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.     

Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial fimbria-dependent invasion

Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis. Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E-/- mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-type P. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-type P. gingivalis by fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purified P. gingivalis antigens. Following a wild-type P. gingivalis challenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonists Staphylococcus aureus lipoteichoic acid (SLTA; TLR2 ligand) and Escherichia coli lipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-type P. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA and E. coli LPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited by E. coli LPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasive P. gingivalis stimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.     

Endothelial cell-derived foam cells fail to express adhesion molecules (ICAM-1 and VCAM-1) for monocytes

The purpose of this study was to assess the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cell-derived foam cells. Hamster aortic endothelial cells (HAEC) in culture were exposed to hypercholesterolemic or normal homologous serum for 24 h. At the end of the incubation period, HAEC exposed to hypercholesterolemic serum exhibited numerous lipid droplets and had a general aspect of foam cells. When examined for the expression of ICAM-1 and VCAM-1 (by indirect immunofluorescence) normal HAEC expressed constitutively (to low level) on their surface these adhesion molecules; however HAEC-derived foam cells failed to display any labeling. To further assess these results, HAEC were first incubated with normal or hypercholesterolemic sera (as above) and then exposed to freshly isolated normal hamster blood monocytes. These experiments showed that monocytes adhered in small number to normal cells and failed to adhere to the surface of HAEC-derived foam cells. Together these data indicate that endothelial cell-derived foam cells: a) do not express ICAM-1 and VCAM-1 on their surface; b) have low or no adhesion properties for monocytes and c) may represent an appropriate experimental model to study the cellular alterations that take place in the advanced stages of atherosclerosis.     

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.     

Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.     

Smooth muscle cell phenotype alters cocultured endothelial cell response to biomaterial-pretreated leukocytes

Model in vitro culturing systems were developed to analyze roles of biomaterial-induced leukocyte activation on endothelial cell (EC) and smooth muscle cell (SMC) phenotype, and their crosstalk. Isolated monocytes or neutrophils were pretreated with model biomaterial beads and applied directly to "more secretory" (cultured in media containing 5% fetal bovine serum) or forced contractile (serum and growth factor starved) human aortic SMCs (HASMCs), or to the human aortic EC (HAEC) surface of HAEC/HASMC cocultures (HASMC phenotype varied to be "more or less secretory") for 5 or 24 h of static culture. Surface expression of proinflammatory [ICAM-1, VCAM-1, E-selectin], procoagulant (tissue factor), and anticoagulant (thrombomodulin) markers, as well as HAEC proliferation, were assessed by flow cytometry. Incubation of HAEC with biomaterial-pretreated monocytes (and neutrophils to lesser degree) suppressed HAEC proliferation and induced a proinflammatory/procoagulant HAEC phenotype. This HAEC phenotype was amplified in coculture with "more secretory" HASMCs and subdued in coculture with "less secretory" HASMCs. Direct incubation of biomaterial-pretreated monocytes or neutrophils with "more secretory" HASMCs further increased HASMC ICAM-1 and tissue factor expression. Direct incubation of biomaterial-pretreated monocytes or neutrophils with forced contractile HASMCs upregulated ICAM-1, VCAM-1, and tissue factor expression above the presence of serum-containing media alone.     

Effect of immunization on experimental Bacteroides gingivalis infection in a murine model.

BALB/c mice were immunized with an invasive (A7A1-28) or noninvasive (381) Bacteroides gingivalis strain, Bacteroides intermedius, or Ringer solution. All immunized mice were subsequently challenged with the invasive B. gingivalis strain and examined for septicemia or secondary spread of the infection or both. Mice immunized with the invasive B. gingivalis strain localized the infection to the challenge site. Mice immunized with the noninvasive B. gingivalis strain, B. intermedius, or Ringer solution developed spreading infections. These data suggest that immunization with an invasive B. gingivalis strain can alter the course of subsequent infections.     

Transformation and expression of a cloned fimA gene in Porphyromonas gingivalis

The Porphyromonas gingivalis fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene, fimA381, encoding the fimbrial subunit of P. gingivalis 381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned fimA381 gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the fimA381 gene into the plasmid vector pYH420 and transformed into the restriction-deficient P. gingivalis host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host's native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the fimA381 gene. These results suggested that transformation of fimA gene from a different strain of P. gingivalis followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.     

