Adhesion of human granulocytes and T lymphocytes to vascular endothelial cells after stimulation with Bacteroides fragilis endotoxin and enterotoxin]

The aim of presented study was to estimates the number of human granulocytes and T lymphocytes adhering to 1 mm2 of vascular endothelial cell culture stimulated by Bacteroides fragilis endotoxins (LPS) and enterotoxin (BFT). HMEC-1 cells were activated with bacterial preparations at the concentration of 10 (micrograms/ml for 4 and 24 hours. Granulocytes and T lymphocytes were isolated from peripheral blood of healthy blood donors. The adhesion tests of granulocytes and adhesion tests of resting and activated with PMA (at the concentration of 10 ng/ml) T lymphocytes to the non-stimulated and stimulated by B. fragilis compounds (LPS and BFT) vascular endothelium were performed. The number of viable leukocytes, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The results of experiments indicate that granulocytes and T lymphocytes (resting and after activation with PMA even in greater number) adhere to the endothelial cells stimulated by B. fragilis endotoxins and enterotoxin. B. fragilis toxins are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS. B. fragilis LPS and BFT preparations stimulate endothelial cells to the adhesion of granulocytes in similar manner, whereas the activation of vascular endothelium to the adhesion of T lymphocytes is differentiated.     

Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus

The recruitment of polymorphonuclear leukocytes (PMNs) from the vascular space into the lung interstitium and airspace is an early step in the host innate immune response to bacterial invasion of these sites. To determine the ability of intact bacteria to directly elicit PMN migration across an endothelial monolayer, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli, as well as to purified E. coli LPS. Bacterial induction of PMN migration was dose dependent and elicited by > or =10(4) bacteria/ml of each of the species tested. Pretreatment of PMNs with blocking Abs to CD18 significantly inhibited migration of PMN in response to all stimuli tested, but had the most profound effect on migration to S. pneumoniae and S. aureus. Intact E. coli were 10 times more potent in inducing transmigration of PMNs than a corresponding amount of purified LPS. Bacterial induction of PMN migration did not correlate with up-regulation of surface endothelial ICAM-1 expression (purified LPS > intact E. coli > S. aureus and S. pneumoniae) nor up-regulation of VCAM-1 and E-selectin. Neutralizing Ab to ICAM-1 had no effect on PMN migration to any of the bacteria or to purified LPS. These findings demonstrate that diverse bacterial pathogens induce PMN migration across a pulmonary microvascular endothelial cell monolayer in a fashion that appears to be organism specific. In addition, intact bacteria elicit PMN-endothelial cell interactions distinct from those seen when purified bacterial products are used as agonists.     

Toxins of Bacteroides fragilis and Bacteroides thetaiotaomicron rods as stimulators of adhesion molecule expression on the surface of vascular endothelial cells

Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF), and from three B. thetaiotaomicron strains were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the procedure of van Tassell et al. (1992). The influence of the examined toxins on the expression of adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human dermal microvascular endothelial cells) was assayed in ELISA test with monoclonal antibodies. Four concentrations of toxins were applied: 0.01, 0.1, 1.0 and 10.0 (micrograms/ml). Endothelial cells were activated for 24 hours (ICAM-1 and VCAM-1 expression) and for 4 hours (E-selectin expression). The coloured product of immunoenzymatic reaction was measured by reading the absorbance at wavelength 492 nm. Two controls were performed in each experiment: with resting HMEC-1 and E. coli O55:B5 LPS (Sigma, USA). Bacteroides fragilis and B. thetaiotaomicron lipopolysaccharides stimulated three adhesion molecules under investigation. Their activity was comparable, but weaker than the activity of E. coli O55:B5 LPS. ICAM-1 was the most stimulated molecule. B. fragilis enterotoxin induced two adhesion molecules: VCAM-1 and E-selectin demonstrating weaker stimulatory activity than E. coli LPS. Stimulation of adhesion molecules on vascular endothelial cells should be considered to be a biological activity of B. fragilis and B. thetaiotaomicron endotoxins and B. fragilis enterotoxin.     

