Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-α. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-α–activated aortic endothelium, ~80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti–L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by ~80% monocyte attachment to TNF-α–activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte–endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.     

L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1.

Leukocytes express L-selectin ligands critical for leukocyte-leukocyte interactions at sites of inflammation. The predominant leukocyte L-selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), which displays appropriate sialyl Lewis x (sLex)-like carbohydrate determinants for L-selectin recognition. Among the sLex-like determinants expressed by human leukocytes is a unique carbohydrate epitope defined by the HECA-452 mAb. The HECA-452 Ag is a critical component of L-selectin ligands expressed by vascular endothelial cells. However, HECA-452 Ag expression on human leukocyte L-selectin ligands has not been assessed. In this study, the HECA-452 mAb blocked 88-99% of neutrophil rolling on, or attachment to, adherent cells expressing L-selectin in multiple experimental systems. A function-blocking anti-PSGL-1 mAb also inhibited L-selectin binding to neutrophils by 89-98%. In addition, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin binding to neutrophils. Western blot analysis revealed that PSGL-1 immunoprecipitated from neutrophils displayed HECA-452 mAb-reactive determinants and that PSGL-1 was the predominant scaffold for HECA-452 Ag display. Leukocyte L-selectin ligands also contained sulfated determinants since culturing ligand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent with this, human neutrophils expressed mRNA encoding five different sulfotransferases associated with the generation of selectin ligands: CHST1, CHST2, CHST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrate determinant displayed on PSGL-1 represented the predominant L-selectin and P-selectin ligand expressed by neutrophils.     

Involvement of the CD11b/CD18 integrin, but not of the endothelial cell adhesion molecules ELAM-1 and ICAM-1 in tumor necrosis factor-alpha-induced neutrophil toxicity.

TNF-alpha can incite neutrophil-mediated endothelial cell damage and neutrophil H2O2 release. Both effects require adherent neutrophils. Using specific mAb, we showed in this in vitro study that the CD18 beta 2-chain and the CD11b alpha M-chain of the CD11/CD18 integrin heterodimer have a major role in both TNF-alpha-induced neutrophil-mediated detachment of human umbilical vein endothelial cells and H2O2 release by TNF-alpha-activated human neutrophils. In contrast to anti-CD18 mAb, which consistently prevented neutrophil activation, anti-CD11a mAb and two of three anti-CD11b mAb did not reduce endothelial cell detachment and neutrophil H2O2 release, although they decreased neutrophil adhesion to human umbilical vein endothelial cells. mAb 904, directed against the bacterial LPS binding region of CD11b, reduced endothelial cell detachment for about 40% and neutrophil H2O2 release for more than 50%, demonstrating that CD11b/CD18 is engaged in TNF-induced neutrophil activation. Dependence on CD11b/CD18 could not be overcome by CD18-independent anchoring of neutrophils via PHA. Additionally, neither induction of increased expression of the endothelial cell adhesion molecules ICAM-1 and ELAM-1, nor subsequent addition of specific mAb, influenced endothelial cell injury or H2O2 release by TNF-activated neutrophils. Interaction with ICAM-1 and ELAM-1 therefore appears not to induce additional activation of TNF-stimulated neutrophils. These studies suggest that a specific, CD11b/CD18-mediated signal, instead of adherence only, triggers toxicity of TNF-activated neutrophils.     

Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion.

The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.     

Adhesion promotes transcellular leukotriene biosynthesis during neutrophil-glomerular endothelial cell interactions: inhibition by antibodies against CD18 and L-selectin.

Eicosanoid formation by transcellular routes can amplify the levels and types of lipid mediators within a local milieu. To evaluate the role of adhesion in this process, we assessed the influence of mAb against adhesion molecules on LTC4 generation by PMN-endothelial cell interaction. Transcellular LTC4 generation was initiated by addition of fMLP to coincubations of GM-CSF-primed PMN and TNF-activated endothelial cells cultured from kidney glomeruli. Both PMN-endothelial cell adhesion and transcellular LTC4 generation were inhibited by mAb against leukocyte L-selectin and CD18. These results indicate that cytokine-treated PMN and endothelial cells generate LTC4 via transcellular routes by receptor-triggered mechanisms. They suggest that adhesion promotes transcellular eicosanoid biosynthesis and that adhesion molecules may also be targets for blockade of transcellular biosynthesis of lipid mediators.     

