Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions

The objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti-4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.     

Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1

We compared the abilities of selectins and the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), to support tethering and rolling of eosinophils and neutrophils under physiologic flow conditions. Eosinophils and neutrophils accumulated on P-selectin at similar site densities and rolled at similar velocities, but fewer eosinophils than neutrophils accumulated at any P-selectin density. Compared with neutrophils, few eosinophils accumulated on E-selectin except at high densities, and those cells that did accumulate rolled faster than neutrophils. Examination of the mechanisms for accumulation revealed that eosinophils and neutrophils formed similar numbers of primary tethers to P-selectin, whereas eosinophils formed fewer primary tethers to E-selectin than did neutrophils. Compared with neutrophils, adherent eosinophils also supported fewer leukocyte-leukocyte interactions, resulting in diminished secondary tethers to either P- or E-selectin. Studies with mAbs to L-selectin and PSGL-1 demonstrated that neither cell type used L-selectin to form primary tethers to P- or E-selectin. Both eosinophils and neutrophils used the NH2 terminus of PSGL-1 to form primary tethers to P-selectin, but not to E-selectin. Both cell types used L-selectin and PSGL-1 to promote leukocyte-leukocyte interactions and secondary tethers to P- or E-selectin. However, eosinophils developed significantly fewer secondary interactions, probably because they express less L-selectin than do neutrophils.     

Phosphorylation of the cytoplasmic domain of E-selectin is regulated during leukocyte-endothelial adhesion

E-selectin, a selectin expressed on activated vascular endothelium, supports rolling and stable adhesion of leukocytes at sites of inflammation. Previously, we have reported that leukocyte adhesion to cultured endothelial cells induces association of the cytoplasmic domain of E-selectin with cytoskeletal elements, suggesting that outside-in signaling may occur during E-selectin-mediated adhesion. To investigate this potential signaling function of E-selectin, HUVEC activated with recombinant human IL-1beta (10 U/ml, 4 h) were labeled with [32P]orthophosphate, and E-selectin was immunoprecipitated using mAb H18/7. Autoradiography revealed constitutive phosphorylation of E-selectin in these cells and time-dependent dephosphorylation following adhesion of HL-60 cells. Cross-linking of cell surface E-selectin using H18/7 and a polyclonal secondary Ab induced E-selectin dephosphorylation, as did adhesion of beads coated with recombinant P-selectin glycoprotein ligand-1 (PSGL-1), an E-selectin ligand. Using adenoviral vector-mediated transfection in HUVEC of a tail-less E-selectin and phosphoamino acid analysis, we documented phosphorylation occurring exclusively within the cytoplasmic domain and involving serine residues. Additional experiments using a series of cytoplasmic domain mutants of E-selectin expressed in COS-7 cells localized the regions that were constitutively phosphorylated. Preincubation with okadaic acid and sodium vanadate abrogated adhesion-induced dephosphorylation of E-selectin. Thus, E-selectin, which is constitutively phosphorylated in cytokine-activated human endothelial cells, undergoes an enzymatically regulated dephosphorylation following leukocyte adhesion. This process appears to be triggered by multivalent ligand binding and/or cross-linking of cell surface E-selectin. Ligand-dependent regulation of the phosphorylation of E-selectin's cytoplasmic domain provides additional evidence for a transmembrane signaling function of this molecule during leukocyte-endothelial interactions.     

Regulation of B cell apoptosis by Bcl-2 and Bcl-XL and its role in the development of autoimmune diseases (Review)

Leukocyte rolling is the earliest observable even in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewis(x) (sLe(x)) is required for function. Despite findings that sLe(x) inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin-dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNF alpha)-stimulated mouse cremaster is increased following treatment with either sLe(x) or the sLe(x)-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti-P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin-dependent) rolling is unaffected by either sLe(x) or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.     

