PMX464, a thiol-reactive quinol and putative thioredoxin inhibitor, inhibits NF-kappaB-dependent proinflammatory activation of alveolar epithelial cells.

BACKGROUND AND PURPOSE: Subtle changes in the intracellular reduction-oxidation (redox) state can modulate nuclear factor-kappaB (NF-kappaB) activity. Thioredoxin-1 (Trx) is a small, ubiquitous, redox-active thiol (-SH) protein that, with thioredoxin reductase-1 (TrxR), modifies the redox status of NF-kappaB pathway components. PMX464 is a novel thiol-reactive quinol thought to inhibit the Trx/TrxR system. The aim of this work was to investigate whether PMX464 inhibited NF-kappaB-mediated proinflammatory activation of human type II alveolar epithelial cells (A549). EXPERIMENTAL APPROACH: Intercellular adhesion molecule-1 (ICAM-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and CXCL8, NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity were assessed in A549 cells stimulated with IL-1beta with or without PMX464 pretreatment. Effects of PMX464 on ICAM-1 expression in human lung microvascular endothelial cells (HLMVEC) were also investigated. For comparison, selected measurements (ICAM-1 and IkappaB-alpha phospho-IkappaB-alpha) were made on A549 cells after RNA interference-mediated silencing (siRNA) of Trx. KEY RESULTS: PMX464 reduced ICAM-1, GM-CSF and CXCL8 expression in IL-1beta-stimulated A549 cells and ICAM-1 in HLMVEC. PMX464 inhibited IL-1beta-induced NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit and factors involved in NF-kappaB activation; specifically, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity in A549. By contrast, Trx siRNA did not alter ICAM-1 expression or IkappaBalpha degradation/phosphorylation in IL-1beta-stimulated A549 cells. CONCLUSION AND IMPLICATIONS: PMX464 inhibits a proinflammatory response in A549 cells targeting the NFkappaB pathway above IKK. The lack of effect with Trx siRNA suggests that PMX464 acts on thiol proteins, in addition to Trx, to elicit anti-inflammatory responses in lung epithelial cells.

Extracellular thioredoxin levels are increased in patients with acute lung injury.

BACKGROUND: Acute lung injury (ALI) and its extreme manifestation the acute respiratory distress syndrome (ARDS) complicate a wide variety of serious medical and surgical conditions. Thioredoxin is a small ubiquitous thiol protein with redox/inflammation modulatory properties relevant to the pathogenesis of ALI. We therefore investigated whether thioredoxin is raised extracellulary in patients with ALI and whether the extent of any increase is dependent upon the nature of the precipitating insult. METHODS: Bronchoalveolar lavage (BAL) fluid and plasma samples were collected from patients with ALI (n=30) and healthy controls (n=18, plasma; n=14, BAL fluid). Lung tissue was harvested from a separate group of patients and controls (n=10). Thioredoxin was measured by ELISA in fluids and by immunohistochemistry in tissue. Interleukin (IL)-8 levels were determined by ELISA. Disease severity was assessed as APACHE II and SOFA scores. RESULTS: BAL fluid levels of thioredoxin were higher in patients with ALI than in controls (median 61.6 ng/ml (IQR 34.9-132.9) v 16.0 ng/ml (IQR 8.9-25.1), p<0.0001); plasma levels were also significantly higher. When compared with controls, sections of wax embedded lung tissue from patients with ALI showed greater positive staining for thioredoxin in alveolar macrophages and type II epithelial cells. BAL fluid levels of thioredoxin correlated with IL-8 levels in BAL fluid but not with severity of illness scores or mortality. BAL fluid levels of thioredoxin, IL-8, and neutrophils were significantly greater in patients with ALI of pulmonary origin. CONCLUSIONS: Extracellular thioredoxin levels are raised in patients with ALI, particularly of pulmonary origin, and have a significant positive association with IL-8. Extracellular thioredoxin levels could provide a useful indication of inflammation in ALI.

Lipoteichoic acid inhibits lipopolysaccharide-induced adhesion molecule expression and IL-8 release in human lung microvascular endothelial cells.

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-alpha, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3-30 microg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 microg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-alpha on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 microg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to approximately 2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.