Overlapping roles for L-selectin and P-selectin in antigen-induced immune responses in the microvasculature

Although L-selectin mediates lymphocyte attachment to endothelial venules of peripheral lymph nodes, its role in leukocyte recruitment into tissues following Ag challenge is less well established. The objective of this study was to systematically examine the role of L-selectin in leukocyte rolling in the peripheral microvasculature during the first 24 h of an immune response. A type I hypersensitivity response was elicited in wild-type (C57BL/6) and L-selectin-deficient mice by systemic (i.p.) sensitization and intrascrotal challenge with chicken egg OVA. The cremaster microcirculation was observed in untreated and sensitized mice 4, 8, and 24 h post-Ag challenge by intravital microscopy. Leukocyte recruitment in L-selectin-deficient mice and wild-type mice treated with an L-selectin function-blocking mAb was examined at each time point. Ag challenge induced a significant increase in leukocyte rolling (60 cells/min/venule to approximately 300 cells/min/venule) in wild-type mice at 4-24 h. This response was reduced by approximately 60-70% in L-selectin-deficient mice and in wild-type mice treated with an L-selectin-blocking mAb. P-selectin blockade by Ab completely inhibited leukocyte rolling at 4-24 h in wild-type animals and also blocked the residual rolling seen in L-selectin-deficient mice. Blocking E-selectin function had no effect on leukocyte rolling flux at any time point in wild-type or L-selectin-deficient mice. Despite reduced rolling, leukocyte adhesion and emigration were not measurably reduced in the L-selectin-deficient mice in this vascular bed. In conclusion, leukocyte rolling is L-selectin-dependent post-Ag challenge with L-selectin and P-selectin sharing overlapping functions.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

Granulocyte-macrophage colony-stimulating factor and other cytokines regulate surface expression of the leukocyte adhesion molecule-1 on human neutrophils, monocytes, and their precursors.

There is increasing evidence that cytokines such as granulocyte-macrophage (GM)-CSF can profoundly affect the adhesion, aggregation, and mobility of neutrophils both in vitro and in vivo. However, the mechanisms whereby these factors might alter the adhesive properties of neutrophils are incompletely understood. A new family of cellular adhesion molecules has recently been identified by cDNA cloning. The members of this family include human leukocyte adhesion molecule-1 (LAM-1), the human endothelial-leukocyte adhesion molecule, and the mouse leukocyte homing receptor for high endothelial venules, MEL-14. LAM-1 is the human homologue of murine MEL-14, and is believed to mediate binding of leukocytes to human high endothelial venules. LAM-1 can be identified by mAb TQ-1, Leu 8, or anti-LAM1.1. The expression and regulation of LAM-1 on granulocytes, monocytes, and their precursors was investigated using flow cytometry and the anti-LAM-1.1 mAb. Neutrophils, eosinophils, monocytes, marrow myeloid cells, granulocyte/macrophage colony-forming unit, and burst-forming unit for erythroid cells were LAM-1+ by flow microfluorimetry. The regulation of LAM-1 expression was tested by treating various cell populations with cytokines or other stimuli for 0-90 min. Exposure of neutrophils, monocytes, and marrow myeloid cells to GM-CSF induced rapid and complete loss of LAM-1 from the cell surface, but had no effect on LAM-1 expression by lymphocytes. The loss of LAM-1 was temporally correlated with up-regulation of CD11b (Mo1), an adhesion molecule involved in neutrophil aggregation. Several other factors known to activate neutrophils also caused down-regulation of LAM-1 and up-regulation of CD11b, including TNF, FMLP, and leukotriene B4. Interestingly, granulocyte-CSF and IFN-gamma had minimal effects on neutrophil LAM-1 expression. Similar results were observed on monocytes and myeloid precursor cells. Thus, exposure of neutrophils to GM-CSF results in a profound change in surface expression of adhesion molecules, with coordinated up-regulation of CD11b and down-regulation of LAM-1. These changes in adhesion proteins are likely to alter aggregation and mobility of both mature myeloid cells and their precursors in patients receiving certain types of cytokine therapy.     