Stimulation of adhesion molecules on vascular endothelium by capsular polysaccharide, lipopolysaccharide and components of lipopolysaccharide from Bacteroides thetaiotaomicron

The aim of this study was to assay the influence of capsular polysaccharide (CPS), lipopolysaccharide (LPS) and components of B. thetaiotaomicron lipopolysaccharide--polysaccharide part (PS) and lipid part (lipid A) on the expression of adhesion molecules associated with inflammation (ICAM-1, VCAM-1, E-selectin) on the surface of vascular endothelial cells. Capsular polysaccharide was isolated by the method of Poxton and Ip (1981). Lipopolysaccharides were extracted using the hot phenol-water method (Westphal and Jann, 1965). Components of LPS were prepared by mild acid hydrolysis of lipopolysaccharide. Experiments with bacterial compounds at concentrations 10, 1, 0.1 and 0.01 (mg/ml) were performed on HMEC-1 cell line (human dermal microvascular endothelial cells). Immunoenzymatic ELISA test with mouse monoclonal antibodies against human: ICAM-1, VCAM-1 and E-selectin was applied to determine adhesion molecules. Resting HMEC-1 and E. coli O55:B5 LPS were used as controls in each experiment. Lipopolysaccharides were the strongest stimulants of endothelial adhesion molecules. Capsular polysaccharide caused the expression of three adhesion molecules, but only at the highest concentration (10 mg/ml). The stimulatory activities of LPS lipid components were much higher than the activities of polysaccharide parts. PS preparations did not reveal the property of adhesion molecule stimulation or their activities were weak. The activity of B. thetaiotaomicron cell-surface antigens in the process of adhesion molecule stimulation on vascular endothelium was lower than the activity of E. coli LPS.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Effect of clindamycin on stimulation of cell adhesion molecules by endotoxins and enterotoxin of Bacteroides fragilis strains]

The influence of clindamycin on expression of B. fragilis endotoxins (LPS) and enterotoxin stimulated cell adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human microvascular endothelial cell line) was tested. Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the method described by van Tassel et al. (1992). All bacterial preparations were used for stimulation at concentration 10 micrograms/ml. Clindamycin was used in concentration of 2 micrograms/ml. The influence of antimicrobial agent on the endotoxins and enterotoxin stimulation and expression of adhesion molecules was tested by ELISA, using monoclonal mouse anti-human antibodies (Genzyme, USA). Peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO A/S Denmark) and OPD (Sigma USA) were used. The coloured reaction product was measured by reading the absorbance at 492 nm in SPECTRA II reader (SLT, Austria). It was observed that clindamycin influenced the expression of cell adhesion molecules on resting cell line. HMEC-1 cells stimulated with Bacteroides fragilis LPS preparations have suppressive effect on ICAM-1 expression. ICAM-1 expression was augmented when stimulated with Tox 1 and Tox 2 preparations. Clindamycin augmented the VCAM-1 expression in tests with all bacterial preparations. All used bacterial preparations of Bacteroides fragilis LPS and enterotoxin enhanced the expression of E-selectin with exception of LPS of NTBF strain.     

Characteristics of lipopolysaccharide interaction with human peripheral-blood monocytes.

The interaction between radioiodinated lipopolysaccharide from Escherichia coli 0111:B4 (125I-LPS) and human peripheral-blood monocytes was studied. The association of 125I-LPS with monocytes at 37 degrees C appeared to depend on binding to the cell membrane with subsequent internalization of the molecule, and was not saturable with time (up to 2 h) or 125I-LPS concentration (up to 10 micrograms/ml). There was no apparent difference in the behaviour of unlabelled LPS and 125I-LPS with respect to monocyte association. 125I-LPS association with monocytes was inhibited by LPS and O-polysaccharide from E. coli 0111:B4 and Salmonella typhi 0901, but not by lipid A or polymyxin B. We propose that the mechanism of human monocyte stimulation by LPS involves polysaccharide-dependent binding to the cell membrane followed by internalization of the LPS molecule. We were unable to demonstrate a specific LPS receptor such as that found on murine B-lymphocytes.     