Cytokine-induced airway epithelial ICAM-1 upregulation: quantification by high-resolution scanning and transmission electron microscopy.

The aim of this study was to investigate the variation of intercellular adhesion molecule (ICAM)-1 which occurs between individual airway epithelial cells of distinct phenotype. 16HBE14o- (16HBE) and BEAS-2B cell lines and human airway explants were cultured with medium alone or a mixture of tumour necrosis factor (TNF)-alpha (10 ng x mL(-1)) and interferon (IFN)-gamma (40 ng x mL(-1)) before being immunogold-labelled and examined quantitatively using sensitive high-resolution electron microscopic techniques. By enzyme-linked immunosorbent assay there was a 1.6-fold increase of ICAM-1 in the BEAS-2B cells following the cytokine mix which was not apparent in the 16HBE cells. However, high-resolution scanning electron microscopy demonstrated that an upregulation had occurred; median and ranges for gold particle number per 10 microm2 cell surface were: 7.9 (0-40) for nonstimulated and 19.1 (0-60 for stimulated) (p<0.01, Mann-Whitney U-test). The value for the nonstimulated BEAS-2B cells was 24.2 (0-60), 3-times higher that the constitutive expression in the 16HBE cells (p<0.01), whereas following stimulation, it was 68.5 (20-130) (p<0.01). Values for explant epithelial outgrowths were similar to the 16HBE cells. Immunohistochemistry of the explanted mucosa showed both constitutive and upregulated expression of epithelial ICAM-1 associated with basal and indeterminate cells rather than with ciliated or goblet cells. These results using high-resolution techniques indicate that there is marked cell-to-cell variation in cellular adhesion molecule expression and that it is the basal cells and less well differentiated (indeterminate) epithelial cells which are likely to play key roles in leukocyte retention via intercellular adhesion molecule-1.

A CD18/ICAM-1-dependent pathway mediates eosinophil adhesion to human bronchial epithelial cells.

Eosinophil adhesion to airway epithelium is believed to facilitate eosinophil accumulation and retention in asthmatic airways. Monoclonal antibodies (mAb) against intercellular adhesion molecule-1 (ICAM-1) and its CD18 leukocyte integrin ligands have been shown to inhibit airway eosinophilia in animal models of asthma, although the role of this pathway in eosinophil-epithelial adhesion is not fully understood. To investigate the role in vitro of CD18 and ICAM-1, we measured adhesion of fluorescently labeled human eosinophils to normal human bronchial epithelial cell (NHBEC) monolayers pretreated for 24 h with culture medium (low constitutive ICAM-1) or tumor necrosis factor-alpha (TNF-alpha; 1 ng/ml) and interferon-gamma (IFN-gamma) (10 ng/ml; increased ICAM-1). Stimulation of eosinophils with C5a (10(-7) M) increased adhesion measured at 30 min to unactivated NHBEC from 11.4 +/- 0.7 to 15.5 +/- 0.4% (n = 4), and this increase was CD18/ICAM-1-independent, whereas phorbolmyristate acetate (PMA) (10(-8) M)-induced adhesion (20.7 +/- 1.7%) was abolished by anti-CD18 and reduced by anti-ICAM-1. In contrast, C5a- and PMA-induced adhesion to TNF-alpha/IFN-gamma-activated NHBEC (increased from 11.1 +/- 1.3% to 21.9 +/- 1.0% and 27.6 +/- 1.9%, respectively) was CD18- and ICAM-1-dependent. Eotaxin, but not regulated on activation normal T cells expressed and secreted, macrophage inflammatory protein-1, formyl methionyl leucyl phenylalanine, leukotriene B4 or platelet-activating factor, also induced CD18/ICAM-1-dependent adhesion to activated NHBEC. In the absence of added chemoattractants, eosinophil adhesion to NHBEC increased with time and, at 120 min, was significantly greater (P < 0.01) to activated NHBEC (37.3 +/- 2.4%, n = 5) than to unactivated monolayers (24.3 +/- 1.9%); mAb against CD18 or ICAM-1 abolished increased, but not basal, adhesion. These results suggest that CD18/ICAM-1 mediated eosinophil adhesion to activated NHBEC but that adhesion to resting NHBEC was largely independent of this pathway.