Dextran sulfate inhibits E-selectin-mediated neutrophil adhesion to endotoxin-activated vascular endothelial cells

Effects of heparin-like glycosaminoglycans (h-GAGs) on neutrophil adhesion to endothelial cells (ECs) were investigated under physiological flow conditions using human umbilical vein ECs. Neutrophil adhesion to lipopolysaccharide-activated ECs was assayed under rotating conditions with addition of h-GAGs or monoclonal antibodies (mAbs) against selectins. Neutrophil adhesion to activated ECs under rotating conditions was suppressed completely by anti-E-selectin mAb and partially by anti-L-selectin mAb. Addition of dextran sulfate or heparin also significantly inhibited neutrophil adhesion to ECs under the same conditions. Moreover, effects of h-GAGs on neutrophil adhesion to recombinant E-selectin-coated plates were analyzed. Anti-E-selectin mAb and dextran sulfate, but not heparin, significantly inhibited neutrophil adhesion to E-selectin-coated plates. Results suggest that dextran sulfate inhibited E-selectin-mediated neutrophil adhesion to endotoxin-activated ECs under physiologic flow conditions.     

Endothelial leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 mediate the adhesion of eosinophils to endothelial cells in vitro and are expressed by endothelium in allergic cutaneous inflammation in vivo.

We have compared the adhesion of 51Cr-labeled eosinophils and neutrophils to cultured human umbilical vein endothelial cell (EC) monolayers that have been stimulated with IL-1, TNF, or LPS. Each agent stimulated the adhesion to EC of both eosinophils and neutrophils in a similar dose- and time-dependent manner. F(ab')2 fragments of mAb 1.2B6 (anti-endothelial leukocyte adhesion molecule (ELAM)-1) and mAb 6.5B5 (anti-intercellular adhesion molecule (ICAM)-1) each inhibited partially, and to a similar extent, eosinophil and neutrophil adhesion to EC monolayers prestimulated with TNF (10 ng/ml) for 6 h. Greater inhibition of both eosinophil and neutrophil adhesion was achieved by combining the effects of mAb 1.2B6 with either mAb 6.5B5 or mAb TS1/18 (anti-CD18). These observations indicate that both ELAM-1 and ICAM-1 are involved in the adhesion of eosinophils and neutrophils to EC stimulated with TNF. In order to determine whether these molecules are expressed in vivo during allergen-induced late phase allergic responses in the skin, human skin biopsies were examined at 6 h after Ag or saline challenge with the use of an alkaline phosphatase-staining technique. Both ELAM-1 and ICAM-1 were expressed with greater intensities in Ag-challenged biopsies, suggesting that these molecules may be involved in granulocyte recruitment in vivo. The similarities we have established between mechanisms of eosinophil and neutrophil adhesion to cytokine-stimulated EC suggests that factors other than differential leukocyte-EC adhesion may be responsible for the selective accumulation of eosinophils at sites of allergic inflammation.     

Neutrophil adhesion to TNF alpha-activated endothelial cells potentiates leukotriene B4 production.

Since adhesion of neutrophils (PMN) to endothelial cells may influence PMN activation responses, we examined whether adhesion of PMN to TNF alpha-activated human umbilical vein endothelial cells (HUVEC) stimulates leukotriene B4 (LTB4) production. Endothelial adhesivity towards PMN increased after HUVEC pretreatment with TNF alpha for 4 h. LTB4 production increased markedly in response to stimulation with arachidonic acid (20 microM) when PMN were added to the hyperadhesive HUVEC. In contrast, stimulation of PMN in suspension did not potentiate LTB4 production. LTB4 production persisted when PMN were applied to TNF alpha-pretreated HUVEC fixed with 1% paraformaldehyde excluding the possibility that metabolic activity of endothelium participates in this response. PMN adhesion to plastic and gelatin also enhanced LTB4 indicating that adhesion was a critical event in inducing LTB4 production. We used monoclonal antibodies (mAb) to adhesion molecules on endothelial cells (i.e., endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1)) or on PMN (CD18) to assess the role of PMN adhesion to the activated endothelium on LTB4 potentiation. Both anti-ELAM-1 mAb and anti-ICAM-1 mAb inhibited PMN adhesion (by 55 and 41%, respectively) as well as LTB4 production (by 65 and 50%, respectively). Anti-CD18 mAb also reduced the adhesion (65%) and the LTB4 production (66%). Furthermore, combination of anti-ELAM-1 mAb (H18/7) and anti-ICAM-1 mAb (RR1/1) or of anti-ELAM-1 mAb (H18/7) and anti-CD18 mAb (IB4) had an additive effect in inhibiting both PMN adhesion as well as LTB4 production. PMN adherence to immobilized recombinant soluble rELAM-1 or rICAM-1 also increased LTB4 production, which was prevented with relevant mAbs. However, neither rELAM-1 nor rICAM-1 stimulated LTB4 production of PMN in suspension. We conclude that PMN adhesion to TNF alpha-stimulated endothelial cells enhances LTB4 production by PMN, a response activated by binding of PMN to expressed endothelial cell surface adhesion molecules.     

ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interaction with endothelial cell and extracellular matrix ligands

Leukocyte activation is a complex process that involves multiple cross- regulated cell adhesion events. In this report, we investigated the role of intercellular adhesion molecule-3 (ICAM-3), the third identified ligand for the beta 2 integrin leukocyte function-associated antigen-1 (LFA-1), in the regulation of leukocyte adhesion to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the 38- and 80-kD fragments of fibronectin (FN40 and FN80). The activating anti-ICAM-3 HP2/19, but not other anti-ICAM-3 mAb, was able to enhance T lymphoblast adhesion to these proteins when combined with very low doses of anti-CD3 mAb, which were unable by themselves to induce this phenomenon. In contrast, anti-ICAM-1 mAb did not enhance T cell attachment to these substrata. T cell adhesion to ICAM-1, VCAM-1, FN40, and FN80 was specifically blocked by anti-LFA-1, anti-VLA alpha 4, and anti-VLA alpha 5 mAb, respectively. The activating anti-ICAM-3 HP2/19 was also able to specifically enhance the VLA-4- and VLA-5-mediated binding of leukemic T Jurkat cells to VCAM-1, FN40, and FN80, even in the absence of cooccupancy of the CD3-TcR complex. We also studied the localization of ICAM-3, LFA-1, and the VLA beta 1 integrin, by immunofluorescence microscopy, on cells interacting with ICAM-1, VCAM-1 and FN80. We found that the anti-ICAM-3 HP2/19 mAb specifically promoted a dramatic change on the morphology of T lymphoblasts when these cells were allowed to interact with those adhesion ligands. Under these conditions, it was observed that a large cell contact area from which an uropod-like structure (heading uropod) was projected toward the outer milieu. However, when T blasts were stimulated with other adhesion promoting agents as the activating anti-VLA beta 1 TS2/16 mAb or phorbol esters, this structure was not detected. The anti-ICAM-3 TP1/24 mAb was also unable to induce this phenomenon. Notably, a striking cell redistribution of ICAM-3 was induced specifically by the HP2/19 mAb, but not by the other anti-ICAM-3 mAb or the other adhesion promoting agents. Thus, ICAM-3 was almost exclusively concentrated in the most distal portion of the heading uropod whereas either LFA-1 or the VLA beta 1 integrin were uniformly distributed all over the large contact area. Moreover, this phenomenon was also observed when T cells were specifically stimulated with the HP2/19 mAb to interact with TNF alpha-activated endothelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.

Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N-terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM-1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis.     

Loss of pentameric symmetry of C-reactive protein is associated with promotion of neutrophil-endothelial cell adhesion.

The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1-200 microg/ml) down-regulated neutrophil L-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21(ras) oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.     

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.     