Survey of rheumatic heart disease in school children of Kinshasa town

Leukocyte adhesion under flow is preferentially mediated by the selectins. In this study we used intravital microscopy to investigate whether E-selectin may promote firm leukocyte adhesion in vivo. E-Selectin is expressed by endothelial cells activated with tumor necrosis factor-alpha (TNF-alpha) and causes slow leukocyte rolling. Microinjection of formyl-methionyl-leucyl-phenylalanine (fMLP) or macrophage inflammatory protein-2 (MIP-2) next to a venule of the TNF-alpha-treated mouse cremaster muscle significantly increased the number of adherent leukocytes. In gene-targeted mice homozygous for a null mutation in the E-selectin gene or in wild-type mice treated with an E-selectin monoclonal antibody (mAb), this response was significantly attenuated (by >80%). No such defect was seen in intercellular adhesion molecule-1 (ICAM-1)-deficient mice. E-Selectin-null mice showed more rapid leukocyte rolling than wild-type or ICAM-1-deficient mice, resulting in significantly shortened leukocyte transit times through venules. Topical application of fMLP onto the whole cremaster muscle generated the same number of adherent leukocytes in wild-type and E-selectin-deficient mice. We conclude that slow leukocyte rolling through E-selectin results in long transit times, which are essential for efficient leukocyte adhesion in response to a local chemotactic stimulus.     

The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.     

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.     

The Fcgamma receptor-mediated respiratory burst of rolling neutrophils to cytokine-activated, immune complex-bearing endothelial cells depends on L-selectin but not on E-selectin

Intracellular H2O2 generation, as a measure of the respiratory burst, was determined after stimulation of neutrophils by immune complex (IC)-bearing human umbilical vein endothelial cells. Under static conditions, neutrophils basically responded to the immune deposits on resting endothelial cells. The rotating shear forces of approximately 0.7 dynes/cm2, corresponding to the physiological flow in postcapillary venules, completely abolished this basal H2O2 generation. After activation of the IC-bearing endothelial layers with interleukin-1 (IL-1) or tumor necrosis factor (TNF), or both, for 4 hours, rolling adhesion of the neutrophils was induced, accompanied by considerable H2O2 production. The neutrophil respiratory burst was prominently inhibited by anti-FcgammaRIII MoAb 3G8 (72.4%), and partially by MoAb 2E1 against FcgammaRII (38.5%). Both MoAbs together inhibited the Fc-mediated H2O2 generation by 93. 4%. The respiratory burst and rolling adhesion were markedly blocked by MoAb LAM1-3 against L-selectin (91.3%), whereas the nonfunctional anti-L-selectin MoAb LAM1-14 was ineffective. F(ab)2' fragments of MoAb 7A9 against E-selectin inhibited neutrophil rolling by 98.6%, but not the respiratory burst. Moreover, rolling adhesion of neutrophils and the related oxidative burst were CD11b/CD18- independent. In summary, L-selectin has a unique auxiliary function in triggering the FcgammaR-mediated respiratory burst of rolling neutrophils to IC-bearing endothelial cells, thereby substituting CD11b/CD18 under conditions of flow.     

Antibodies to proteinase-3 mediate expression of intercellular adhesion molecule-1 (ICAM-1, CD 54)

We investigated the role of intercellular adhesion molecule-1 (ICAM-1) in the adhesion of polymorphonuclear neutrophils (PMN) to classic antineutrophil cytoplasmic antibody (C-ANCA)-treated endothelial cells, independently of cytokines. Human umbilical vein endothelial cells (HUVEC) grown to confluence in cytokine-free conditions were stimulated with C-ANCA sera and affinity-purified anti-proteinase 3 antibodies (PR3) from Wegener's granulomatosis (WG) patients. Non-activated PMN were added to treated HUVEC and adhesion was measured. In parallel experiments, treated HUVEC were fixed and ICAM-1 and E-selectin were assayed by cyto-ELISA; in other experiments anti-ELAM-1 and anti-ICAM-1 antibodies were assessed. In this in vitro model, adhesion of non-activated PMN to anti-PR3-stimulated HUVEC was enhanced. Adhesion was greater with anti-PR3 antibodies than with control and normal immunoglobulins, and correlated with the level of anti-PR3 antibodies. Neutralization of anti-PR3 antibodies by neutrophil azurophilic granule proteins abolished adhesion. This adhesion increased at the fourth hour after simulation, peaked at the twelfth hour and then decreased. This phenomenon occurred mainly through endothelial expression of ICAM-1 (the main counter-receptor for integrins, involved in firm PMN adhesion and migration) and E-selectin on HUVEC membranes. Anti-adhesion molecule antibodies inhibited this adhesion. This work supports the hypothesis of a direct effect of C-ANCA in endothelial stimulation, namely, on endothelium-PMN adhesion, and strengthens the major role of ICAM-1, directly involved in firm sticking of PMN to HUVEC, besides E-selectin. C-ANCA upregulate endothelial adhesiveness and thus participate in inflammatory reactions by providing endothelial adhesive structures for neutrophils. This might be one of the first steps leading to clinical expression of the disease. These results provide new insights into the pathogenesis of C-ANCA-related diseases.     