IL-1 induction-capacity of defined lipopolysaccharide partial structures

Natural and synthetic lipid A as well as natural and synthetic oligosaccharide partial structures of LPS were examined in dose-response experiments to define the minimal structure necessary for IL-1 induction and release in cultures of human mononuclear cells. Wild type LPS (S. abortus equi) and rough mutant LPS was active in minimal-doses of 1 to 100 pg/ml, whereas synthetic heptaacyl and hexaacyl lipid A (Salmonella minnesota and Escherichia coli lipid A, respectively) induced IL-1 in minimal-doses of 100 to 1,000 pg/ml and 10 to 1,000 pg/ml, respectively. Nanogram amounts (0.1 to 10 ng/ml) of synthetic monodephospho partial structures of E. coli lipid A were necessary for IL-1 induction. Synthetic pentaacyl partial structures induced IL-1 very weakly. Synthetic tetraacyl and bisacyl partial structures lacking non-hydroxylated fatty acids were not active. Compared to LPS million-fold higher doses of natural and synthetic 3-deoxy-D-manno-octulosonic acid containing core oligosaccharides were necessary for IL-1 induction. Dose-response investigations with LPS and natural or synthetic partial structures established the following hierarchy in IL-1 induction-capacity: LPS greater than lipid A much greater than lipid A partial structures greater than core oligosaccharides greater than oligoacyl lipid A. Lipid A was shown here to be the portion of LPS mainly responsible for induction of IL-1 activity. The high potency of lipid A in inducing IL-1 release and the failure of the precursor Ia of lipid A to induce IL-1 production and release was also observed measuring intracellular IL-1 activity after freeze-thawing the cells. Levels of IL-1 beta mRNA in extracts of mononuclear cells correlated with biologic activity. In co-incubation experiments, precursor Ia of lipid A produced dose-dependent inhibition of production and release of IL-1 activity induced by lipid A or LPS, but not by Staphylococcus epidermidis or PHA. Incubation of cells with precursor Ia for 1h, followed by a medium change and further incubation of stimulus without precursor Ia of lipid A also resulted in inhibition. We conclude that lipid A is the main portion of LPS responsible for induction of IL-1, and that specific activation- and/or binding-mechanisms are involved in stimulation of cells with LPS and/or lipid A.     

Effects of bacterial lipopolysaccharide on the binding of lymphocytes to endothelial cell monolayers

Preincubation of human umbilical vein endothelial cell (EC) monolayers with 1 ng to 10 micrograms/ml lipopolysaccharide (LPS) increased the binding of T lymphocytes to EC. The effect was maximal at LPS concentrations of 0.1 to 10 micrograms/ml, and occurred with LPS derived from Escherichia coli (serotypes 0111:B4 and 0127:B8), Shigella flexneri (serotype 2a), Serratia marcescens (serotype 0:3), and Yersinia entercolitica (serotype 0:3). The increased binding appeared to be mediated primarily through an action on EC; preincubation of T cells rather than EC with LPS did not lead to enhanced binding. The onset of enhanced binding was very rapid, being observed after 2 to 3 min of preincubation and becoming maximal after 1 hr. EC were unresponsive to LPS after fixation with 2% paraformaldehyde-L-lysine-periodate and also when the LPS was incubated with EC at 4 degrees C. Enhanced binding was seen with lipid A and with LPS from Salmonella minnesota Re 595 (mainly lipid A) and was abolished by conjugation with polymyxin B. The observed increase in the binding of lymphocytes to EC exposed to LPS suggests that the lymphocytopenia induced by endotoxemia may result from augmentation of the adherence of lymphocytes to altered endothelium.     