Porphyromonas gingivalis strain-dependent activation of human endothelial cells

Porphyromonas gingivalis is an important bacterium involved in periodontal diseases. Colonization by periodontopathogens has been associated with severe local inflammatory reactions in the connective tissue. In this study we characterized P. gingivalis-mediated infection and activation of human umbilical vein endothelial cells by using two strains of different virulence capacities, strains ATCC 53977 and DSMZ 20709. Both strains were able to adhere to and infect endothelial cells with an infection rate of 0.48% for ATCC 53977 and 0.007% for DSMZ 20709. The triggering of two signal transduction pathways in P. gingivalis-infected endothelial cells was demonstrated for both strains, with a rapid increase of p38 mitogen-activated protein kinase phosphorylation and a more delayed degradation of IkappaBalpha, followed by nuclear translocation of NF-kappaB. In addition, both strains induced enhanced expression of endothelial adhesion molecules E-selectin and intracellular adhesion molecule 1 (ICAM-1). Target cell activation was independent of bacterial fimbriae expression since the fimA knockout strain A7436 DeltafimA induced the same level of ICAM-1 as the corresponding wild type (A7436-WT). Thus, two P. gingivalis strains, ATCC 53799 and DSMZ 20709, infect endothelial cells and trigger signaling cascades leading to endothelial activation, which in turn may result in or promote severe local and systemic inflammation.     

Keratinocyte growth factor (KGF) decreases ICAM-1 and VCAM-1 cell expression on bronchial epithelial cells

Activation of leucocytes during airway inflammatory reaction involves adhesion to bronchial epithelial cells (BEC), a process implicating specific interactions between glycoproteins with epithelial cell surface proteins, mainly intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In this study, the effect of keratinocyte growth factor (KGF), a growth factor involved in pulmonary epithelium repair, was evaluated on adhesion molecule expression with BEAS-2B cells and BEC and granulocyte adherence to BEAS-2B. The modulation by KGF of membrane and mRNA expression of ICAM-1 and VCAM-1 was studied on confluent cells stimulated or not with tumour necrosis factor-alpha (TNF) (200 UI/ml) or TNF and interleukin (IL)-4 (50 UI/ml and 10 ng/ml). Levels of soluble-(s)ICAM-1 and sVCAM-1 were measured by ELISA. Although moderately, KGF significantly decreased membrane ICAM-1 expression in unstimulated BEAS-2B cells (24% inhibition at 100 ng/ml) or in TNF- or TNF + IL-4-stimulated cells (22.5 and 18.7% inhibition, respectively). Treatment with KGF tended to decrease VCAM-1 expression in TNF- and TNF + IL-4-stimulated BEAS-2B (P = n.s. and P < 0.05, 14 and 15% inhibition, respectively). In primary culture of BEC, adhesion molecule expression was also reduced. ICAM-1 and VCAM-1 mRNA expression were also inhibited by KGF. Levels of sICAM-1 and sVCAM-1 were not significantly increased in supernatants from KGF-treated cells (30% and 24% increase at 100 ng/ml, respectively) compared to controls. Moreover, KGF decreased by 31% the adherence of neutrophils to TNF-activated BEAS-2B. In conclusion, KGF decreases ICAM-1 and VCAM-1 expression and neutrophil adherence in BEC. These suggest its involvement in the resolution of the inflammatory reaction.     

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.     

Effect of ultraviolet light on the expression of adhesion molecules and T lymphocyte adhesion to human dermal microvascular endothelial cells

In order to determine the effect of ultraviolet radiation (UVR) on the cell adhesion molecules expressed in human dermal microvascular endothelial cells (HDMEC), the cells were exposed to varying UVR doses and the cell surface was examined for expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM- 1), and E-selectin. The effect of UVB irradiation on the binding of T lymphocytes to HDMEC was also examined. UVA irradiation did not affect the surface expression of ICAM-1, VCAM-1, or E-selectin on the HDMEC. However, following UVB exposure, ELISA demonstrated a significant increase in the baseline ICAM-1 cell surface expression on the HDMEC. However, no induction of either E-selectin or VCAM-1 was noted. UVB also significantly augmented ICAM-1 induction by IL-1alpha and TNF-alpha. VCAM-1 was induced by stimulating HDMEC with IL-1alpha following a UVB irradiation dose of 100 mJ/cm2. Flow cytometric analysis of the HDMEC stimulated with IL-1alpha for 24h demonstrated that 12% of the cells expressed VCAM-1 but either IL-1alpha or UVB irradiation alone failed to induce VCAM-1 expression. Enhancement of T cell-HDMEC binding by IL-1alpha or TNF-alpha treatment was not significantly affected after UVB irradiation. This study demonstrated that UVB irradiation can alter ICAM-1 and VCAM-1 expression on the HDMEC surface and that augmentation of ICAM-1 expression and the IL-1alpha-dependent induction of VCAM-1 following UVB exposure might be important steps in the pathogenesis of sunburn.     

