Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells

E. coli lipopolysaccharide (LPS) induces cytokine and adhesion molecule expression via the toll-like receptor 4 (TLR4) signaling complex in human endothelial cells. In the present study, we investigated the mechanism by which Porphyromonas gingivalis LPS antagonizes E. coli LPS-dependent activation of human endothelial cells. P. gingivalis LPS at 1 micro g/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 micro g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1beta) stimulation. P. gingivalis LPS (1 micro g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1beta-dependent activation of NF-kappaB in human microvascular endothelial (HMEC-1) cells, consistent with antagonism occurring upstream from the TLR/IL-1 receptor adaptor protein, MyD88. Surprisingly, P. gingivalis LPS weakly but significantly activated NF-kappaB in HMEC-1 cells in the absence of E. coli LPS, and the P. gingivalis LPS-dependent agonism was blocked by transient expression of a dominant negative murine TLR4. Pretreatment of HUVECs with P. gingivalis LPS did not influence the ability of E. coli LPS to stimulate E-selectin mRNA expression. Taken together, these data provide the first evidence that P. gingivalis LPS-dependent antagonism of E. coli LPS in human endothelial cells likely involves the ability of P. gingivalis LPS to directly compete with E. coli LPS at the TLR4 signaling complex.     

Cimetidine inhibits the adhesion of gastric cancer cells expressing high levels of sialyl Lewis x in human vascular endothelial cells by blocking E-selectin expression

Cimetidine has been shown to have anti-metastatic activity and improves the survival of patients with colorectal cancer. One hypothesis is its modulation of the expression of the cell adhesion molecule by target organ endothelial cells. Because of the inconclusive results in clinical trials of gastric cancer, we investigated the effects of cimetidine on the adhesion of gastric cancer cells to activated endothelial cells and on the expression of some cell adhesion molecules. Human endothelial cells were pre-incubated with cimetidine for 6 h, incubated with the cytokine tumor necrosis factor for 4?h, and the endothelial surface expression of E-selectin was evaluated by flow cytometry, immunostaining and ELISA. Further, we investigated E-selectin mRNA expression by RT-PCR. Three gastric cancer cell lines (SGC-7901, MGC-803, BGC-823) and a normal gastric epithelial cell line, GES-1, were studied for the surface expression of sialyl Lewis x by flow cytometry and immunostaining. Adherence of CFSE-labeled gastric cancer cells and GES-1 cells to endothelial cell monolayers was determined. Cimetidine significantly reduced E-selectin expression of activated endothelial cells, but did not influence E-selectin expression at the mRNA level. Three gastric cancer cell lines expressed high levels of sialyl Lewis x, whereas GES-1 did not. Cimetidine also significantly decreased gastric cancer cell adherence to stimulated endothelial cells. The inhibition of E-selectin expression corresponded to the reduction of tumor cell adherence. The effects of cimetidine on tumor adhesion were almost nullified by pre-incubation with E-selectin and sialyl Lewis x antibody. Furthermore, there was no significant change of GES-1 adherence to endothelial cells by TNF-α, cimetidine, E-selectin and sialyl Lewis x antibody. The inhibiton of gastric cancer cell adherence to cytokine-stimulated endothelial cells treated with cimetidine appears to result from blocking endothelial E-selectin expression. These data support the hypothesis that cimetidine may exert its anti-metastatic effects in gastric cancer, in part, by inhibiting E-selectin/sialyl Lewis x-mediated adherence of gastric cancer cells to endothelial cells in the metastasis target organs.     