Cyclo-oxygenase-2 regulates inducible ICAM-1 and VCAM-1 expression in human vascular smooth muscle cells.

Prostaglandins are well characterised inflammatory mediators, whose formation is regulated by constitutive (COX-1) or inducible (COX-2) isoforms of cyclo-oxygenase. We have previously demonstrated that IL-1 beta causes an induction of COX-2 in human vascular smooth muscle (1). This present study investigates the ability of different cytokines to induce ICAM-1 and VCAM-1 on human vascular smooth muscle, and tests whether co-induced COX-2 would regulate their expression. IL-1 beta induced ICAM-1, and COX activity, while it had no affect on VCAM-1. Conversely, IL-4 induced VCAM-1, while it had no effect on PGE2 release or ICAM-1 expression. Inhibition of IL-1 beta induced COX-2 and elevated ICAM-1 expression, an effect reversed by exogenous PGE2. Furthermore, IL-1 beta inhibited IL-4 induced VCAM-1 expression, which was also reversed by COX-2 inhibition. These results demonstrate that COX-2 limits adhesion molecule expression on human vascular smooth muscle cells and suggest that COX-2 can play a protective role in cardiovascular and inflammatory diseases.

Modulation of cell adhesion molecule expression and function on human lung microvascular endothelial cells by inhibition of phosphodiesterases 3 and 4.

1. Expression of cell adhesion molecules (CAM) on the lung microvascular endothelium is believed to play a key role in the recruitment of leukocytes in pulmonary inflammation. Moreover, regulation of CAM expression may be an important mechanism through which this inflammation may be controlled. Experimental evidence has suggested that combined phosphodiesterase (PDE) 3 and 4 inhibitors increase cyclic AMP levels within cells greater than inhibition of either isoenzyme alone. In the present study we assessed the effect of combinations of rolipram (PDE4 inhibitor), ORG 9935 (PDE3 inhibitor) and salbutamol (beta-agonist) on CAM expression and neutrophil or eosinophil adhesion to human lung microvascular endothelial cells (HLMVEC). 2. Tumour necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin expression were measured on HLMVEC monolayers at 6 h by a specific ELISA technique in the presence of different combinations of medium, rolipram, ORG 9935 and salbutamol. 3. Rolipram in combination with salbutamol, but neither agent alone, inhibited TNF-alpha-induced E-selectin expression, whilst ICAM-1 and VCAM-1 expression were not affected. ORG 9935 had no significant effect on CAM expression alone. However, in combination with rolipram a syngergistic inhibition of VCAM-1 and E-selectin, but not ICAM-1, expression was observed. No further inhibition was seen in the additional presence of salbutamol. 4. Neutrophil adhesion to TNF-alpha-stimulated (6 h) HLMVEC was mainly E-selectin dependent in this model, as ENA2 an anti-E-selectin monoclonal antibody (mAb) abrogated neutrophil adhesion. Eosinophil adhesion was E-selectin-, ICAM-1- and VCAM-1-dependent, as assessed by the inhibitory activity of ENA2 and the ability of a mAb to the ICAM-1 ligand, CD18, and a mAb to the VCAM-1 ligand, VLA4, to attenuate adhesion. 5. Rolipram in the presence of salbutamol or ORG 9935 significantly inhibited neutrophil adherence to TNF-alpha-stimulated HLMVEC. Eosinophil adherence to monolayers was inhibited only when HLMVEC were activated in the presence of rolipram and ORG 9935. 6. Collectively, the findings presented in this manuscript suggest that inhibition of PDE4 with appropriate activation of adenylate cyclase is sufficient to inhibit induction of E-selectin expression on HLMVEC to a level that has functional consequences for neutrophil adhesion. In contrast, combined inhibition of PDE3 and 4 isoenzymes is necessary to inhibit VCAM-1 and to have inhibitory effects on eosinophil adhesion to activated HLMVEC. Upregulation of ICAM-1 expression on HLMVEC does not appear to be modulated by PDE3 and 4 inhibition. These data may have implications for the use of selective PDE4 inhibitors in lung inflammation.