Cutting edge: evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils

Leukocyte urokinase plasminogen activator receptors (uPARs) cluster at adhesion interfaces and at migratory fronts where they participate in adhesion, chemotaxis, and proteolysis. uPAR aggregation triggers activation signaling even though this glycolipid-anchored protein must associate with membrane-spanning proteins to access the cell interior. This study demonstrates a novel partnership between uPAR and L-selectin in human polymorphonuclear neutrophils. Fluorescence resonance energy transfer demonstrated a direct physical association between uPAR and L-selectin. To examine the role of L-selectin in uPAR-mediated signaling, uPAR was cross-linked and intracellular Ca(2+) concentrations were measured by spectrofluorometry. A mAb reactive against the carbohydrate binding domain (CBD) of L-selectin substantially inhibited uPAR-mediated Ca(2+) mobilization, whereas mAbs against the beta(2) integrin complement receptor 3 (CR3), another uPAR-binding adhesion protein, had no effect. Similarly, fucoidan, a sulfated polysaccharide that binds to L-selectin CBD, inhibited the Ca(2+) signal. We conclude that uPAR associates with the CBD region of L-selectin to form a functional signaling complex.     

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.     

Canine neutrophil margination mediated by lectin adhesion molecule-1 in vitro.

The contributions of the canine neutrophil lectin adhesion molecule-1 (LECAM-1) (canine homologue of the murine MEL-14 Ag) in neutrophil-endothelial cell adhesion and transendothelial migration were studied using anti-LECAM-1 mAb, CL2/6, and SL1 under static conditions and at wall shear stresses of up to 1.85 dynes/cm2 (dpc). Both mAb were found to inhibit attachment of neutrophils to cytokine-stimulated canine jugular vein endothelium. The inhibitory effects of the anti-LECAM-1 mAb were more evident at a wall shear stress of 1.85 dpc (greater than 50%) than at 0.23 dpc or under static conditions (approximately 30%). In contrast the anti-CD18 mAb, R15.7, exhibited higher inhibitory ability at the lower shear stress and under static conditions with marginal inhibition of adhesion at 1.85 dpc. Anti-LECAM-1 and anti-CD18 mAb showed additive inhibitory effects at the lower wall shear stress and under static conditions. Chemotactic stimulation of the neutrophils caused rapid down-regulation of LECAM-1 from the neutrophil surface and reduced adhesion by 60% at a wall shear stress of 1.85 dpc. This inhibition was not additive to anti-LECAM-1 mAb. Pretreatment with CL2/6 or SL1 did not affect trans-endothelial migration of adherent neutrophils under any experimental conditions tested. Anti-CD18 mAb, however, blocked transendothelial migration by 98% and 56% under static condition and at a wall shear stress of 0.23 dpc, respectively. The results in this report indicate that canine LECAM-1 is involved in the initial adhesion of unstimulated neutrophils to cytokine-stimulated endothelial cells under flow, but in contrast to CD18-integrins, plays no role in the transendothelial migration.     

LOX-1 supports adhesion of Gram-positive and Gram-negative bacteria

Adhesion of bacteria to vascular endothelial cells as well as mucosal cells and epithelial cells appears to be one of the initial steps in the process of bacterial infection, including infective endocarditis. We examined whether lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), a member of scavenger receptor family molecules with C-type lectin-like structure, can support adhesion of Gram-positive and Gram-negative bacteria. Chinese hamster ovary-K1 (CHO-K1) cells stably expressing LOX-1 can support binding of FITC-labeled Staphylococcus aureus and Escherichia coli, which was suppressed by poly(I) and an anti-LOX-1 mAb. Adhesion of these bacteria to LOX-1 does not require divalent cations or serum factors and can be supported under both static and nonstatic conditions. Cultured bovine aortic endothelial cells (BAEC) can also support adhesion of FITC-labeled S. aureus, which was similarly suppressed by poly(I) and an anti-LOX-1 mAb. In contrast, binding of FITC-labeled E. coli to BAEC was partially inhibited by the anti-LOX-1 mAb, and poly(I) did not block FITC-labeled E. coli adhesion to BAEC, but, rather, enhanced it under a static condition. TNF-alpha increased LOX-1-dependent adhesion of E. coli, but not that of S. aureus, suggesting that S. aureus adhesion to BAEC may require additional molecules, which cooperate with LOX-1 and suppressed by TNF-alpha. Taken together, LOX-1 can work as a cell surface receptor for Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli, in a mechanism similar to that of class A scavenger receptors; however, other unknown molecules may also be involved in the adhesion of E. coli to BAEC, which is enhanced by poly(I).