补肾宁心方对人单核-血管内皮细胞粘附的影响

目的:探讨补肾宁心方对人单核-血管内皮细胞粘附的影响及机理。方法:以培养人脐静脉内皮细胞(HUVECs)作为靶细胞,在内皮细胞培养基中加入氧化的低密度脂蛋白(ox-LDL)或在试验体系中加入灌服补肾宁心方的兔血清,以孟加拉玫瑰红活细胞染色法测定人单核细胞系U937与HUVECs的粘附,并用流式细胞仪检测内皮细胞表面粘附分子细胞间粘附分子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1)以及E-选择素的表达。结果:ox-LDL显著增强单核U937细胞与内皮细胞之间的相互粘附,如在试验体系中加入灌服补肾宁心方的动物血清,则粘附率明显降低(P＜0.01)。流式细胞仪分析结果显示ox-LDL能明显促进内皮细胞表面ICAM-1、VCAM-1以及E-选择素的表达,补肾宁心方中药灌服血清可显著下调内皮细胞表面ICAM-1、VCAM-1以及E-选择素的表达(P＜0.01)。结论:补肾宁心方含药血清可能通过下调内皮细胞表面粘附分子的表达抑制单核-血管内皮细胞粘附,从而发挥对血管内皮细胞的保护作用。     

Ability of bacteria associated with chronic inflammatory disease to stimulate E-selectin expression and promote neutrophil adhesion.

Porphyromonas gingivalis, Pseudomonas aeruginosa, and Helicobacter pylori have been shown to be associated with adult periodontal disease, chronic lung infections, and peptic ulcers, respectively. The ability of these bacteria to stimulate E-selectin expression and promote neutrophil adhesion, two components necessary for the recruitment of leukocytes in response to infection, was investigated. Little or no stimulation of E-selectin expression was observed with either P. gingivalis or H. pylori when whole cells, lipopolysaccharide (LPS), or cell wall preparations added to human umbilical cord vein endothelial cells were examined. P. aeruginosa was able to induce E-selectin to near-maximal levels; however, it required approximately 100 to 1,000 times more whole cells or LPS than that required by E. coli. Neutrophil-binding assays revealed that LPS and cell wall preparations obtained from these bacteria did not promote endothelial cell adhesiveness by E-selectin-independent mechanisms. In addition, P. gingivalis LPS blocked E-selectin expression by LPS obtained from other bacteria. We propose that lack of E-selectin stimulation and the inability to promote endothelial cell adhesiveness are two additional indications of low biologically reactive LPS. We suggest that this property of LPS may contribute to host tissue colonization. In addition, the ability of P. gingivalis to inhibit E-selectin expression may represent a new virulence factor for this organism.     

Fimbria-Dependent Activation of Cell Adhesion Molecule Expression in Porphyromonas gingivalis-Infected Endothelial Cells

Porphyromonas gingivalis is an oral pathogen that has recently been associated with chronic inflammatory diseases such as atherosclerosis. The strength of the epidemiological associations of P. gingivalis with atherosclerosis can be increased by the demonstration that P. gingivalis can initiate and sustain growth in human vascular cells. We previously established that P. gingivalis can invade aortic, heart, and human umbilical vein endothelial cells (HUVEC), that fimbriae are required for invasion of endothelial cells, and that fimbrillin peptides can induce the expression of the chemokines interleukin 8 and monocyte chemotactic protein. In this study, we examined the expression of surface-associated cell adhesion molecules on endothelial cells in response to P. gingivalis infection by fluorescence-activated cell sorting FACS analysis and confocal microscopy. Coculture of HUVEC with P. gingivalis strain 381 or A7436 resulted in the induction in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P- and E-selectins, which was maximal at 48 h postinfection. In contrast, we did not observe induction of ICAM-1, VCAM-1, or P- or E-selectin expression in HUVEC cultured with the noninvasive P. gingivalis fimA mutant DPG3 or when P. gingivalis was incubated with fimbrillin peptide-specific anti-sera prior to the addition to HUVEC. Furthermore, the addition of a peptide corresponding to the N-terminal domain of fimbrillin to HUVEC resulted in an increase in ICAM-1, VCAM-1, and P- and E-selectins, which was maximal at 48 h and similar to that observed for live P. gingivalis. Treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also resulted in the inhibition of ICAM-1, VCAM-1, or P- and E-selectin expression. Taken together, these results indicate that active P. gingivalis invasion of HUVEC mediated via the major fimbriae stimulates surface-associated cell adhesion molecule expression. Stimulation of adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by P. gingivalis may play a role in the pathogenesis of systemic inflammatory diseases associated with this microorganism, including atherosclerosis.     