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.     

Eosinophil tethering to interleukin-4-activated endothelial cells requires both P-selectin and vascular cell adhesion molecule-1

We examined the mechanisms used by eosinophils to tether and accumulate on interleukin-4 (IL-4)-stimulated human umbilical vein endothelial cells (HUVECs) under flow conditions. As previously reported, HUVECs treated for 24 hours with 20 ng/mL IL-4 had increased expression of P-selectin and vascular cell adhesion molecule-1 (VCAM-1) but not E-selectin. We found that eosinophils tethered and rolled on IL-4-stimulated HUVECs at physiologic shear stresses. Eosinophil rolling was quickly followed by firm adhesion. Treatment with either an anti-P-selectin monoclonal antibody (MoAb) or an anti-VCAM-1 MoAb decreased both eosinophil tethering and accumulation at 2 dyn/cm2. VCAM-1 interacts with 4-integrins expressed on eosinophils. We found that an anti-4-integrin MoAb also blocked eosinophil tethering and accumulation at 2 dyn/cm2. None of these MoAbs alone had an impact on eosinophil accumulation at lower shear stresses, but when either an anti-VCAM-1 or an anti-4-integrin MoAb was used in combination with an anti-P-selectin MoAb, all eosinophil tethering and accumulation on IL-4-stimulated HUVECs were blocked. This was true at both high and low shear stresses. These data show that both P-selectin and VCAM-1 are required to tether eosinophils at high shear stresses, but at low shear stresses these adhesion proteins can act independently to recruit eosinophils to IL-4-stimulated HUVECs.     

CD4+ T cells migrate into inflamed skin only if they express ligands for E- and P-selectin

Previous data suggested a role of endothelial selectins in skin homing of lymphocytes. In the current study, we have analyzed the expression and functional role of E-and P-selectin ligands on CD4+ T cells induced in vivo upon skin sensitization, using soluble selectin-Ig chimera and blocking Abs. Only low numbers of CD4+ cells expressing significant levels of E- or P-selectin ligands were present in s.c. lymph nodes of untreated mice (0.5-1.5% and 2-4%, respectively). Induction of a delayed-type hypersensitivity reaction increased the percentage of E-selectin-binding CD4+ cells in the draining lymph nodes up to 6 to 9% and that of P-selectin-binding cells up to 14%. The majority of E- and P-selectin-binding cells displayed an activated phenotype as judged by the increase in IL-2R, CD71, or cell size. The populations of E- and P-selectin-binding cells were largely overlapping; all E-selectin-binding cells also bound to P-selectin, whereas only a subfraction of P-selectin-binding cells reacted with E-selectin. Both E- and P-selectin-binding CD4+ cells, isolated by FACS, efficiently migrated into inflamed, but not normal skin, whereas P- or E-selectin ligand-negative CD4+ T cells did not. Abs against one of the two endothelial selectins partially inhibited the entry of isolated, ligand-positive cells, whereas a combination of Abs against both selectins almost completely abrogated skin homing. These data indicate that the expression of functional ligands for E- and for P-selectin is essential for homing of CD4+ T cells into the inflamed skin.     