Biological function of CD40 on human endothelial cells: costimulation with CD40 ligand and interleukin-4 selectively induces expression of vascular cell adhesion molecule-1 and P-selectin resulting in preferential adhesion of lymphocytes

The expression of adhesion molecules on vascular endothelial cells determines the pattern of migration and extravasation of leucocytes in inflammation and immunity. Here we show that costimulation with CD40 ligand (CD40L) and interleukin (IL)-4 (or IL-13) gives rise to a unique pattern of adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). CD40 ligation alone enhanced expression of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin whereas IL-4 and IL-13 increased expression of VCAM-1 and P-selectin but not ICAM-1 or E-selectin. When IL-4 and CD40L were combined there was an additional increase of both VCAM-1 and P-selectin, but ICAM-1 and E-selectin were both inhibited. The combined effects of IL-4 and CD40L signalling were not the result of altered response kinetics, enhanced sensitivity of the endothelium, or increased expression of CD40 or the IL-4 receptor. The rise in VCAM-1 expression induced by combined IL-4 and CD40L stimulation was slower and more sustained than with tumour necrosis factor-alpha (TNF-alpha) and occurred only on a subset (75-80%) of the endothelial cell population compared to 100% with TNF-alpha. Costimulation with IL-4 and CD40L increased adhesion of T cells and B cells above levels obtained with either signal alone, but decreased adhesion of neutrophils. Furthermore, CD40 and IL-4 synergistically increased IL-6 but decreased IL-8 production by HUVEC. These results show that interactions between IL-4 and CD40 on endothelial cells give rise to specific patterns of adhesion molecule expression and cytokine production that may have important implications for lymphocyte and neutrophil migration and function at sites of inflammation.     

Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 cooperatively contribute to the cutaneous Arthus reaction

Immune complex (IC)-induced inflammation is mediated by inflammatory cell infiltration, a process that is highly regulated by expression of multiple adhesion molecules. The roles and interactions of ICAM-1 and VCAM-1, the major regulators of leukocyte firm adhesion, were examined in the cutaneous reverse-passive Arthus reaction using ICAM-1-deficient (ICAM-1-/-) mice and blocking mAb against VCAM-1. Within 8 h, IC challenge of wild-type mice induced edema, hemorrhage, interstitial accumulation of neutrophils and mast cells, as well as production of TNF-alpha and IL-6. All of these inflammatory parameters were reduced significantly in ICAM-1-/- mice. The blockade of VCAM-1 in wild-type mice did not affect any inflammatory parameters. In contrast, ICAM-1-/- mice treated with anti-VCAM-1 mAb had significantly reduced edema, hemorrhage, and neutrophil infiltration. Furthermore, VCAM-1 blockade in ICAM-1-/- mice suppressed cutaneous TNF-alpha and IL-6 production. Thus, VCAM-1 plays a complementary role to ICAM-1 in the cutaneous Arthus reaction by regulating leukocyte accumulation and proinflammatory cytokine production.     

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and function on cultured human glomerular epithelial cells.

Glomerular epithelial cells are involved in extracapillary inflammation (crescents) but the mechanisms of this extracapillary accumulation of macrophages, epithelial cells and occasional lymphocytes are unknown. Human glomerular parietal epithelial cells express ICAM-1 and VCAM-1 on immunohistological stains of renal biopsies. We studied the expression of these cell adhesion molecules on cultured human glomerular epithelial cells (HGEC), their regulation by pro-inflammatory cytokines, and their role in mediating the adhesion of concanavalin A (Con A)-activated peripheral blood mononuclear cells. Human glomerular epithelial cells in culture constitutively express ICAM-1 and VCAM-1. The expression of ICAM-1 was not significantly altered by tumour necrosis factor-alpha (TNF-alpha) (P = 0.32), IL-1 beta (P = 0.24), interferon-gamma (IFN-gamma) (P = 0.66) or IL-4 (P = 0.85). VCAM-1 expression was increased by all four cytokines, but only significantly so by IL-4 (P = 0.0001). Con A-stimulated, monocyte-depleted peripheral blood lymphocytes bound to human glomerular epithelial cells, median 28.9% (range 14.5-37.9%). This adherence was significantly inhibited by anti-ICAM-1 (P = 0.03) and anti-LFA-1 (P = 0.02), but not by anti-VCAM-1 (P = 0.13) or by antibody to von Willebrand factor (P = NS). The interaction between ICAM-1 on HGEC and LFA-1 on mononuclear cells may be important in the pathogenesis of extracapillary inflammation in glomerulonephritis.     

