Upregulation Of Endothelial Adhesion Molecules Research Articles

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell–cell adhesion of colorectal cancer cells

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Upregulation of endothelial adhesion molecules by lysophosphatidylcholine

Lysophosphatidylcholine induces expression of adhesion molecules; however, the underlying molecular mechanisms of this are not well elucidated. In this study, the intracellular signaling by which lysophosphatidylcholine upregulates vascular cell adhesion molecule-1 and P-selectin was delineated using YPEN-1 and HEK293T cells. The results showed that lysophosphatidylcholine dose-dependently induced expression of vascular cell adhesion molecule-1 and P-selectin, accompanied by the activation of transcription factor nuclear factor kappaB. However, the nuclear factor kappaB inhibitor caffeic acid phenethyl ester (CAPE) and the antioxidant N-acetylcysteine only partially blocked lysophosphatidylcholine-induced adhesion molecules. Subsequently, we found that the lysophosphatidylcholine receptor G protein-coupled receptor 4 (GPK4) was expressed in YPEN-1 cells and triggered the cAMP/protein kinase A/cAMP response element-binding protein pathway, resulting in upregulation of adhesion molecules. Further evidence showed that overexpression of human GPK4 enhanced lysophosphatidylcholine-induced expression of adhesion molecules in YPEN-1 cells, and enabled HEK293T cells to express adhesion molecules in response to lysophosphatidylcholine. In conclusion, the current study suggested two pathways by which lysophosphatidylcholine regulates the expression of adhesion molecules, the lysophosphatidylcholine/nuclear factor-kappaB/adhesion molecule and lysophosphatidylcholine/GPK4/cAMP/protein kinase A/cAMP response element-binding protein/adhesion molecule pathways, emphasizing the importance of the lysophosphatidylcholine receptor in regulating endothelial cell function.

Tetrahydrobiopterin (BH4) attenuates neutrophil adhesion/transmigration in myocardial ischemia/reperfusion injury

Attenuating endothelial dysfunction during early reperfusion (i.e., within the first 5 min) inhibits upregulation of endothelial adhesion molecules, attenuates PMN-endothelium interaction, and restores post-reperfusion cardiac contractile function. BH4 is an essential cofactor of endothelial nitric oxide synthase to produce nitric oxide. We hypothesize that BH4, not dihydrobiopterin (BH2, i.e., oxidized BH4), given during reperfusion will attenuate PMN vascular adherence and infiltration and restore cardiac function in isolated rat hearts subjected to ischemia (20 min) and reperfusion (45 min)and reperfused with PMNs. In BH4 treated I/R+PMN hearts (10uM, n=6), left ventricular developed pressure (LVDP) at 45 min post reperfusion significantly recovered to 85±8% of baseline, whereas I/R+PMN hearts and BH2-perfused hearts only recovered to 53±7% and 64±9% of baseline (P<0.01). IR/PMN perfused BH4 hearts were associated with decreased PMN coronary vascular adherence, tissue infiltration, and attenuated ICAM-1 expression, compared to IR/PMN hearts or BH2-perfused hearts. This preliminary data suggest that BH4 attenuates PMN induced post-I/R LVDP dysfunction, in part, by attenuating PMN vascular adherence and tissue infiltration, possibly by inhibiting ICAM-1 expression. This study was supported by NHLBI Grant 1R15HL-76235-01 and Center For Chronic Disorders Of Aging. Philadelphia College of Osteopathic Medicine.

Pitavastatin prevents NMDA‐induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.

Anti‐angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte‐endothelium interactions and infiltration in tumors

Tumor escape from immunity, as well as the failure of several anti-cancer vaccination and cellular immunotherapy approaches, is suggested to be due to the angiogenesis-mediated suppression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions. We hypothesized that inhibition of angiogenesis would overcome this escape from immunity. We investigated this in vivo by means of intravital microscopy and ex vivo by immunohistochemistry in two mouse tumor models. Angiogenesis inhibitors anginex, endostatin, and angiostatin, and the chemotherapeutic agent paclitaxel were found to significantly stimulate leukocyte-vessel wall interactions by circumvention of EC anergy in vivo, i.e., by the up-regulation of endothelial adhesion molecules in tumor vessels. This was confirmed by in vitro studies of cultured EC at the protein and mRNA levels. The new angiostatic designer peptide anginex was most potent at overcoming EC anergy; the enhanced leukocyte-vessel interactions led to an increase in the numbers of tumor infiltrating leukocytes. While anginex inhibited tumor growth and microvessel density significantly, the amount of infiltrated leukocytes (CD45), as well as the number of CD8+ cytotoxic T lymphocytes, was enhanced markedly. The current results suggest that immunotherapy strategies can be improved by combination with anti-angiogenesis.

FK778: New Cellular and Molecular Mechanisms of Action

Purpose The new malononitrilamide FK778 is currently being evaluated as an immunosuppressant for organ transplantation. Its main mechanism is inhibition of a pivotal enzyme of pyrimidine biosynthesis. This report revealed new mechanisms of action on different cell types involved in acute and chronic allograft rejection. Methods Purified Brown-Norway rat aortic endothelial cell (EC) cultures were pretreated with several concentrations of FK778. Endothelial adhesion molecule expression (ICAM-1/VCAM-1) stimulated with TNF-α was quantified by immunofluorescence. Purified Lewis rat lymphocytes (LC) incubated with FK778 were stimulated via TCR/CD28 signals, and CD25 expression was quantified using FACS analysis. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Lymphocyte–EC interaction was assessed by micromanipulator-assisted single-cell adhesion assays. Finally, smooth muscle cell (SMC) proliferation and migration was analyzed. Uridine addition was used in all assays to reverse the pyrimidine synthesis blockade. Results TNF-α stimulation and TCR/CD28 co-stimulation significantly increased EC ICAM-1/VCAM-1-expression and LC CD25 surface expression, respectively. These effects were dose-dependently inhibited by FK778 and were not reversed by the addition of uridine. FK778 dose-dependently attenuated LC adhesion to allogeneic EC. The dose-dependent inhibition of SMC proliferation by FK778 was abolished by uridine addition, whereas the inhibitory effect on SMC migration was not affected by uridine supplementation. Conclusions FK778 directly reduced endothelial adhesion molecule up-regulation, inhibited lymphocyte activation, and attenuated lymphocyte-endothelium interactions, critical early steps in graft rejection. These effects were separate from the blockade of pyrimidine synthesis. The antiproliferative potency of FK778 on SMC may be an important mechanism to inhibit the fibroproliferative lesions of chronic organ rejection.

Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants

In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).

Gaining More From Gamma Globulins

The use of intravenous immunoglobulin (IVIg), which is immunoglobulin G pooled from thousands of healthy donors, in the treatment of immunodeficient and autoimmune diseases has grown during the past 2 decades. Although its initial application was largely limited to replacement therapy in hypogammaglobulinemia, IVIg is gaining acceptance as therapy for autoimmune thrombocytopenia purpura, and a number of other autoimmune diseases such as multiple sclerosis.1 Although the exact mechanisms underlying the protection conferred by IVIg in these immune disorders remain undefined, several potential molecular and cellular targets have been proposed. For example, IVIg can block Fc receptors on macrophages and effector cells to reduce the phagocytic capacity of these cells. IVIg may also regulate the immune response by reacting with a number of membrane receptors on T cells, B cells, and monocytes that are pertinent to autoreactivity and induction of tolerance to self.1 Recent work has also revealed a beneficial effect of IVIg in systemic inflammatory disorders such as sepsis and asthma. It has been suggested that IVIg may exert its antiinflammatory effects by attenuating complement-mediated attack,2 inducing antiinflammatory cytokines, and reducing the production of proinflammatory cytokines such as tumor necrosis factor-α, interferon-γ and interleukin-13 (Figure). Many of these mechanistic studies of IVIg effects on the inflammatory response are based on in vitro models and in vivo data are lacking. Differential effects of IVIg on several steps in the inflammatory cascade: Numbers denote potential antiinflammatory actions of IVIg; letters denote possible adverse effects of IVIg that may worsen the inflammatory response. 1, Attenuation of complement deposition on vascular endothelium; 2, reduction of chemokine and cytokine generation; 3, attenuation of leukocyte recruitment at least in part via downregulation of adhesion molecules on leukocytes and inhibition of endothelial adhesion molecule upregulation; 4, abrogation of protein extravasation/vascular leak; 5, prevention …

Monocyte Adhesion to Xenogeneic Endothelium during Laminar Flow Is Dependent on α-Gal-Mediated Monocyte Activation

Monocytes are the predominant inflammatory cell recruited to xenografts and participate in delayed xenograft rejection. In contrast to allogeneic leukocytes that require up-regulation of endothelial adhesion molecules to adhere and emigrate into effector tissues, we demonstrate that human monocytes adhere rapidly to unstimulated xenogeneic endothelial cells. The major xenoantigen galactosealpha(1,3)galactosebeta(1,4)GlcNAc-R (alpha-gal) is abundantly expressed on xenogeneic endothelium. We have identified a putative receptor for alpha-gal on human monocytes that is a member of the C-type family of lectin receptors. Monocyte arrest under physiological flow conditions is regulated by alpha-gal, because cleavage or blockade results in a dramatic reduction in monocyte adhesion. Recruitment of human monocytes to unactivated xenogeneic endothelial cells requires both alpha(4) and beta(2) integrins on the monocyte; binding of alpha-gal to monocytes results in rapid activation of beta(2), but not alpha(4), integrins. Thus, activation of monocyte beta(2) integrins by alpha-gal expressed on xenogeneic endothelium provides a mechanism that may explain the dramatic accumulation of monocytes in vivo.

Upregulation of endothelial adhesion molecules in hearts with congestive and ischemic cardiomyopathy: immunohistochemical evaluation of inflammatory endothelial cell activation

In recent years many data emphasized, that inflammatory reactions seem to be involved in the pathogenesis of ischemic (ICM) and congestive (CCM) heart disease. Since, it is well known that endothelial adhesion molecules play a pivotal role in the initiation and maintenance of inflammatory reactions we therefore, evaluated the endothelial expression of a wide variety of different adhesion molecules in hearts suffering from ICM and CCM. Tissue samples from coronary arteries, and left and right ventricle myocardium originating form heart with ICM and CCM were evaluated. Tissue samples from healthy human donor hearts, which were not transplanted, served as controls. Evaluated adhesion molecule expression: selectin-family ELAM-1, CD62, immunoglobulin-supergene-family PECAM-1, ICAM-1, VCAM-1, integrin-family VLA-1,-2,-3,-4,-5, and -6, complementary-adhesion-molecules CD34, CD44 and the von-Willebrand-factor (vWF). While endocardial surfaces and coronary arteries revealed only little differences when comparing tissue samples originating from healthy donor hearts and those suffering from ICM and CCM, significant differences were found within the myocardial microvasculature. Both kinds of diseased hearts showed stronger expressions for CD62, ELAM-1, ICAM-1 and VCAM-1 (only CCM) than controls. More and above, integrin molecules showed differential expressions too. Whereas, VLA-1 showed stronger expressions in diseased hearts, VLA-3,-5, and -6 were expressed much weaker in those hearts. Complementary adhesion molecules (CD34/CD44) did not show significant differences and the vWF was not found in any sample. Inflammatory reactions play a pivotal role in the propagation and maintenance of both these cardiac detoriating diseases.

ABCA1 Gene Deletion Protects against Cerebral Malaria: Potential Pathogenic Role of Microparticles in Neuropathology

The ATP-binding cassette transporter A1 (ABCA1) modulates the transbilayer distribution of phosphatidylserine at the outer leaflet of the plasma membrane. This external exposure of phosphatidylserine is a hallmark of microparticle production and is impaired in ABCA1(-/-) mice. In this study, we report about the complete resistance to cerebral malaria of these mice. On analysis of histological and systemic parameters we evidenced an impairment of cellular responses to Plasmodium berghei ANKA infection in ABCA1(-/-) mice, as shown by lower plasma tumor necrosis factor levels, a weaker up-regulation of endothelial adhesion molecules in brain microvessels, a reduced leukocyte sequestration, as well as an ablated platelet accumulation. Besides, the number and the procoagulant activity of microparticles were dramatically reduced in the plasma of ABCA1(-/-) compared to ABCA1(+/+) mice. Moreover, microparticles derived from Plasmodium berghei ANKA-infected ABCA1(+/+) mice induced a significant increase of tumor necrosis factor release by noninfected macrophages. In ABCA1(-/-) mice platelet and macrophage responses to vesiculation agonists were ablated and reduced, respectively. Altogether, by pointing out the ABCA1 transporter as a major element controlling cerebral malaria susceptibility, these data provide a novel insight into its pathophysiological mechanisms and are consistent with a pathogenic role of microparticles in this neurological syndrome.

Antioxidants Inhibit Sickle Cell Adhesion to Activated Endothelium in an Ex Vivo Preparation.

In sickle cell anemia (SS), intravascular sickling and attendant transient occlusive events are implicated in the generation of reactive oxygen species (ROS) and vascular endothelial activation. Oxidants (as well as cytokines) result in up-regulation of endothelial adhesion molecules that may lead to increased adhesion of sickle red blood cells (SS RBC) and contribute to vaso-occlusion. We hypothesize that endothelial activation will augment the expression of adhesion molecules involved in SS RBC-endothelium interaction, and that antioxidants will have an inhibitory effect on this interaction. To this end, we have tested selected antioxidants for their efficacy to inhibit SS RBC adhesion in the ex vivo mesocecum vasculature of the rat stimulated by platelet-activating factor (PAF). PAF, a potent inflammatory agent, results in endothelial ROS generation (Suematsu et al. Am. J. Physiol. 1993), as well as in increased adhesion of SS RBCs (Kaul et al. Blood, 2000). Here, we have tested the efficacy of superoxide dismutase (SOD), catalase and polynitroxylated albumin (PNA). While SOD and catalase remove superoxide (O2 ·−) and H2O2, respectively, nitroxides covalently attached to albumin (i.e., PNA) act as intra-vascular SOD mimetic and also facilitate heme-mediated catalytic removal of H2O2. To directly demonstrate the involvement of ROS (O2 •- and H2O2), the ex vivo vasculature was pre-treated with PAF (200 pg/ml in Ringer-albumin solution) followed by infusion with SOD (1500 U in 5 ml Ringer-albumin) or catalase (10,000 U in 5 ml) (each 15-min incubation). The control preparations were incubated with PAF and Ringer-albumin. For experiments involving PNA, the vasculature was first treated with PAF and followed by infusion of PNA or control human serum albumin (HSA) (each 33 mg/ml) diluted in Ringer-albumin solution (incubation 30 min). In PAF or PAF/HSA-treated preparations, infusion of SS RBCs (Hct 30 in Ringer-albumin) caused widespread adhesion of these cells exclusively in postcapillary venules. The adhesion was inversely correlated with vessel diameter, resulting in frequent blockage of small-diameter venules. In contrast, preparations treated with PAF/SOD or PAF/catalase showed a marked decrease in adhesion in venules of all diameters resulting in a significantly lower Y-intercept of the regression line as compared with the control group (p<0.00001). On the other hand, PNA caused almost complete inhibition of SS RBC adhesion in PAF-treated preparation, demonstrating even a greater anti-adhesive efficacy as evidenced by lower Y-intercept (p<0.001–0.0001 vs. SOD and catalase). The decrease in adhesion was accompanied by a significantly lower peripheral resistance (p<0.03–0.018). These results show that antioxidants inhibit SS RBC adhesion to endothelium stimulated by PAF, probably by scavenging and/or blocking PAF-generated oxidants. Thus, blockade of oxidant generation may constitute an effective therapeutic strategy to prevent SS RBC adhesion.

Stress modulates peptidergic innervation and alters the cutaneous immune response: exacerbating pathomechanisms in atopic dermatitis?

Stress is said to induce itchiness of the skin and exacerbate inflammatory skin diseases such as atopic dermatitis. In this context, stress mediators such as the neuropeptide substance P play a role as immunmodulators and in a wider sense growth factors. For example, we were recently able to show that stress or treatment of mice with substance P is associated with mast cell degranulation, increased cutaneous inflammation and increased apoptosis in the hair follicle. However, local interactions between the nervous and immune systems, especially under perceived stress, have rarely been reported. Here, we show for the first time, that 24 and 48 h after sonic stress exposure, the number of SP‐immunoreactive nerve fibres in the back skin of C57BL/6 mice with all there hair follicles in the resting phase of the hair cycle (telogen, low numbers of cutaneous nerve fibres) increased significantly over non‐stressed mice with the strongest increase after 24 h. Such substance P immunoreactive nerve fibres contacted mast cells more frequently, which became significant after 48 h. At the same time, the percentage of degranulated mast cells increased significantly after 24 and 48 h with the strongest increase after 48 h when apoptotic cells also became significantly upregulated. The same stressor increased dermal infiltration, e.g. by eosinophils in C57BL/6 mice with experimentally induced allergic dermatitis over mice that were either stressed or had allergic dermatitis as well as over untreated controls. Increased infiltration was associated with increased epidermal thickness in stressed mice with allergic dermatitis and with an increased number of VCAM‐immunoreactive blood vessels. At the same time, the percentage of degranulated mast cells increased significantly, and the number of substance P‐immunoreactive peptidergic sensory nerve fibres decreased in the acute allergic dermatitis lesions. By semiquantitative RT‐PCR, allergic dermatitis increased cutaneous IL‐4 and to a lesser degree IFN‐γ production, but this was not affected by stress. Ultrastructural investigation showed unmyelinated peptidergic nerve fibres in a state of deterioration close to degranulating mast cells and eosinophils in the skin of stressed mice with allergic dermatitis, suggesting a decreased number of substance P‐immunoreactive nerve fibres due to active release of SP. This may lead to an upregulation of endothelial adhesion molecules and increased infiltration by immunocytes to the skin but at mRNA level does not alter the production of classical atopy‐related cytokines in skin. These data provide first evidence for stress‐induced exacerbation of cutaneous allergic diseases such as atopic dermatitis by local interaction of the peripheral nervous system with substance P.

FK778 attenuates lymphocyte-endothelium interaction after cardiac transplantation: in vivo and in vitro studies.

The malononitrilamide FK778 is a novel derivate of leflunomide and interacts with T- and B-cell function by inhibiting de novo pyrimidine synthesis. We investigated the effects of FK778 upon acute cardiac allograft rejection and upon adhesion molecule upregulation in experimental transplantation and by using in vitro cell culture. Heterotopic, abdominal cardiac transplantations were performed in the Brown Norway (BN) to Lewis (Lew) rat model. The study groups received daily low- or high-dose FK778 immunosuppression. FK778 plasma levels were quantified by HPLC. Grafts were harvested on the fifth postoperative day for histologic and immunohistologic examinations using computerized morphometry. Purified BN aortic endothelial cell cultures were pretreated with low- or high-dose FK778 according to FK778 plasma levels and were stimulated with tumor necrosis factor (TNF)-alpha. Adhesion molecule expression was quantified by immunofluorescence, FACS analysis, and Western blotting. Lymphocyte-endothelium adhesion assays were performed using purified Lew lymphocytes and radiolabeled TNF-alpha was used for receptor binding assays. FK778 treatment dose-dependently reduced graft mononuclear infiltration of CD4(+), CD8(+), and ED1(+) cells, but only high-dose FK778 treatment significantly reduced early upregulation of ICAM-1 and VCAM-1 in vivo. FK778 also dose-dependently reduced TNF-alpha-stimulated endothelial adhesion molecule upregulation in vitro, whereas the effect on VCAM-1 was more dominant. We did not find evidence that FK778 interferes with surface receptor binding of TNF-alpha. Lymphocyte adhesion to endothelial cell monolayers was significantly attenuated by FK778. Besides its inhibitory effect on pyrimidine synthesis, FK778 directly reduces endothelial adhesion molecule upregulation and attenuates lymphocyte-endothelium interaction, which is a critical step in graft rejection.

Extracellular role of HMGB1 in inflammation and sepsis.

High mobility group box 1 (HMGB1), a 30 kDa nuclear and cytosolic protein widely studied as a transcription factor and growth factor, has recently been identified as a cytokine mediator of lethal systemic inflammation (e.g. endotoxaemia and sepsis), arthritis and local inflammation. It is released by activated macrophages, and serum levels increase significantly during endotoxaemia, sepsis and arthritis with significant delayed kinetics in comparison with tumour necrosis factor (TNF) and interleukin-1beta. Recently identified biological activities of HMGB1 include activation of macrophages/monocytes to release proinflammatory cytokines, upregulation of endothelial adhesion molecules, stimulation of epithelial cell barrier failure, and mediation of fever and anorexia. Passive immunization with anti-HMGB1 antibodies confers significant protection against lethal endotoxaemia, sepsis, arthritis and lipopolysaccharide-induced acute lung injury, even when antibody administration is delayed until after the early TNF responses have resolved. Strategies to inhibit HMGB1 activity and release are being investigated in these and other preclinical models of acute and chronic inflammation.

Pressure distension stimulates the expression of endothelial adhesion molecules in the human saphenous vein graft

Background Mechanical trauma occurring during saphenous vein graft harvesting plays a major role in graft failure after coronary bypass surgery. There is increasing evidence that neutrophil–endothelial interaction is involved in the pathogenesis of early graft occlusion. This study evaluates the effect of pressure distension on the expression of endothelial adhesion molecules in human saphenous vein. Methods Segments of saphenous vein graft (SVG) were collected from 20 patients undergoing coronary bypass surgery. We evaluated the expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and P-selectin on SVG endothelium under basal conditions and after pressure distension at 300 mm Hg. In the same experimental setting we also evaluated adhesion of both unstimulated and activated neutrophils to the endothelium of SVG. Results Control endothelial cells exhibited only a weak staining for intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and P-selectin, whereas the levels of adhesion molecules increased significantly in the distended veins. Similarly, significantly greater adhesion of both unstimulated and activated neutrophils was observed in distended veins compared with control veins. Conclusions Pressure distension of SVG before coronary bypass surgery induces upregulation of endothelial adhesion molecules, with subsequent increase in neutrophil adhesion to the endothelium. Neutrophil adhesion to endothelial cells may contribute to early failure of SVG.

Stimulation of adhesion molecule expression by Helicobacter pylori and increased neutrophil adhesion to human umbilical vein endothelial cells

Helicobacter pylori upregulates endothelial adhesion molecules but the pattern is unclear. Human umbilical vein endothelial cells (HUVEC) were exposed to control medium or H. pylori 60190. Binding of monoclonal antibodies against P-selectin, E-selectin, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was determined using enzyme-linked immunosorbent assay. Binding of polymorphonuclear leukocytes to HUVEC was determined on cells exposed as above. After 6 h exposure to H. pylori, there were 30%, 124%, 167% and 100% increases in P-selectin, E-selectin, VCAM-1 and ICAM-1 levels and a 400% increase in polymorphonuclear leukocyte adhesion in HUVEC exposed to H. pylori. Effects of incubation for other intervals between 0 and 18 h are also described. H. pylori exerts some of its effects on gastric mucosa via gastric vasculature. This study gives insight into the pattern of H. pylori-associated endothelial adhesion molecule upregulation.

Mast cells induce upregulation of P-selectin and intercellular adhesion molecule 1 on carotid endothelial cells in a new in vitro model of mast cell to endothelial cell communication.

It is suggested that mast cells contribute to cell recruitment in inflammation through the upregulation of endothelial adhesion molecules. P-selectin and intercellular adhesion molecule(ICAM)-1 are two key adhesion molecules that have been associated indirectly with mast cell activity. The canine C2 mastocytoma cell line and primary cultures of canine carotid endothelial cells were used to establish a new in vitro model to help study the interaction between mast cells and endothelial cells. Carotid endothelial cells were incubated with mast cell mediators to uncover their effect on endothelial ICAM-1 and P-selectin expression. To assess the relative contributions of tumour necrosis factor (TNF)-alpha and histamine to such effect, an H1 antihistamine and a TNF-alpha blocking antibody were used. Prior to activation by mast cell mediators, P-selectin was expressed only within the cytoplasm, and ICAM-1 was constitutively expressed on the surface of the canine carotid endothelial cells. Both adhesion molecules were enhanced significantly and strongly upon mast cell activation at various time points. Unstored TNF-alpha was fully responsible for ICAM-1 upregulation. P-selectin was up-regulated by both preformed and newly synthesized mast cell mediators, but neither histamine nor TNF-alpha accounted for such an effect. Therefore,a new model is proposed in which the pro-inflammatory effect of mast cells on endothelial cells can be studied in vitro. In this model, it has been demonstrated that only TNF-alpha accounts for the overexpression of ICAM-1 induced by mast cells, and that mast cells up-regulate P-selectin expression through a histamine-independent mechanism.

Chemokines in ischemia/reperfusion injury

Ischemia/reperfusion injury is an unavoidable consequence of organ transplantation that results in the generation of reactive oxygen species and the initiation of an inflammatory response. The characteristic features of ischemia/reperfusion injury involve the activation of endothelium by reactive oxygen species and inflammatory cytokines, induction of adhesion molecules (for example, P-selectin and E-selectin), adherence of platelets, and infiltration by leukocytes consisting initially of neutrophils with subsequent infiltration by monocytes, macrophages, and T lymphocytes. In this review, we consider the current evidence for involvement of chemokines during the period after ischemia/reperfusion injury and examine the temporal relation between chemokine expression and leukocyte infiltration. Changes in the profile of chemokines after ischemia/reperfusion injury, in association with upregulation of endothelial adhesion molecules, may contribute to the immunogenicity of a transplanted organ by increasing its ability to recruit particular leukocyte subsets at specific time points after ischemia/reperfusion injury.

Adhesion molecules and cellular infiltrate: histology of urticaria.

Urticarial reactions are characterized by dermal capillary dilatation and edema, associated with a variably intense mixed inflammatory infiltrate consisting of neutrophils, eosinophils, T-helper lymphocytes, and activated macrophages. Mast cell numbers are moderately increased by a factor of 2.4, in contrast to mastocytosis where numbers are much higher (5-48-fold increase). In urticarial vasculitis there is in addition endothelial damage, leukocytoclasia, and fibrin and complement deposition. The emigration of leukocytes is regulated by vasoactive and chemotactic mediators released firom mast cells, inducing a sequential upregulation of endothelial adhesion molecules (P-selectin, E-selectin, ICAM-1, and VCAM-1), of beta2-integrins on leukocytes, and of cytokines on endothelial, epithelial, and infiltrating cells. In nonlesional skin, there is also an increase of mast cells and an upregulation of endothelial adhesion molecules, probably due to molecules in the circulation of which P-selectin and TNFalpha have so far been demonstrated. Whereas these data provide a molecular basis for the understanding of variations in mast cell-dependent pathology, they underline the fact that they are not diagnostic for different types of urticaria, except for urticarial vasculitis and mastocytosis.