Expression of leukocyte-endothelial cell adhesion molecules on monocyte adhesion to human endothelial cells on plasma treated PET and PTFE in vitro

We used a coculture model to evaluate the inflammatory potential of ammonia gas plasma modified PET and PTFE by flow cytometry and immunohistochemistry. In these studies, human endothelial cells from umbilical cord (HUVEC) and promonocytic U937 cells were used. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-) were used as controls. U937 adhesion to endothelium on each surface was evaluated at day 1 and day 7. To further investigate the role of leukocyte–endothelial cell adhesion molecules (CAMs) in cell-to-cell interaction on material surfaces, the expression of the leukocyte–endothelial CAMs: ICAM-1, VCAM-1, PECAM-1, and E-selectin on HUVECs were evaluated after U937 cell adhesion. The results demonstrated that plasma treated PET (T-PET) and treated PTFE (T-PTFE) did not increase U937 cell adhesion compared to the negative control. Maximal adhesion of U937 cells to HUVEC was observed on TNF- stimulated endothelium with significant differences between day 1 and day 7, which is consistent with our prior observation that T-PET and T-PTFE did not cause HUVECs to increase the expression of adhesion molecules. After U937 cell adhesion, the expression of ICAM-1 and VCAM-1 of HUVECs were not different on T-PET and T-PTFE compared with the negative control. However, the expression of E-selectin was reduced on day 1, but not on day 7. The effects of plasma treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, the cell number increased significantly on the surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion and expression of leukocyte–endothelial CAMs. The monocyte adhesion to endothelial cells on surfaces can be used as a tool for the evaluation of material surface modification and further to study the mechanisms of cell-to-cell interactions in response to surfaces.     

Hemagglutinin B is involved in the adherence of Porphyromonas gingivalis to human coronary artery endothelial cells

Porphyromonas gingivalis is a periodontopathogen that may play a role in cardiovascular diseases. Hemagglutinins may function as adhesins and are required for virulence of several bacterial pathogens. The aim of this study was to determine the role of hemagglutinin B (HagB) in adherence of P. gingivalis to human coronary artery endothelial (HCAE) cells. P. gingivalis strain 381, a P. gingivalis 381 HagB mutant, Escherichia coli JM109 expressing HagB (E. coli-HagB), and E. coli JM109 containing pUC9 (E. coli-pUC9) were tested for their ability to attach to HCAE cells. Inhibition assays were performed to determine the ability of purified recombinant HagB (rHagB) as well as antibodies to HagB, including the polyclonal antibody (PAb) A7985 and the monoclonal antibody (MAb) HL1858, to inhibit the attachment of P. gingivalis to HCAE cells. As expected, when the attachment of P. gingivalis and the HagB mutant were compared, no statistical significance was observed between the two groups (P = 0.331), likely due to the expression of the hagB homolog hagC. However, E. coli-HagB adhered significantly better to HCAE cells than did E. coli-pUC9, the control strain. In a competition assay, the presence of purified rHagB decreased bacterial adhesion of P. gingivalis or E. coli-HagB to HCAE cells. The presence of PAb A7985 or MAb HL1858 also significantly decreased attachment of P. gingivalis and E. coli-HagB to host cells. These results indicate that HagB is involved in the adherence of P. gingivalis to human primary endothelial cells.     

