Human monocyte adherence to cultured vascular endothelium: monoclonal antibody-defined mechanisms

We have evaluated the binding of human peripheral blood monocytes to cultured vascular endothelium as an in vitro model of monocyte interaction with the vessel wall. Monocytes were purified (91% +/- 4 SE esterase positive) by elutriation to avoid contact with surfaces before assay. Adherence of 51Cr-labeled monocytes after 45 min (36% +/- 11 SE) was significantly higher than that observed with autologous radiolabeled neutrophils (9% +/- 5 SE) and was greater on monolayers of human umbilical vein endothelium than on bovine aortic endothelium. Peripheral blood mononuclear cells treated with monoclonal antibody (MoAb) 60.3, a reagent that binds leukocyte membrane complex CDw18, implicated in multiple adherence-dependent functions, failed to adhere and flatten on artificial surfaces. Mononuclear cells treated with MoAb 60.3 simulated cells from a patient with recurrent infections whose phagocytes failed to react with MoAb 60.3 and failed to emigrate to extravascular sites in vivo. Incubation of monocytes with MoAb 60.3 inhibited (by 32 to 61%) monocyte adherence to endothelium in a dose-dependent manner for periods up to 24 hr, but had negligible effects on basal (unstimulated) neutrophil adherence. Basal monocyte adherence in the presence of MoAb 60.3 remained significantly greater than basal neutrophil adherence. Augmentation of phagocyte adherence to endothelial monolayers by autologous plasma or phorbol ester (PMA) was abrogated by incubation with MoAb 60.3. Studies with immunofluorescence flow cytometry indicated that PMA stimulation of monocytes resulted in a specific 40% increase in monocyte surface expression of the epitope recognized by MoAb 60.3. These in vitro findings, in conjunction with observations from two patients, support the hypothesis that monocyte adherence to endothelium and emigration to tissues is mediated by mechanisms both dependent upon and independent of the CDw18 complex and the epitope recognized by MoAb 60.3.     

Neutrophils, monocytes, and lymphocytes bind to cytokine-activated kidney glomerular endothelial cells through L-selectin (LAM-1) in vitro.

The role of L-selectin (LAM-1) as a regulator of leukocyte adhesion to kidney microvascular glomerular endothelial cells was assessed in vitro by using L-selectin-directed mAb and an L-selectin cDNA-transfected cell line. The initial attachment of neutrophils, monocytes, and lymphocytes to TNF-activated bovine glomerular endothelial cells was significantly inhibited by the anti-LAM1-3 mAb. Under static conditions, anti-LAM1-3 mAb inhibited neutrophil adhesion by 15 +/- 5%, whereas the anti-LAM1-10 mAb, directed against a functionally silent epitope of L-selectin, was without effect. The binding of a CD18 mAb inhibited adhesion by 47 +/- 6%. In contrast, when the assays were carried out under nonstatic conditions or at 4 degrees C, the anti-LAM1-3 mAb generated significantly greater inhibition (approximately 60%). CD18-dependent adhesion was minimal (approximately 10%) under these conditions. TNF-activated glomerular endothelial cells also supported adhesion of a mouse pre-B cell line transfected with L-selectin cDNA, but not wild-type cells. This process was also inhibited by the anti-LAM1-3 mAb. Leukocyte adhesion to unstimulated endothelial cells was independent of L-selectin, but, after TNF stimulation, L-selectin-mediated adhesion was observed at 4 h, with maximal induction persisting for 24 to 48 h. Leukocyte adhesion was not observed if glomerular endothelial cells were exposed to TNF in the presence of RNA or protein synthesis inhibitors. Leukocyte attachment to TNF-activated glomerular endothelial cells was also partially inhibited by treatment of the cells with mannose-6-phosphate or phosphomannan monoester, a soluble complex carbohydrate, or by prior treatment of glomerular endothelial cells with neuraminidase, suggesting that the glomerular endothelial cell ligand shares functional characteristics with those expressed by lymph node and large vessel endothelial cells. These data suggest that TNF activation induced the biosynthesis and surface expression of a ligand(s) for L-selectin on glomerular endothelial cells, which supports neutrophil, monocyte, and lymphocyte attachment under nonstatic conditions.     

Mac-1 Directly Binds to the Endothelial Protein C-Receptor: A Link between the Protein C Anticoagulant Pathway and Inflammation?

Objective

The endothelial protein C-receptor (EPCR) is an endothelial transmembrane protein that binds protein C and activated protein C (APC) with equal affinity, thereby facilitating APC formation. APC has anticoagulant, antiapoptotic and antiinflammatory properties. Soluble EPCR, released by the endothelium, may bind activated neutrophils, thereby modulating cell adhesion. EPCR is therefore considered as a possible link between the anticoagulant properties of protein C and the inflammatory response of neutrophils. In the present study, we aimed to provide proof of concept for a direct binding of EPCR to the β₂ –integrin Mac-1 on monocytic cells under static and physiological flow conditions.

Measurements and Main Results

Under static conditions, human monocytes bind soluble EPCR in a concentration dependent manner, as demonstrated by flow cytometry. Binding can be inhibited by specific antibodies (anti-EPCR and anti-Mac-1). Specific binding was confirmed by a static adhesion assay, where a transfected Mac-1 expressing CHO cell line (Mac-1+ cells) bound significantly more recombinant EPCR compared to Mac-1+ cells blocked by anti-Mac-1-antibody and native CHO cells. Under physiological flow conditions, monocyte binding to the endothelium could be significantly blocked by both, anti-EPCR and anti-Mac-1 antibodies in a dynamic adhesion assay at physiological flow conditions. Pre-treatment of endothelial cells with APC (drotrecogin alfa) diminished monocyte adhesion significantly in a comparable extent to anti-EPCR.

Conclusions

In the present study, we demonstrate a direct binding of Mac-1 on monocytes to the endothelial protein C receptor under static and flow conditions. This binding suggests a link between the protein C anticoagulant pathway and inflammation at the endothelium side, such as in acute vascular inflammation or septicaemia.     

Diminished lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 on neonatal neutrophils underlies their impaired CD18-independent adhesion to endothelial cells in vitro.

To define further the molecular basis for abnormal interactions of cord blood or neonatal neutrophils with endothelial cells in vitro, we studied neutrophil adhesion and migration under experimental conditions specifically designed to evaluate CD18-independent mechanisms. Unstimulated cord blood neutrophils of healthy term neonates demonstrated significantly diminished adhesion to IL-1-stimulated endothelial cell monolayers under conditions of shear stress (congruent to 1.85 dynes/cm2); overall levels of migration by neonatal cells were also significantly diminished, although the adherent subpopulation of these cells migrated relatively normally. A mAb (DREG-56) against the human homologue of the murine MEL-14 antigen (termed lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 (LECAM-1), a member of the LEC-CAM family of adhesion molecules) markedly inhibited adhesion of healthy adult but not cord blood neutrophils. In additional assessments of endothelial cell adhesion or migration in the absence of shear forces, cord blood neutrophils demonstrated significantly diminished values compared to adult controls. Moreover, mAb DREG-56 significantly diminished adhesion of healthy adult but not cord blood suspensions in the presence or absence of the anti-CD18 mAb R15.7. Immunofluorescence assessments of unstimulated cord blood neutrophils or neutrophils of neonates 12 to 48 h of age showed dramatically diminished levels of surface LECAM-1 compared to adult neutrophils. Chemotactic stimuli (FMLP, 10 nM, 15 min) consistently "down-regulated" surface LECAM-1 on adult neutrophils to levels approximately 10% of unstimulated suspensions and comparable to those of most unstimulated neonatal suspensions. Moreover, FMLP stimuli elicited little or no down-regulation of LECAM-1 on neonatal cells. In comparative studies, endothelial cell adhesion of unstimulated cord blood or adult control neutrophils (assessed under conditions of flow) was directly related to levels of neutrophil surface LECAM-1. Although FMLP stimulation significantly diminished both adhesion and LECAM-1 surface levels of adult control cells, the adhesion and LECAM-1 expression observed with cord blood cells were not significantly influenced by this stimulus. The mechanisms underlying diminished LECAM-1 expression and LECAM-1-dependent adhesion of neonatal neutrophils and the physiologic significance of these abnormalities deserve investigation.     

CD14 contributes to the adherence of human monocytes to cytokine-stimulated endothelial cells.

Monocyte adherence to endothelial cells (EC) is selectively increased during inflammation. The mechanisms underlying monocyte-EC interaction indicated the involvement of surface-adhesion molecules on monocytes and EC. In earlier studies we noticed that the monocyte-specific mAb, designated mAb 63D3, in contrast to mAb against the beta 2-integrin molecules, inhibited the monocyte binding to monolayers of rIL-1 alpha-stimulated venous EC. The aim of the present study was to further characterize the Ag recognized by mAb 63D3 and to investigate the specific contribution of this Ag to the adherence of monocytes to cultured human macrovascular venous or arterial EC. Flow cytometric analysis demonstrated that the 63D3 Ag is expressed exclusively on the surface of peripheral blood monocytes. SDS-PAGE analysis of mAb 63D3 immunoprecipitates of 125I-labeled human monocyte surface proteins revealed that the target Ag for mAb 63D3 is a 52- to 55-kDa molecule identical to the myeloid differentiation protein CD14. Stimulation of EC with rIL-1 alpha or rTNF-alpha for 4 or 24 h or rIFN-gamma for 24 h increased (p less than 0.005) the number of monocytes bound to both types of EC. This cytokine-induced increase in monocyte adherence was significantly (p less than 0.0005) inhibited when the monocytes were coated with various mAb against CD14. The binding of monocytes to nonstimulated venous or arterial EC was not inhibited by anti-CD14 mAb. Our results lead to the conclusion that CD14 molecules, which on basis of their structure and m.w. are not related to the beta 2-integrin family of heterodimeric leukocyte adhesion molecules, participate in the binding of monocytes to cytokine-stimulated EC.     

CD18 dependency of transendothelial neutrophil migration differs during acute pulmonary inflammation

Neutrophil extravasation during inflammation can occur either by a mechanism that requires the neutrophil integrin complex, CD18, or by an alternative CD18-independent route. Which of the two pathways is used has been shown to depend on the site and nature of the inflammatory insult. More recent evidence suggests that selection may also depend on whether inflammation is chronic or acute, but why this is the case remains unknown. Using an in vitro model that supports both migratory mechanisms, we examined the CD18 dependency of migration of neutrophils isolated from patients with either chronic or acute pulmonary infection. Chronic neutrophils were found to behave like normal neutrophils by migrating to IL-8 and leukotriene B(4) using the CD18-independent pathway, but to the bacterial product, FMLP, using the CD18-dependent route. In contrast, migration of acute neutrophils to all of these stimuli was CD18 dependent. Normal neutrophils could be manipulated to resemble acute neutrophils by exposing them to FMLP before migration, which resulted in a "switch" from the CD18-independent to -dependent mechanism during migration to IL-8 or leukotriene B(4). Although treatment of normal neutrophils with FMLP caused selective down-regulation of the IL-8 receptor, CXCR2, and acute neutrophils were found to have less CXCR2 than normal, a functional relationship between decreased CXCR2 and selection of CD18-dependent migration was not demonstrated. Results indicate that selection of the CD18-dependent or -independent migration mechanism can be controlled by the neutrophil and suggest that the altered CD18 requirements of acute neutrophils may be due to priming in the circulation during acute infection.     

Polymorphonuclear leucocyte (PMN) inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism

Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm, inhibits CD11b/CD18-dependent neutrophil adhesion by binding to CD11b. We studied the effects of NIF on neutrophil-dependent endothelial cell injury using bovine pulmonary microvessel endothelial cells grown on microporous filters. Endothelial injury was determined as an increase in the transendothelial ¹²⁵I-albumin clearance rate (a measure of transendothelial permeability). Layering of neutrophils on the endothelial cell monolayer (ratio of 10 neutrophils: 1 endothelial cell) followed by activation of neutrophils with 500 nM of phorbol 12-myristate 13-acetate (PMA) increased transendothelial permeability of albumin by 3- to 4-fold over control monolayers. Pretreatment of neutrophils with NIF at concentrations of 100 nM and above prevented the increased permeability. Pretreatment of neutrophils with the anti-CD18 monoclonal antibody (mAb) IB4 similarly prevented the increase of permeability. Pretreatment of neutrophils with OKM-1, a control isotype-matched mAb directed against an irrelevant epitope on CD11b mAb, did not affect the neutrophil-dependent increase in permeability. NIF reduced the adhesion of neutrophils at concentrations of ≥100 nM and this effect was abolished by an anti-NIF polyclonal Ab. However, NIF did not prevent the generation of superoxide anions following PMA-induced activation of neutrophils layered on endothelial cell. These findings indicate that NIF inhibits the neutrophil-dependent endothelial injury by preventing CD11b/CD18-mediated neutrophil adhesion, but without altering the oxidant generating capacity of neutrophils interacting with the endothelial cell monolayer. J. Cell. Physiol. 171:212–216, 1997. © 1997 Wiley-Liss, Inc.     

CD11/CD18 cell surface adhesion glycoproteins: Discordance of monocyte function and expression in response to stimulation

The adherence of blood leukocytes to vascular endothelium precedes their diapedesis into the extravascular space. These processes require the expression of adherence glycoproteins on the cell surface of the leukocyte. The relative importance of these adherence molecules is so far poorly understood. However, there is evidence to suggest that a disparity exists between the surface receptor expression of these glycoproteins and leukocyte adherence to vascular endothelial cells in culture. We have investigated the importance of each of the adhesion glycoproteins CD11a, CD11b, and CD11c in mediating the adherence of human monocytes to endothelial cells in culture. We have also investigated the chronological relationship between changes in monocyte adherence to endothelial cells and the surface expression of CD11a, CD11b, and CD11c following stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The increase in adherence occurred within 1 minute, but declined if monocytes were preincubated with fMLP for up to 30 minutes. The surface expression of adherence molecules demonstrated a significant increase in CD11a and CD11b in the presence of fMLP after 10 min and was maintained while monocyte adherence to endothelium declined. These changes in surface receptor expression were quantitated using an immunolabeling technique. It is suggested that fMLP stimulation of monocyte adherence is unlikely to be solely dependent on increased surface receptor expression of adhesion molecules.     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

The cellular location and specificity of bacterial cytochrome c peroxidases.

We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils.     

LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ

To exit blood vessels, most (～80%) of the lumenally adhered monocytes and neutrophils crawl toward locations that support transmigration. Using intravital confocal microscopy of anesthetized mouse cremaster muscle, we separately examined the crawling and emigration patterns of monocytes and neutrophils in blood-perfused unstimulated or TNF-α-activated venules. Most of the interacting cells in microvessels are neutrophils; however, in unstimulated venules, a greater percentage of the total monocyte population is adherent compared with neutrophils (58.2 ± 6.1% versus 13.6 ± 0.9%, adhered/total interacting), and they crawl for significantly longer distances (147.3 ± 13.4 versus 61.8 ± 5.4 μm). Intriguingly, after TNF-α activation, monocytes crawled for significantly shorter distances (67.4 ± 9.6 μm), resembling neutrophil crawling. Using function-blocking Abs, we show that these different crawling patterns were due to CD11a/CD18 (LFA-1)- versus CD11b/CD18 (Mac-1)-mediated crawling. Blockade of either Mac-1 or LFA-1 revealed that both LFA-1 and Mac-1 contribute to monocyte crawling; however, the LFA-1-dependent crawling in unstimulated venules becomes Mac-1 dependent upon inflammation, likely due to increased expression of Mac-1. Mac-1 alone was responsible for neutrophil crawling in both unstimulated and TNF-α-activated venules. Consistent with the role of Mac-1 in crawling, Mac-1 block (compared with LFA-1) was also significantly more efficient in blocking TNF-α-induced extravasation of both monocytes and neutrophils in cremaster tissue and the peritoneal cavity. Thus, mechanisms underlying leukocyte crawling are important in regulating the inflammatory responses by regulating the numbers of leukocytes that transmigrate.     

Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.     

Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

The lectin-like domain of complement receptor 3 protects endothelial barrier function from activated neutrophils

The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of beta(2) integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the beta(2) integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist beta-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates beta-glucan protection while activation of the LLD by VIM12 cross-linking mimics the beta-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. beta-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.     

Characterization of hyaluronan-binding proteins on guinea pig polymorphonuclear leukocytes: possible involvement of complement receptor type 3 (CR3, CD11b/CD18) in the hyaluronan-leukocyte interaction

Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.     

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

E-selectin (endothelial-leukocyte adhesion molecule-1) is not required for the migration of neutrophils across IL-1-stimulated endothelium in vitro.

Treatment of vascular endothelial cells with inflammatory cytokines stimulates surface expression of E-selectin (previously known as endothelial-leukocyte adhesion molecule-1) and promotes the transendothelial migration of neutrophils. To assess participation of E-selectin in cytokine-mediated neutrophil migration, an in vitro model consisting of monolayers of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue was used. When HUVEC-amnion cultures were stimulated for 4 h with relatively low concentrations of IL-1 (0.1 to 0.15 U/ml), mAb BB11 or H18/7 to E-selectin partially inhibited migration of subsequently added neutrophils. However, when the cultures were stimulated with 15 U/ml of IL-1 for 4 or 24 h, little to no inhibition was observed. mAb to E-selectin also failed to inhibit migration of neutrophils across HUVEC-amnion cultures treated with low doses of IL-1 when the leukocytes were additionally stimulated by the chemoattractant leukotriene B4. In contrast, migration of neutrophils across IL-1-treated HUVEC was profoundly inhibited by mAb to CD11/CD18 leukocytic integrins under all conditions tested. Results of these studies suggest that participation of E-selectin is not essential for migration of neutrophils across cytokine-stimulated HUVEC in vitro; rather, E-selectin can be bypassed in favor of CD11/CD18-dependent mechanisms under appropriate circumstances.     

Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst.

We studied neutrophil responses to LPS using three methodologic refinements: Teflon bags or serum-coated glass tubes that did not directly trigger neutrophils, LPS-free cytochrome c to measure O2- release, and heat-inactivated serum to inhibit inactivation of LPS by neutrophils. Neutrophils incubated in uncoated glass or plastic tubes adhered to the glass and released O2-, but were not primed for enhanced release of O2- in response to triggering by FMLP. Triggering by the glass or plastic surface did not occur if the neutrophils were stirred to prevent adherence. Adherence to glass or plastic and O2- release were not affected by a mAb (IB4) directed against the beta-chain of the leukocyte adhesion family of surface glycoproteins (CD11/CD18). Neutrophils incubated in glass or plastic did not show enhanced expression of alkaline phosphatase on their surface. When neutrophils were incubated in serum-coated glass tubes or in Teflon bags, there was no O2- release. However, adherence, expression of alkaline phosphatase, and release of O2- were triggered by adding 1 ng/ml LPS plus 1% serum, but not by either LPS or serum alone. In the presence of LPS and serum, O2- release was much higher when the cells were unstirred (adherent) rather than stirred. However, both unstirred and stirred cells expressed a similar elevated level of alkaline phosphatase. LPS-triggered O2- release and adherence were inhibited by antibody IB4. In contrast, priming by LPS for enhanced FMLP-triggered O2- release was greater in stirred cells than in unstirred cells. The antibody enhanced priming of unstirred neutrophils. These results suggested that uncoated glass or plastic triggered O2- release without involvement of leukocyte adhesion glycoproteins. However, neutrophils incubated with LPS and serum expressed alkaline phosphatase and IB4-inhibitable adherence glycoproteins that allowed neutrophils to interact with serum-coated glass or Teflon to trigger O2- release. Priming by LPS for enhanced response to FMLP was suppressed in adherent neutrophils, and this suppression was partly released by IB4. Thus, triggering and priming were reciprocally regulated by neutrophil glycoproteins interacting with surfaces.