IL-1 and TNF independent pathways mediate ICAM-1/VCAM-1 up-regulation in ischemia reperfusion injury

In vitro studies have suggested that targeting interleukin (IL)-1 and tumor necrosis factor (TNF) can be used to regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and potentially treat kidney inflammation. We therefore evaluated ICAM-1 and VCAM-1 regulation in knockout (KO) mice deficient in both IL-1 receptor 1 (R1) and TNF-R1 during renal ischemia reperfusion injury. ICAM-1 and VCAM-1 mRNA expression was measured with specific murine probes and Northern blotting (n =4/group). Protein expression was measured using immunohistochemistry. Serum creatinine (SCr), tubular histology, and neutrophil infiltration into postischemic kidneys were also quantified. ICAM-1 and VCAM-1 mRNA expression increased in both wild-type (WT) and KO mice at 2, 6, and 24 h. Protein expression of ICAM-1 and VCAM-1 was also increased at 24 h postischemia. SCr levels and tubular necrosis scores were comparable in WT and KO mice at 24 and 48 h. Neutrophil migration in KO mice was decreased at 24 h but comparable to WT at 48 h. These data demonstrate that IL-1 and TNF are not essential for postischemic increases in ICAM-1 and VCAM-1.     

Effects of mixed leukocyte reaction,hydrocortisone and cyclosporine on expression of leukocyte adhesion molecules by endothelial and mesangial cells.

We investigated the effects of mixed leukocyte reaction (MLR), hydrocortisone (HC) and cyclosporine A (CsA) on the expression of leukocyte adhesion molecules on the mesangial (MC) and endothelial cells (EnC). Cell surface enzyme immunoassay showed that INFnu, IL-1beta, or TNF alpha stimulated expression of ICAM-1, or VCAM-1 on MC after 24 hours. Flow cytometric analysis demonstrated that MLR supernatant induced a marked increase in mean fluorescence of or % of cells highly expressing intercellular adhesion molecule(ICAM)-1 or vascular cell adhesion molecule (VCAM)-1 on both cells after 24 hours (p<0.001). HC treatment(300 ng/ml) during MLR effectively inhibited MLR-induced upregulation of ICAM-1 and VCAM-1 on both cells (p<0.005). When MLR supernatant with HC was added to adhesion molecule assay, there was no inhibitory effect of HC on VCAM-1. CsA treatment (500 ng/ml) during MLR caused a modest decrease in upregulation of VCAM-1 on EnC (p<0.05), but had no effects on ICAM-1 on both cells. CsA directly decreased expression of VCAM-1 on MC. In conclusion, alloreactive lymphocytes and monocytes upregulate the expression of VCAM-1 and ICAM-1 on target cells probably by the mediation of cytokines. HC effectively prevents MLR-induced upregulation of VCAM-1 and ICAM-1. CsA does not increase the expression of VCAM-1 and ICAM-1.     

促炎症细胞因子刺激人类关节滑膜B型细胞表达粘附分子

目的透析相关性淀粉样变（DRA）病人关节滑膜组织粘附分子表达增强,并与局部炎细胞浸润密切相关。旨在探讨DRA时诱导滑膜细胞粘附分子表达上调的机制。方法分离正常人关节滑膜B型细胞,与晚期糖基化终产物修饰的β     

Proteasome inhibitors block VCAM-1 and ICAM-1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor-ϰB

Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up-regulation of cell surface adhesion molecules, including VCAM-1 and ICAM-1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)-1β induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n-acetyl-leucinyl-leucinyl-norleucinal (norLEU), as well as specific protease inhibitors, n-tosyl-Lys-chloromethylketone and n-tosyl-Phe-chloromethylketone, blocked IL-1β induction of VCAM-1 and ICAM-1 promoter-driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM-1 and ICAM-1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)-?B in response to IL-1β stimulation. In contrast, norLEU did not prevent IL-1β-induced nuclear translocation of NF-?B. The effects of norLEU were specific because it did not inhibit the IL-1β induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL-1β induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells.