Plasmodium coatneyi-infected rhesus monkeys: a primate modelfor human cerebral malaria

Although several animal models for human cerebral malaria have been proposed in the past, name have shown pathological findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied the pathology of brains of Plasmodium coatneyi (primate malaria parasite)-infected rhesus monkeys. Our study demonstrated parazitized erythrocyte (PRBC) sequestration and cytoadherence of knobs on PRBC to endothelial cells in cerebral microvessels of these monkeys. This similar to the findings een in human cerebral malaria. Crebral microvessels with sequestred PRBC were shown by immunohistochemistry to possess CD36, TSP and ICAM-1. These proteins were not evident in cerebral microvessels of uninfected control monkeys. Our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Systemic modulation of peripheral eosinophilia (air pouch model) in Schistosoma mansoni infection

Schistosoma mansoni infection induces in their hosts a marked and sustained eosinophilia, which is influenced or modulated by complex mechanisms, that vary according to the phase of infection. To address this phenomenon, we used the air pouch (AP) model in control and infected Swiss webster mice, analyzing the cellular, tissue response and local expression of adhesion molecules [CD18 (beta 2-chain), CD44, ICAM-1 (CD54), L-selectin (CD62L), CD49d (alpha 4-chain), LFA1 (CD11a)]. Infected animals were studied at 3 (pre-oviposition phase), 7 (acute phase), and 14 (chronic phase) weeks after infection (5-6 mice/period of infection). Normal mice were age-matched. Results showed that after egg stimulation, compared with matched controls, the infected mice, at each point of infection, showed a lower eosinophil response in the acute (7 weeks) and chronic phase (14 weeks) of infection. However, when the infected mice were in pre-oviposition phase (3 weeks) their eosinophil response surpassed the control ones. In the AP wall of infected mice, a significant decrease in the expression of ICAM-1 and CD44 in fibroblastic-like cells and a reduction in the number of CD18 and CD11a in migratory cells were observed. The other adhesion molecules were negative or weakly expressed. The results indicated that in the air pouch model, in S. mansoni-infected mice: (1) eosinophil response is strikingly down-regulated, during the acute ovular phase; (2) in the pre-oviposition phase, in contrast, it occurs an up-regulatory modulation of eosinophil response, in which the mechanisms are completely unknown; (3) in the chronic phase of the infection, the down modulation of eosinophil response is less pronounced; 4) Down-regulation of adhesion molecules, specially of ICAM-1 appear to be associated with the lower eosinophil response.

Differential expression of adhesion moleculesshaping the T-cell subset prevalence during the early phase of autoimmune and Trypanosoma cruzi-elicited myocarditis

The participation of cell adhesion molecules (CAMs) in the establishment of autoimmune and infectious myocarditis is an important matter of investigation and may have therapeutic implication. Trypanosoma cruzi infection induces a CD8-mediated myocarditis in patients with severe cardiomyopathy and experimental animals. Previously, we have proposed that this predominance of CD8+ T-cells is, at least in part, consequence of the differential expression of CAMs on circulating CD8+ lymphocytes. In the present study we investigated the participation of CAMs in shaping the phenotypic nature of the autoimmune CD4-mediated myosin-induced and the CD8-mediated T. cruzi-elicited myocarditis. We provide evidence that the prevalence of a certain T-cell subset inside the inflamed heart reflects the differential profile of the adhesion molecules VLA-4, LFA-1, and ICAM-1 displayed on a large proportion of this particular T-cell population in peripheral blood during the early phase of inflammation. Further, the expression of VCAM-1, ligand for VLA-4, and ICAM-1, counter-receptor for LFA-1, was up-regulated on vascular endothelium and paralleled the entrance of inflammatory cells into the cardiac tissue. Thus, this up-regulated expression of receptors-counter-receptors that regulate T-cell transmigration through the vascular endothelium may have an important role in the pathogenesis of the early phase of both autoimmune and infectious myocarditis.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Endoglycan mediates malignant leukocyte rolling on e-selectin and signals through SYK and the MAPK pathway

Selectins play a key role regulating leukocyte migration into tissues by mediating leukocyte tethering (capture) and rolling on inflamed endothelium and/or on adherent leukocytes or platelets. During leukocyte rolling, endothelial E- or P-selectin bind to glycoprotein ligands carrying sialyl Lewis χ (sLex) determinant. P-selectin glycoprotein ligand-1 (PSGL-1) is a common ligand for L-, P- and E-selectin, which sequentially cooperates with CD44 and E- selectin ligand-1 (ESL-1) to roll on E-selectin. During rolling on endothelial selectins, PSGL-1 and CD44 signal through Src family kinases and Syk, leading to αι_β2 integrin partial activation and slow rolling on intercellular adhesion molecule-1 (ICAM-1). Leukocyte exposure to chemokines then leads to firm adhesion. Little information is available on ligands that mediate malignant leukocyte rolling on E- selectin. We defined these ligands on U937 monoblasts by immunoadsorbtion and immunoblotting using mAb raised against CD43, CD44, PSGL-1, sLex/CLA determinants and E-selectin/IgM chimera. Immunoblotting and blot rolling assays demonstrated that PSGL-1, CD43, CD44 and a -125 kDa sLex/CLA positive ligand contribute to support E-seiectin- dependent rolling. This -125 kDa ligand is endoglycan, a member of the CD34 family of sialomucins. Endoglycan was frequently detected by flow cytometry on primary leukemia, lymphoma and multiple myeloma ceils (in -50% of cases). Endoglycan, immunopurified from U937 cells, as well as endoglycan/IgG chimera efficiently supported E-selectin dependent rolling. Membrane fractionation on sucrose gradient demonstrated that endoglycan is expressed in lipid rafts. We tested the hypothesis that it signals, like PSGL-1 and CD44, through Src kinases and the MAPK pathway. Indeed, endoglycan engagement induced Syk and ERK phosphorylation in a iipid raft-dependent manner. Syk activation was dependent on Src kinase activity. Downstream of Syk, endoglycan activated PI3K and Akt as well as Bruton's tyrosine kinase and p38 MAPK. Thus, endoglycan is a ligand for endothelial selectins which may contribute to regulate leukemia, lymphoma and multiple myeloma cell trafficking and interactions with bone marrow microenvironment. - Les sélectines contrôlent la migration tissulaire des leucocytes en assurant leur capture et leur roulement sur l'endothélium vasculaire enflammé et/ou sur des plaquettes ou des leucocytes adhérant à la paroi vasculaire. Lors du roulement leucocytaire, les sélectines endothéliales (E- et P-sélectine) se lient à des ligands porteurs du saccharide sialyl Lewis χ (sLex). PSGL-1 est un ligand commun des sélectines qui coopère avec CD44 et ESL-1 pour permettre la capture et le roulement des neutrophiles. Lorsque PSGL-1 et CD44 se lient aux sélectines endothéliales, elles induisent la phosphorylation des kinases Src et de Syk conduisant à l'activation partielle de l'intégrine aLp2 et au ralentissement des leucocytes sur les sélectines et ICAM-1. Les chimiokines induisent ensuite l'adhésion ferme des leucocytes. Les ligands des sélectines qui assurent le roulement, sur la E-sélectine, des cellules issues d'hémopathies malignes sont peu connus. Nous avons caractérisé ces ligands en les purifiant avec des anticorps dirigés contre CD43, CD44, PSGL-1, sLex/CLA et en utilisant la chimère E-sélectine/IgM. Des tests d'adhésion ont montré que PSGL-1, CD43, CD44 et une glycoprotéine de ~125 kDa soutiennent les interactions cellulaires dépendant de la E- sélectine. Le ligand de -125 kDa a été identifié comme étant l'endoglycan. Il a été détecté, par cytométrie de flux, sur les cellules leucémiques, les cellules de lymphomes ou de myélome multiple, dans ~50% des cas analysés. Sa forme membranaire, immunopurifiée, ou recombinante (endoglycan/lgG) soutient les interactions cellulaires dépendant de la E- sélectine. Nous avons montré qu'il réside dans les rafts lipidiques membranaires puis avons testé l'hypothèse que l'endoglycan, comme PSGL-1 et CD44, induit une signalisation via les kinases de type Src et la voie des MAPK. Nous avons pu observer que son engagement induit la phosphorylation de Syk et de ERK pour autant que la structure des rafts soit préservée. En aval de Syk, l'endoglycan active la PI3K, Akt, Btk et la MAPK p38. Ces résultats montrent que l'endoglycan est un ligand des sélectines endothéliales qui pourrait participer au contrôle du trafic et des interactions des cellules leucémiques, de lymphomes ou de myélomes multiples avec leur microenvironnement. - Le sang est un élément clé du fonctionnement de notre corps. La circulation sanguine permet la communication et le transfert de molécules et cellules entre divers organes. Lors d'une inflammation aiguë due à une réaction allergique, une infection ou une blessure, on observe un oedème local accompagné de rougeur, de chaleur et souvent de douleurs. Au sein des tissus enflammés, on observe des globules blancs (leucocytes) et diverses molécules inflammatoires qui attirent les leucocytes dans les tissus lésés (chimiokines). Le sang est composé de globules rouges, de plaquettes et de leucocytes spécialisés dans les défenses immunes. Pour atteindre le site d'inflammation, les leucocytes doivent quitter la circulation sanguine. Ils utilisent pour cela des molécules d'adhésion présentes à leur surface qui se lient à d'autres molécules d'adhésion de la paroi sanguine. Leurs interactions permettent aux leucocytes de rouler à la surface du vaisseau sanguin. Lorsqu'ils roulent au voisinage d'un site d'inflammation, les leucocytes sont exposés à des chimiokines qui induisent leur arrêt et les dirigent dans les tissus enflammés. Ce processus physiologique est aussi impliqué dans des pathologies telles que l'infarctus, l'artériosclérose ou la thrombose. Il peut être détourné à des fins moins louables par des cellules cancéreuses pour permettre leur dissémination (métastatisation). Dans ce travail de thèse, nous avons caractérisé une molécule d'adhésion qui soutient l'adhésion des leucocytes aux sélectines endothéliales: l'endoglycan. Nous avons observé que cette molécule d'adhésion est fréquemment exprimée par les cellules malignes de nombreuses maladies du sang comme les leucémies, les lymphomes et le myélome multiple. Nous avons également pu montrer que l'endoglycan envoie des signaux à l'intérieur des cellules malignes lorsqu'elles se lient aux sélectines endothéliales. Ces signaux pourraient jouer un rôle déterminant dans la régulation des interactions des cellules malignes avec leur microenvironnement. Elles pourraient peut-être aussi favoriser leur survie et leur prolifération.

Peripheral inflammation increases the damage in animal models of nigrostriatal dopaminergic neurodegeneration: possible implication in Parkinson's disease incidence.

Inflammatory processes described in Parkinson’s disease (PD) and its animal models appear to be important in the progression of the pathogenesis, or even a triggering factor. Here we review that peripheral inflammation enhances the degeneration of the nigrostriatal dopaminergic system induced by different insults; different peripheral inflammations have been used, such as IL-1β and the ulcerative colitis model, as well as insults to the dopaminergic system such as 6-hydroxydopamine or lipopolysaccharide. In all cases, an increased loss of dopaminergic neurons was described; inflammation in the substantia nigra increased, displaying a great activation of microglia along with an increase in the production of cytokines such as IL-1β and TNF-α. Increased permeability or disruption of the BBB, with overexpression of the ICAM-1 adhesion molecule and infiltration of circulating monocytes into the substantia nigra, is also involved, since the depletion of circulating monocytes prevents the effects of peripheral inflammation. Data are reviewed in relation to epidemiological studies of PD.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis.

Fas ligand (FasL) causes apoptosis of epidermal keratinocytes and triggers the appearance of spongiosis in eczematous dermatitis. We demonstrate here that FasL also aggravates inflammation by triggering the expression of proinflammatory cytokines, chemokines, and adhesion molecules in keratinocytes. In HaCaT cells and in reconstructed human epidermis (RHE), FasL triggered a NF-kappaB-dependent mRNA accumulation of inflammatory cytokines (tumor necrosis factor-alpha, IL-6, and IL-1beta), chemokines (CCL2/MCP-1, CXCL1/GROalpha, CXCL3/GROgamma, and CXCL8/IL-8), and the adhesion molecule ICAM-1. Oligomerization of Fas was required both for apoptosis and for gene expression. Inhibition of caspase activity abolished FasL-dependent apoptosis; however, it failed to suppress the expression of FasL-induced genes. Additionally, in the presence of caspase inhibitors, but not in their absence, FasL triggered the accumulation of CCL5/RANTES (regulated on activation normal T cell expressed and secreted) mRNA. Our findings identify a novel proinflammatory role of FasL in keratinocytes that is independent of caspase activity and is separable from apoptosis. Thus, in addition to causing spongiosis, FasL may play a direct role in triggering and/or sustaining inflammation in eczemas.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours. Adhesion assays of human T cell lines demonstrated that all T cell lines were capable of adhesion to HUVEC and that adhesion of T cells to HUVECs was primarily mediated by CD11a/CD18 and ICAM-1 interactions. Furthermore, transendothelial migration of CD 3+ enriched human peripheral blood T cells was inhibited by pretreating the T cells with anti-CD 18 monoclonal antibodies. The inability of malignant T cells to migrate through HUVEC layers in the absence of chemoattractants was not due to poor motility per se, since both normal and malignant T cells migrated well on extracellular matrix components as determined by using Boyden chambers. Crosslinking of alpha 1 beta 2 and alpha 4 beta 1 with immobilized monoclonal antibodies induced motile behaviour in activated CD 3 enriched human peripheral blood T cells but not in malignant T cell lines. In conclusion, the differences in the ability of transendothelial migration between normal and malignant human T cells in the absence of chemoattractants is primarily due to the differences in the capacity of alpha 1 beta 2 and alpha 4 beta 1 to trigger motile behaviour in the separate cell types.

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.

Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T Lymphocytes during cell polarization

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane-cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, ß-actin and ¿-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti-ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin-ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin-ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Heparin attenuates TNF-alpha induced inflammatory response through a CD11b dependent mechanism

Background In addition to its anticoagulant properties, heparin has anti-inflammatory effects, the molecular and mechanistic bases of which are incompletely defined. AIMS The current studies were designed to test the hypothesis that heparin abrogates the expression or function of leucocyte-endothelial adherence molecules which are fundamental to the acute inflammatory response. Methods The effects of heparin on tumour necrosis factor alpha (TNF-¿) induced leucocyte rolling, adhesion, and migration as well as vascular permeability were assessed in rat mesenteric venules using intravital microscopy. Expression of adhesion molecules was quantitated using a double radiolabelled monoclonal antibody (mAb) binding technique in vivo (P-selectin, intercellular cell adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1)) or flow cytometry (CD11a, CD11b, and L-selectin). Ex vivo binding of heparin to neutrophils was assessed by flow cytometry. RESULTS TNF-alpha induced a significant increase in leucocyte rolling, adhesion, and migration, and vascular permeability, coincident with a significant increase in expression of P-selectin, ICAM-1, and VCAM-1. Ex vivo assessment of blood neutrophils showed significant upregulation of CD11a and CD11b and significant downregulation of L-selectin within five hours of TNF-¿ administration. Heparin pretreatment significantly attenuated leucocyte rolling, adhesion, and migration but did not affect expression of cell adhesion molecules or vascular permeability elicited by TNF-¿ administration. Binding of heparin was significantly increased on blood neutrophils obtained five hours after TNF-¿ administration. Preincubation with an anti-CD11b mAb but not with an anti-CD11a or anti-L-selectin antibody significantly diminished heparin binding ex vivo.