Acacetin inhibits expression of E-selectin on endothelial cells through regulation of the MAP kinase signaling pathway and activation of NF-κB

Abstract

Since E-selectin-mediated adhesion of leukocytes or tumor cells to the vascular endothelium is a key early event in the initiation of inflammatory response and cancer metastasis, E-selectin inhibition is thought to be a good target for therapeutic intervention. Several flavones have been shown to have anti-inflammatory and anticancer properties. In the present study, we investigated the effects of plant flavones on expression of E-selectin in human umbilical vein endothelial cells. Among 11 flavones, acacetin strongly inhibited TNF-α-induced E-selectin expression in HUVECs. Acacetin suppressed the TNF-α-induced phosphorylation of p38 but did not inhibit TNF-α-induced phosphorylations of JNK and ERK. Acacetin also inhibited the activation of NF-κB by stimulation with TNF-α. Furthermore, adhesion of monocytes to TNF-α-treated endothelial cells was inhibited by cotreatment with acacetin. These results suggest that acacetin inhibits the expression of E-selectin by regulation of the p38 MAPK signaling pathway and activation of NF-κB.     

Lymphocyte-facilitated tumour cell adhesion to endothelial cells: the role of high affinity leucocyte integrins

AIM: Lymphocytes transiently express an active form of the beta_2 integrin LFA-1 (LFA-1Af) which has conformational changes in extracellular domains enabling higher affinity binding to the ligand ICAM-1. In this study, we investigated the role of lymphocytes bearing LFA-1Af as potential mediators of binding of ICAM-1-positive tumour cells to endothelium. METHODS: LFA-1 expression on 51Cr-PBLs was modulated in order to express high affinity LFA-1Af and conjugates were formed with 35S-labelled COLO526. The binding of the conjugates to resting or IL-1beta-stimulated human umbilical vein endothelial cells (HUVECs) was then assessed via a modified radioactive HUVEC binding assay. In addition, the binding of PBL-COLO526 conjugates to HUVECs was demonstrated by confocal microscopy. RESULTS: The binding of COLO526 to endothelial cells did not change significantly between unstimulated and stimulated HUVECs. In addition, pre-incubating the COLO526 with fresh PBLs did not significantly alter the binding of COLO526 to resting or activated HUVECs; whereas, in the presence of PBLs with LFA-1Af, the COLO526 conjugate binding dramatically increased from basal levels to 41% on resting HUVECs and 81% on stimulated HUVECs. COLO526-PBL(LFA-1Af) conjugate adhesion to stimulated HUVECs was inhibited by blocking antibody to LFA-1 (50%), VLA-4 (32%) or L-selectin (40%). Antibodies to the HUVEC adhesion molecules ICAM-1, VCAM-1 and E-selectin also inhibited COLO526-PBL(LFA-1Af) conjugate binding to activated HUVECs by 79, 60 and 73%, respectively. CONCLUSIONS: PBLs bearing LFA-1Af can enhance COLO526 adhesion to both resting and activated HUVECs. Furthermore, blocking studies demonstrate that a range of pathways are involved in this phenomenon (LFA-1/ICAM-1, VLA4/VCAM-1, L-selectin/E-selectin). These studies have identified a novel alternative pathway for lymphocyte-facilitated tumour cell adhesion to endothelial cells.     

蛋白激酶C在脂多糖诱导内皮细胞β-1,4-半乳糖基转移酶-Ⅰ表达中的作用

目的 研究蛋白激酶c(PKC)对脂多糖(LPS)诱导内皮细胞β-1,4-半乳糖基转移酶-Ⅰ(β-1,4-galactosyltransferase-Ⅰ,β-1,4-GalT-Ⅰ)表达的调节作用,以及对内皮细胞骨架结构改变及其黏附能力的影响.方法 分别用PKC激动剂或几种不同类型的PKC抑制剂预处理人脐静脉内皮细胞(HUVECs)30 min,LPS刺激HUVECs 4 h后,RT-PCR、Western blot方法 检测β-1,4一GalT-Ⅰ表达变化,细胞荧光染色观察β-1,4-GalT-Ⅰ催化的糖链表达变化及细胞骨架结构的改变,通过内皮-单核细胞黏附试验观察HUVECs黏附能力的改变.结果 几种不同类型的PKC抑制剂均能不同程度地抑制LPS刺激HUVECs引起的β-1,4-GalT-Ⅰ表达的上调,PKC激动剂能够使上调的β-1,4-GalT-Ⅰ的表达进一步增加;在HUVECs中β-1,4-GalT-Ⅰ与细胞骨架有共同定位,PKC抑制剂显著抑制LPS诱导的内皮细胞骨架蛋白的重构和β-1,4-GalT-Ⅰ细胞内的再分布;PKC抑制剂显著抑制LPS诱导的内皮-单核细胞黏附能力的上调.结论 PKC可能参与调节LPS诱导的HUVECs β-1,4-GalT-Ⅰ的表达,可能多种类型的PKC参与了这一调节过程;PKC可能通过对β-1,4-GalT-Ⅰ的调节进而影响炎症过程中内皮细胞骨架蛋白的重构及内皮细胞与单核细胞的黏附能力.     

VE-statin/Egfl7 siRNA inhibits angiogenesis in malignant glioma in vitro

This study investigated the role of VE-statin/Egfl7 and its mechanism in angiogenesis in malignant glioma. Transwell culture plates were used to establish an U251-HUVEC co-culture system, which was used to mimic the interaction between malignant glioma and endothelial cells. Lentiviral vectors expressing VE-statin/Egfl7 siRNA were constructed, and U251 cells and HUVECs were transfected to inhibit VE-statin/Egfl7 expression. The proliferation, adherence, migration, and lumen formation of endothelial cells were assayed to investigate the influence of VE-statin/Egfl7 on angiogenesis in malignant glioma in vitro. Data showed that HUVEC growth was temporarily slowed after silencing the VE-statin/Egfl7 gene but rapidly returned to normal. Although endothelial cell migration was not influenced, cell adherence was markedly inhibited. Furthermore, the endothelial cells failed to generate a capillary-like lumen after VE-statin/Egfl7 gene silencing. Therefore, it can be concluded that VE-statin/Egfl7 may regulate the adherence of endothelial cells, thus playing an important role in endothelium-induced lumen formation during angiogenesis in malignant glioma.     

A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells.     

Flow cytometric analysis of adherence of Porphyromonas gingivalis to oral epithelial cells

By using fluorescence microscopy, fluorescently labeled Porphyromonas gingivalis W50 was shown to adhere to oral epithelial (KB) cells as discrete cells or small cell aggregates, whereas P. gingivalis ATCC 33277 bound as large cell aggregates. Flow cytometric analysis showed that for P. gingivalis W50 there was a logarithmic relationship between the bacterial cell ratio (BCR), that is the number of bacterial cells to KB cells, and the percentage of KB cells with W50 cells attached. This percentage of KB cells with W50 attached reached a plateau of approximately 84% cells at a BCR of 500:1. In contrast, a quadratic relationship was observed between BCR and the percentage of KB cells with P. gingivalis ATCC 33277 attached, reaching a maximum of 47% at a BCR of 100:1 but decreasing to 7% at a BCR of 1,000:1. The lower binding of ATCC 33277 at high cell concentrations was attributed to autoaggregation. P. gingivalis W50 cells treated with an inhibitor (Nalpha-p-tosyl-L-lysine chloromethyl ketone [TLCK]) of its RgpA-Kgp proteinase-adhesin complex exhibited significantly reduced binding to KB cells than to untreated cells, suggesting a role for proteinase activity in binding to KB cells. Competitive inhibition with purified proteinase-active and TLCK-inactivated RgpA-Kgp complex significantly decreased the adherence of P. gingivalis W50 cells to KB cells. Furthermore, isogenic mutants of P. gingivalis W50 lacking the kgp gene product, but not the rgpA or rgpB gene products, exhibited significantly decreased adherence to KB cells compared to the wild type.     

Discrete protein determinant directs the species-specific adherence of Porphyromonas gingivalis to oral streptococci

For pathogens to survive in the human oral cavity, they must identify a suitable niche in the complex multispecies biofilm that exists on oral tissues. The periodontal pathogen Porphyromonas gingivalis adheres to Streptococcus gordonii by interacting with a specific region of the streptococcal SspB polypeptide, designated BAR. However, it does not adhere to Streptococcus mutans, which expresses SpaP, a highly conserved homolog of SspB. Comparison of the predicted secondary structure of BAR with the corresponding region of SpaP suggested that the substitution of Asn for Gly1182 and Val for Pro1185 in SspB may confer a unique local structure that is not conserved in SpaP. A synthetic peptide of 26 amino acids that encompassed residues 1167 to 1193 of SspB promoted avid adherence of P. gingivalis, whereas a peptide derived from the region corresponding to BAR in SpaP was inactive. Substitution of Gly1182 and Pro1185 for Asn1182 and Val1185 in SspB by site-specific mutation generated proteins that were predicted to assume an SpaP-like secondary structure, and the purified proteins did not promote P. gingivalis adherence. Furthermore, Enterococcus faecalis strains expressing the site-specific mutants did not support adherence of P. gingivalis cells. In contrast, P. gingivalis adhered efficiently to E. faecalis strains expressing intact SspB or SspB-SpaP chimeric proteins containing BAR. These results suggest that a region of SspB consisting of 26 amino acids is sufficient to mediate the adherence of P. gingivalis to S. gordonii and that the species specificity of adherence arises from its interaction with a discrete structural determinant of SspB that is not conserved in SpaP.     

溶血磷脂酰胆碱对内皮细胞表面粘附分子表达的影响

大量的单核细胞募集是动脉粥样损伤形成的早期表现之一,相关的内皮细胞粘附分子在其中具有积极作用。本文研究了溶血磷脂酰胆碱（Ｌｙｓｏｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ,Ｌｙｓｏ－ＰＣ）对培养的人脐静脉内皮细胞（ＨＵＶＥＣｓ）膜上细胞间粘附分子－（Ｉｎｔｅｒｃｅｌｌｕｌａｒ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ－１,ＩＣＡＭ－１）、Ｅ选择素（Ｅｎｄｏｔｈｅｌｉａｌ－ｌｅｕｋｏｃｙｔｅ ａｄｈｅｓｉｏｎ     

Alkali-Treated LPS of Yersinia enterocolitica does not Induce Expression of E-Selectin, ICAM-1 or VCAM-1 on Endothelial Cells but may Mediate Antibody- and Complement-Dependent Cell Injury

Lipopolysaccharide (LPS) prepared from a rough mutant of Salmonella typhimurium and deacylated enzymatically (dLPS) does not promote neutrophil adherence to human umbilical vein endothelial cells (HUVECs). This paper reports that similarly, a smooth form of LPS prepared from Yersinia enlerocolitica O:3, a serotype known to trigger reactive arthritis in humans, and treated with alkali (yersinia LPS-OH) failed to augment neutrophil adherence to HUVECs. Studies of the mechanism underlying the poor augmentation revealed that neither enzymatically deacylated LPS from Escherichia coli J5 (J5 dLPS) nor yersinia LPS-OH stimulated expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, whereas both Intact J5 LPS and yersinia LPS were stimulatory. Impaired up-regulation could not be explained by decreased binding of yersinia LPS-OH to HUVECs. Furthermore, ⁵¹Cr-labelled HUVECs treated with different concentrations of yersinia LPS-OH released ⁵¹Cr in the presence of anti-yersinia anti-0 antibody and complement. J5 dLPS and yersinia LPS-OH inhibited up-regulation of the adhesion molecules induced by J5 LPS and yersinia LPS but not that induced by tumour necrosis factor alpha.
Taken together, the results suggest that although yersinia LPS-OH can depress development of acute inflammation by inhibiting up-regulation of etidothelial-cell adhesion molecules, sufiicient LPS-OH is bound to induce cell injury and thereby inflammation in the prescence of specific antibody and complement. The findings may have pathogenetic implications in yersinia-triggered reactive arthritis characterized by dissemination of yersinia LPS throughout the body.     

Anti-metastatic prostacyclins inhibit the adhesion of colon carcinoma to endothelial cells by blocking E-selectin expression

Prostacyclins have long been shown to have anti-metastatic activity. One hypothesis is their modulation of cell adhesion molecule (CAM) expression by target organ endothelial cells. We have postulated that prostacyclin, its analogs, and mechanistic mimics decrease colon carcinoma adhesion to cytokine-stimulated endothelial cells by blocking endothelial expression of the adhesion molecule E-selectin, but not the vascular cell adhesion molecule-1 (VCAM-1). Cultured human microvascular endothelial cells (HDMEC) were pre-incubated with prostacyclin (PGI₂), dibutyrl-CAMP (dbcAMP), forskolin (FOR), and/or iso-methylbutylxanthine (IBMX) for 15 min, then co-incubated with the cytokine tumor necrosis factor (TNF) for 4 h. HDMEC surface expression of E-selectin and VCAM-1 was evaluated by flow cytometry and ELISA. Adherence of ⁵¹Cr-labeled colon carcinoma cells to HDMEC monolayers was then determined. In parallel assays, HDMECs were incubated with anti-E-selectin and anti-VCAM-1 monoclonal antibody (1:100) prior to the addition of tumor cells. Prostacyclins, its analogs, and mimics significantly reduced E-selectin expression by HDMEC, while the reduction of VCAM-1 expression was much less pronounced. Prostacyclins also significantly decreased colon carcinoma adherence to stimulated HDMECs. The inhibition of E-selectin expression, but not VCAM-1 expression, corresponded to the reduction of tumor cell adherence. Prostacyclin's effects on tumor adhesion were nullified by pre-incubation with E-selectin antibody. The inhibition of colon carcinoma adherence to cytokine-stimulated endothelial cells treated with prostacyclin, its analogs, and mimics appears to result from blocking endothelial E-selectin, but not VCAM-1, expression. These data support the hypothesis that prostacyclins may exert their anti-metastatic effect, in part, by inhibiting CAM-mediated adherence of colon carcinoma to endothelial cells in metastatic target organs.     

Aspergillus fumigatus stimulates leukocyte adhesion molecules and cytokine production by endothelial cells in vitro and during invasive pulmonary disease

Invasive aspergillosis is characterized by hyphal invasion of the blood vessels, which contributes to the pathogenesis of this disease. During this angioinvasion, Aspergillus fumigatus interacts with the endothelial cell lining of the blood vessels. We investigated the response of vascular endothelial cells to A. fumigatus infection in vitro and in mouse models of invasive pulmonary aspergillosis. Infection with hyphae, but not with conidia, stimulated endothelial cells to synthesize E-selectin, vascular cell adhesion molecule 1 (VCAM-1), interleukin 8, and tumor necrosis factor alpha (TNF-alpha) in vitro. Killed hyphae induced approximately 40% less stimulation than did live hyphae. Endothelial cell stimulation required contact between the hyphae and endothelial cells but not endocytosis of the organisms. Studies with DeltagliP and DeltastuA null mutants of A. fumigatus indicated that the extent of endothelial cell stimulation was not influenced by gliotoxin or other StuA-dependent factors synthesized by A. fumigatus. In neutropenic mice infected with wild-type A. fumigatus, increased pulmonary expression of E-selectin, cytokine-induced neutrophil chemoattractant (KC), and TNF-alpha occurred only when neutropenia had resolved. In nonneutropenic mice immunosuppressed with corticosteroids, A. fumigatus stimulated earlier pulmonary expression of E-selectin, VCAM-1, and KC, while expression of intercellular adhesion molecule 1 and TNF-alpha was suppressed. In both mouse models, expression of E-selectin and KC was associated with high pulmonary fungal burden, angioinvasion, and neutrophil adherence to endothelial cells. Therefore, the expression of leukocyte adhesion molecules and secretion of proinflammatory cytokines by endothelial cells in response to A. fumigatus could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of angioinvasion.