Effect of lipopolysaccharides on cultured human endothelial cells. Relationship between tissue factor activity and prostacyclin release

Exposure to lipopolysaccharides (LPS; 10 micrograms/ml derived from either S. enteritidis or E. coli or to their lipid A moiety alone induced procoagulant activity in cultured human endothelial cells. This exclusively cell-associated activity was identified as tissue factor activity by two criteria: Firstly, the presence of Factor VII was required for its expression and, secondly, clotting was abolished by the addition of the IgG fraction of anti-human tissue factor antibodies. Concomitant analysis of prostacyclin (PGl2) formation by the cells showed a substantial increase in the production of this potent platelet inhibiting substance during exposure to endotoxin. LPS-induced release of PGl2 did not result in refractoriness of the cells to generate new PGl2 as indicated by the retained response to stimulation with 20 microM arachidonic acid. While the release of PGl2 could be inhibited by pretreatment of the cells with 100 microM acetylsalicylic acid (ASA), the induction of tissue factor activity remained unaffected by ASA. In contrast to LPS-free control cultures, ASA did not completely prevent PGl2 formation by human endothelial cells after exposure to LPS suggesting the induction of a cyclooxygenase-independent pathway by LPS.     

Adhesion of human T lymphocytes and granulocytes to endothelial cells stimulated by cell-surface antigens of Bacteroides thetaiotaomicron

The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 micrograms/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA (in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS.     

Stimulation of adhesion molecule expression in human vascular endothelium by Bacteroides fragilis toxins--influence of polymyxin B

The aim of this study was to examine the influence of polymyxin B on the level of expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 on human vascular endothelium activated with B. fragilis endotoxins or enterotoxin. Lipopolysaccharides were extracted by phenol-water method from one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) B. fragilis strains. LPS preparations were purified with nucleolytic enzymes and ultracentrifugation. Enteotoxin (BFT) was prepared from the supernatant of reference B. fragilis ATCC 43858 culture by precipitation with ammonium sulphate. BFT preparations were purified with the application of ion-exchange chromatography and hydrophobic chromatography. Adhesion molecule expression on the surface of human vascular endothelial cells (HMEC-1 cell line) was determined after simultaneous stimulation with bacterial compounds at the concentration of 10 micrograms/ml and polymyxin B at the concentration of 20 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) or for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with the use of mouse, monoclonal antibodies against human ICAM-1, VCAM-1, and E-selectin. The results of performed experiments suggest, that polymyxin B changes the level of adhesion molecule expression on human vascular endothelium. This antibiotic causes changes in the expression of endothelial ICAM-1, VCAM-1, and E-selectin during simultaneous stimulation of endothelium with B. fragilis endotoxins or enterotoxin. In the majority of cases the addition of polymyxin B leads to the up-regulation of examined adhesion molecules.     

IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.     

The effect of metronidazole on stimulation of adhesion molecule expression on the surface of vascular endothelial cells by Bacteroides fragilis endotoxins and enterotoxin]

The influence of metronidazole on the level of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells activated with B. fragilis endotoxins and enterotoxin was examined. Three enterotoxigenic (ETBF) strains and one nonenterotoxigenic (NTBF) strain were used for lipopolysaccharide extraction. Enterotoxin was prepared from the culture supernatant of the reference B. fragilis ATCC 43858 strain. Expression of adhesion molecules on vascular endothelial cells (HMEC-1 cell line) was determined after their stimulation with bacterial compounds at the concentration of 10 micrograms/ml in the presence of metronidazole at the concentration of 4 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) and for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with mouse, monoclonal antibodies against human ICAM-1, VCAM-1 and E-selectin. The results of experiments suggest, that metronidazole enhances the expression of examined adhesion molecules on endothelial cells. This antimicrobial agent causes some changes in the expression of endothelial ICAM-1, VCAM-1 and E-selectin stimulated by B. fragilis endotoxins and enterotoxin.     

S-form lipopolysaccharide (LPS), but not lipid A or R-chemo-type LPS, induces interleukin-6 production in vitamin D3-differentiated THP-1 cells

Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A. We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity. Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E. coli-type lipid A (compound 506), E. coli Re mutant LPS (ReLPS), or alkali-treated LPS. The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406). An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production. These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells.     

DNA damage by 5-nitro-2-furylacrylic acid, a nitrofuran derivative

5-Nitro-2-furylacrylic acid (5-NFA) caused dose dependent inhibition of growth of Escherichia coli K-12 strain AB 2480 (uvr-, rec-), the 37% (D37) and 10% (D10) survival doses being 1.0 microgram/ml.h and 1.75 micrograms/ml.h, respectively. Although much higher doses of drug were required to achieve comparable inhibition of growth of E. coli strain 1157 (repair proficient), significant filamentation of these cells was produced by treatment with 1.0 microgram/ml 5-NFA for 4 hr. Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that 5-NFA treatment of E. coli strain AB 2480 produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the initial part of the reaction obeyed a first order relation. 5-NFA also produced dose-dependent increase of prophage induction in E. coli strain GY 5027: envA, uvrB, ampA1, strA (lambda). The implications of the action of 5-NFA on DNA in relation to the induction of 'SOS' functions and carcinogenesis were discussed.     

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.     

Studies on the adhesive interaction between purified human eosinophils and cultured vascular endothelial cells

Many recent studies have established the eosinophil as a primary effector cell in the pathology of allergic diseases. However, relatively little is known about the mechanisms by which eosinophils accumulate and are activated at local sites of tissue inflammation in allergic or other eosinophil-dependent pathologic states. Because the adherence of leukocytes to vascular endothelial cells (VEC) is a critical initial event in eosinophil infiltration, we have studied the interaction of purified human eosinophils with cultured human umbilical vein endothelial cells. Treatment of VEC with stimuli known to activate endothelial cells, including purified human IL-1, rTNF-alpha, bacterial endotoxin LPS, and the tumor-promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate resulted in time- and dose-dependent increases (from two- to fourfold) in adhesiveness for eosinophils. Adherence induced by optimal concentrations of IL-1 (2 U/ml), TNF (1 micrograms/ml), and LPS (1 microgram/ml) is dependent upon the CD18 leukocyte cell surface adherence glycoproteins, because a mAb (60.3) directed against the common beta-subunit of the complex inhibits adherence induced by these stimuli. Several agents directly activated eosinophils to display increased adhesiveness to both VEC and gelatinized plates. The bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine (10(-8) to 10(-6) M), TNF (1 to 1000 ng/ml), and 12-O-tetradecanoyl-phorbol-13-acetate (0.3 to 3 ng/ml) all increased eosinophil binding to VEC by two to fivefold. Platelet-activating factor (PAF; 10(-8) to 10(-6) M), but not lyso-PAF, caused approximately a twofold increase in eosinophil binding to both VEC and gelatinized tissue culture plates, suggesting that activation of eosinophils may be responsible for the known ability of PAF to induce eosinophilic responses. These results suggest that the initiation of an eosinophilic infiltrate in vivo can result from activation of endothelial cells, activation of eosinophils, or activation of both cell types.     

Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells

Cultured endothelial cells secrete a platelet-derived growth factor-like molecule (PDGFc). We examined the effects of purified human alpha-thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of alpha-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of alpha-thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha-thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell-mediated conversion of PDGFc from an inactive to an active form.     

Gangliosides block the inhibition of macrophage Fc-dependent phagocytosis by lipopolysaccharide

The mechanism whereby bacterial lipopolysaccharide (LPS) exerts its biologic effects on mammalian cells is unknown. Plasma membrane gangliosides bind bacterial toxins and have been implicated in modulating the effects of a variety of immunoregulatory substances. We investigated the possibility that gangliosides can inhibit the effect of lipopolysaccharide on Fc-dependent phagocytosis by murine peritoneal macrophages. Protein-free lipopolysaccharide preparations significantly inhibited Fc-mediated phagocytosis (less than 71% of control) at concentrations of 100 ng/ml or greater after 90 min of incubation. The inhibitory effect of LPS (1 micrograms/ml) was blocked when macrophages were incubated with mono-, di-, or trisialogangliosides (25-50 micrograms/ml). Neither asialoganglioside nor sialic acid alone were capable of blocking the effect of LPS. When chromatographed separately on a Sepharose 4B column, LPS and trisialoganglioside had different elution profiles. LPS and trisialoganglioside coeluted, however, when premixed at 37 degrees C for 60 min and then applied to the column. Therefore, abrogation of the effect of LPS on Fc-dependent phagocytosis may occur as a consequence of direct interaction between LPS and gangliosides. These data suggest that gangliosides may modulate the response of macrophages to bacterial lipopolysaccharide.