cytokine release without accessory molecules. BALF obtained 24 h after segmental ragweed antigen challenge in 11 asthmatics allergic to ragweed contained increased levels of two LPS accessory molecules compared with preantigen BALF, 158-fold more LPS-binding protein (LBP) 4.83 +/- 2.02 vs. 742 +/- 387 ng/ml; P < 0.03) and 31.6-fold more soluble CD14 (sCD14) (3.45 +/- 1.04 vs. 110 +/- 51.6 ng/ml; P < 0.02). Postantigen BALF enhanced binding of fluorescein-conjugated LPS to CD14-bearing THP-1 cells and supported LPS-induced non-CD14-bearing endothelial cell expression of intercellular adhesion molecule-1 and interleukin-6, indicating functional LBP and sCD14. We suggest that extravasation of LBP and sCD14 into the bronchoalveolar compartment after antigen inhalation may enhance the capacity of inhaled or aspirated LPS to activate an inflammatory cascade that may amplify the inflammatory response to inhaled antigen in some asthmatics.

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

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.

Cerebral mucormycosis associated with intravenous drug use: three case reports and review.

We describe three cases of cerebral mucormycosis in intravenous drug users and review 22 previously reported cases. Involvement of the basal ganglia was demonstrated in all but two cases. Seven of the 10 patients tested for antibodies to the human immunodeficiency virus (HIV) were seronegative. Eight of the 25 patients survived and were discharged from the hospital; for 7 of 10 patients, cultures of brain lesions yielded Rhizopus arrhizus. The radiographic findings varied, and in most cases, no or minimal contrast enhancement was seen in the initial computed tomography scans. Although uncommon, the diagnosis of cerebral mucormycosis should be considered when basal ganglia lesions are present in an intravenous drug user, regardless of previous exposure to HIV.

SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function.

SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of injury 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 > or = 0.5 microgram/ml. At a fixed dose (15 micrograms/ml), exposure times > or = 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 of F-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.

Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

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.

Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: concurrent F-actin depolymerization and new actin synthesis.

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.

TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction.

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.

Interleukin-1 alpha and -beta augment pulmonary artery transendothelial albumin flux in vitro.

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.