Interleukin-4 and lipopolysaccharide synergize to induce vascular cell adhesion molecule-1 expression in human lung microvascular endothelial cells.

Recent studies suggest that increased vascular cell adhesion molecule-1 (VCAM-1) expression on vascular endothelium in bronchial mucosa biopsies correlates with interleukin-4 (IL-4) levels in bronchiolar lavage fluid of allergic asthmatics. The severity of asthma in patients allergic to house dust mite has also been shown to correlate with lipopolysaccharide (LPS), rather than allergen, concentration in dust. We hypothesized that to induce effective VCAM-1 expression in human lung microvascular endothelial cells (HLMVEC), IL-4 may require the presence of a co-stimulus such as LPS. To test this hypothesis we measured, by enzyme-linked immunosorbent assay, induction of cell adhesion molecule expression on, and human eosinophil adhesion to, cultured HLMVEC monolayers pretreated with IL-4 alone or combined with LPS. IL-4 synergized with LPS to induce VCAM-1 expression at 24, 48, or 72 h, whereas IL-4 alone induced expression at 72 h only. IL-4 did not induce expression of intercellular adhesion molecule-1 or E-selectin or alter LPS-induced expression of either. Pre-exposure of HLMVEC to LPS or IL-4 (1 h), followed by IL-4 or LPS, respectively (23 h), also induced VCAM-1 expression. Eosinophil adhesion to HLMVEC monolayers treated with IL-4 and LPS together, but not alone, significantly (P < 0.001) increased from 9.6 +/- 1.5% (control) to 26.9 +/- 3.3% and was inhibited by a monoclonal antibody against the VCAM-1 ligand, very late antigen-4. Analysis of VCAM-1 mRNA revealed synergism between IL-4 and LPS which may, in part, contribute to enhanced VCAM-1 expression. These results suggest that the presence of a co-stimulus such as LPS may be necessary for IL-4 to effectively induce VCAM-1 expression in lung microvasculature.

Endogenous nitric oxide limits cytokine-induced damage of murine lung epithelial cells.

This study investigated whether endogenous nitric oxide (NO) limits cytokine-induced damage to the murine lung epithelial cell line LA-4. NO production was assessed as nitrite using the Griess reaction, and cell damage was assessed using ethidium homodimer-1. Cytotoxicity was first detected after a 24-h incubation with a combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma (cytomix). Nitrite production increased to 78.0 +/- 0.5 nmol/10(6) cells at 24 h. Coincubation of LA-4 with cytomix and NO synthase inhibitors, aminoguanidine (3-1,000 microM) and N(G)-monomethyl-L-arginine (10-1,000 microM), but not N(G)-monomethyl-D-arginine, or a soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one, reduced cytomix-induced nitrite production and increased cytotoxicity up to twofold (24 h). Removal of L-arginine from the medium increased damage; reintroduction of 1,000 microM L-arginine, but not D-arginine, reversed this. In aminoguanidine-treated cells, replacement of NO with an NO donor, S-nitrosoglutathione (30 microM), reversed, in part, the cell damage observed in aminoguanidine/cytomix-treated cells. These results suggest that endogenous NO limits cytokine-induced lung epithelial damage.

Tumour necrosis factor-alpha-induced ICAM-1 expression in human vascular endothelial and lung epithelial cells: modulation by tyrosine kinase inhibitors.

1. Tumour necrosis factor-alpha (TNF alpha) increases the expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) on cultured endothelial and epithelial cells and modulation of this may be important in controlling inflammation. Activation of tyrosine kinase(s) is known to be involved in the signal transduction pathways of many cytokines. In this study we have investigated the effects of the tyrosine kinase inhibitors, ST638, tyrphostin AG 1288 and genistein, on TNF alpha-induced ICAM-1 expression in human alveolar epithelial (A549) and vascular endothelial (EAhy926) cell lines and also normal human lung microvascular endothelial cells (HLMVEC). 2. ICAM-1 expression on cultured cells was determined by a sensitive enzyme-linked immunosorbant assay (ELISA). Endothelial or epithelial monolayers were exposed to increasing doses of TNF-alpha (0.01-10 ng ml-1), in the presence or absence of either ST638 (3-100 microM), AG 1288 (3-100 microM) or genistein (100 microM) and ICAM-1 expression was measured at 4 and 24 h. Control experiments examined the effect of ST638 on phorbol 12-myristate 13-acetate (PMA, 20 ng ml-1, 4 h)-stimulated ICAM-1 and compared it to that of a specific protein kinase C inhibitor, R031-8220 (10 microM). Also, functional consequences of changes in ICAM-1 expression were assessed by measuring adhesion of 111 In-labelled human neutrophils to EAhy926 endothelial and A549 epithelial monolayers treated with TNF alpha, in the presence or absence of ST638. 3. ST638 caused a concentration-dependent reduction in TNF alpha- (0.1-10 ng ml-1)-induced ICAM-1 on EAhy926 endothelial (at 4 h) and A549 epithelial monolayers (at 4 and 24 h). In contrast, ST638 caused a concentration-dependent increase in TNF alpha- (0.1-10 ng ml-1)-induced ICAM-1 on EAhy926 endothelial cells at 24 h. Similar effects were seen with AG 1288 or genistein. ST638 (100 microM) significantly (P < 0.01) inhibited ICAM-1 expression on HLMVEC endothelial cells induced by 0.01 ng ml-1 TNF alpha at 4 or 24 h or 0.1 ng ml-1 at 4 h, but increased ICAM-1 expression induced by 0.1 ng ml-1 TNF alpha at 24 h. ST638 did not significantly change the expression of PMA-stimulated ICAM-1 on either A549 epithelial, EAhy926 or HLMVEC endothelial cells. However, PMA-induced ICAM-1 expression was inhibited by Ro31-8220. Also, treatment of epithelial or endothelial monolayers with TNF alpha and ST638 altered adhesion of human neutrophils to A549 epithelial or EAhy926 endothelial cells in a manner that corresponded to the alteration in ICAM-1 expression. 4. These results show that tyrosine kinase inhibitors alter TNF alpha-induced ICAM-1 expression, but that the cell type, concentration of TNF alpha and time of exposure to this cytokine determine whether expression is decreased or increased by the inhibitor. An increased understanding of the signal transduction pathway(s) involved in TNF alpha-induced ICAM-1 expression on lung epithelial and vascular endothelial cells may be of potential therapeutic value in the treatment of inflammatory disease.

Eotaxin stimulates eosinophil adhesion to human lung microvascular endothelial cells.

Effects of the human C-C chemokines eotaxin, MIP-1 alpha, and RANTES on human eosinophil or neutrophil adhesion to human lung microvascular endothelial cells (LMVEC) were investigated. Basal adhesion of unstimulated eosinophils to LMVEC was increased following pretreatment of LMVEC with TNF alpha (10ng/ml) for 6h. Stimulation of eosinophils with eotaxin (30 and 100ng/ml) resulted in increased adhesion to LMVEC pretreated with TNF alpha but not culture medium. Neutrophil adhesion was not increased by eotaxin under similar conditions. Neither MIP-1 alpha (3-100 ng/ml) nor RANTES (3-100 ng/ml) increased eosinophil or neutrophil adhesion to LMVEC pretreated for 6 h with either TNF alpha (10 ng/ml) or culture medium. Monoclonal antibodies (mAb) against eosinophil adhesion molecule VLA-4 (2B4; 30 micrograms/ml) but not CD18 (6.5E; 10 micrograms/ml) inhibited eotaxin-induced eosinophil adhesion to TNF alpha-activated LMVEC. 2B4 in combination with 6.5E reduced adhesion to basal levels. These data show that eotaxin, but not MIP-1 alpha or RANTES, stimulates eosinophil adhesion to LMVEC and that this effect can be abolished by anti-VLA-4 and CD18 mAb in combination. These results suggest that eotaxin may facilitate eosinophil migration from blood vessels in the lung by increasing eosinophil adhesion to endothelial cells.

Regulation of ICAM-1 by dexamethasone in a human vascular endothelial cell line EAhy926.

Regulation by dexamethasone of intercellular adhesion molecule-1 (ICAM-1) in cultured monolayers of the human umbilical vein endothelial cell line EAhy926 was investigated. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in combination or lipopolysaccharide (LPS) alone gave time- and dose-dependent increases in ICAM-1. Sustained expression of ICAM-1 was observed after short exposure (30 min) to TNF-alpha + IFN-gamma, but not to LPS. LPS-induced ICAM-1 expression was not inhibited by interleukin-1 (IL-1) receptor antagonist (0.01-100 micrograms/ml). Dexamethasone (1,000 nM) did not inhibit TNF-alpha + IFN-gamma-induced ICAM-1 expression or mRNA induction. In contrast, dexamethasone dose dependently (0.1-1,000 nM) inhibited LPS-induced ICAM-1 expression; however, its effect on mRNA was not established, because ICAM-1 mRNA induced by LPS was not detected at the time points investigated in this study (3 and 20 h). Adhesion of unstimulated human neutrophils to EAhy926 monolayers activated with TNF-alpha + IFN-gamma or LPS was increased in the presence of dexamethasone at low doses, whereas neutrophil adhesion to LPS- but not cytokine-stimulated endothelial cells was significantly reduced (P < 0.05) in the presence of a high dose of dexamethasone (1,000 nM). In conclusion, dexamethasone was demonstrated to regulate the expression and function of ICAM-1 in a stimulus-dependent manner.

Does TNF-alpha directly increase endothelial cell monolayer permeability?

The effects of dexamethasone (DEX) and N omega-nitro-L-arginine methyl ester (L-NAME) on the tumour necrosis factor-alpha (TNF-alpha)-induced increase in permeability of human umbilical vein endothelial cell (HUVEC) monolayer to [125I] labelled bovine serum albumin (BSA) were examined. Preincubation of HUVEC monolayers with DEX (1 microM, 2h) completely abolished the effect of TNF-alpha (5 ng/ml, 18 h). Administration of DEX 2 h after TNF-alpha also reduced the effect of TNF-alpha while L-NAME (5 ng/ml, 1 mM, 18 h) had no significant effect. The observed inhibition of the TNF-alpha-induced permeability increase on preincubation with DEX would suggest a role for nitric oxide (NO) in mediating the permeability response. However, this is not confirmed by the experiments with L-NAME. The inhibition caused by DEX administered after TNF-alpha would suggest alternative mechanisms by which DEX may be acting in addition to inhibition of NO synthase induction.

Modulation of human endothelial cell permeability by combinations of the cytokines interleukin-1 alpha/beta, tumor necrosis factor-alpha and interferon-gamma.

The permeability of human umbilical vein endothelial cell (HUVEC) monolayers to [125I]-labelled bovine serum albumin (BSA) was examined following pretreatment of the cells with various cytokines. The electrical resistance measured across untreated, confluent, intact HUVEC monolayers was 18.2 +/- 3.8 omega.cm2 (mean +/- S.D. of 4 observations). Human recombinant (hr) interleukin-1 alpha/beta (IL-1 alpha/beta), hr tumor necrosis factor-alpha (TNF-alpha), and hr interferon-gamma (IFN-gamma) each increased HUVEC monolayer permeability in a time- and dose-dependent manner. These effects were inhibitable by neutralizing antibodies (nAb) to the corresponding cytokines, and were not due to contamination by endotoxin (abolition of cytokine effect by heat treatment, and no effect on cytokine action of the endotoxin inhibitor polymyxin B). The effects of these cytokines were not due to endothelial cell (EC) interleukin-6 (IL-6) induction (IL-6 shown not to increase permeability) and the effect of hrTNF-alpha could not be accounted for by induction of IL-1 (effect not inhibited by hrIL-1 alpha/beta nAb). The effects of three different combinations of the cytokines (each combination at two different concentrations) on HUVEC monolayer permeability were also examined. hrIFN-gamma with hrTNF-alpha or hrIL-1 alpha/beta gave an increase in permeability (at both concentration combinations) greater than that seen with either cytokine alone. hrTNF-alpha and hrIL-1 alpha/beta in combination however produced an enhanced effect only at low concentrations, high concentrations in combination producing an effect no greater than either agent alone. These results highlight the importance of investigating actions of cytokine combinations on in vitro models of endothelial cell activation.