Survival in lung reperfusion injury is improved by an antibody that binds and inhibits L- and E-selectin

The selectins are a three-member family of leukocyte, platelet, and endothelial cell adhesion proteins that mediate leukocyte traffic into normal and inflamed tissues. P-selectin is expressed by endothelial cells and platelets, E-selectin by endothelial cells, and L-selectin by circulating leukocytes. To determine if selectin-mediated leukocyte adhesion influences the development of lung reperfusion injury, we studied hemodynamics and respiratory and inert gas exchange in sheep subjected to 3-hour in situ left lung ischemia followed by 6-hour left lung reperfusion with the right lung excluded. Ten minutes before reperfusion, eight animals received EL-246 (1 mg/kg intravenously), a novel antihuman selectin antibody that recognizes and blocks both L- and E-selectin and cross-reacts in sheep. Eight control animals with ischemia received no treatment, whereas three received an isotype-matched antihuman L-selectin antibody that does not cross-react in sheep (DREG-56, 1 mg/kg intravenously). Eight sham control sheep underwent an identical operative procedure but were never subjected to ischemia. Volume-cycled, pressure-limited (20 cm H2O) mechanical ventilation was consistent in all animals throughout the experiment. Six-hour survival in EL-246 recipients (100%) was significantly higher than in either ischemic control sheep (37.5%) or DREG-56 recipients (33.3%), but gravimetric lung water was equivalent in EL-246 recipients (5.9 +/- 1.7 ml/kg), ischemic control sheep (8.3 +/- 3.0 ml/kg), and DREG-56 recipients (9.1 +/- 2.6 ml/kg).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor-alpha induces leukocyte recruitment by different mechanisms in vivo and in vitro

It is well established that E-selectin is the endothelial adhesion molecule that is primarily responsible for mediating leukocyte rolling on TNF-alpha-stimulated cultured endothelial cells. Despite this, few studies in in vivo inflammatory models have observed reduced leukocyte accumulation using mAbs against E-selectin. The objective of this study was to compare the function of E-selectin on endothelial cells in vitro with its role in TNF-alpha-induced leukocyte recruitment in vivo using EL246, a mAb that blocks the function of E-selectin on activated feline endothelial cells. In vitro experiments using feline endothelial cells showed that EL246 functionally inhibits E-selectin-dependent leukocyte recruitment induced by TNF-alpha, without affecting the function of other rolling mechanisms. Intravital microscopy of single 25- to 40-microm venules in the feline mesentery was then used to examine leukocyte rolling and adhesion in response to superfusion with TNF-alpha. TNF-alpha treatment significantly increased the number of both rolling and adherent leukocytes and significantly decreased leukocyte rolling velocity. Treatment with EL246 (1 mg/kg), either i.v. at the start of the TNF-alpha protocol or directly into the superior mesenteric artery after 3 h of TNF-alpha treatment, had no effect on leukocyte rolling, adhesion, or rolling velocity. However, treatment with the selectin-binding carbohydrate, fucoidan, reduced leukocyte rolling to below baseline levels. These results suggest that in contrast to its prominent role on cultured endothelial cells, E-selectin does not contribute to leukocyte recruitment in TNF-alpha-stimulated feline mesenteric venules in vivo.     

Effects of antirheumatic drugs on adhesiveness of endothelial cells and neutrophils

Because disease-modifying antirheumatic drugs might exert part of their effects on adhesion of polymorphonuclear neutrophils (PMN) to endothelial cells, this being the first step for PMN migration to inflammatory lesions, we evaluated such drug effects in vitro. Gold sodium thiomalate (GSTM) impaired the ability of interleukin 1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) to express E-selectin and to bind PMN but had no effect on the expression of intercellular adhesion molecule 1 (ICAM-1) or on hyperadhesivity of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN. Auranofin (AF) interacted with HUVEC and PMN adhesiveness but in opposite directions: this drug hampered IL-1beta-induced HUVEC hyperadhesiveness and expression of E-selectin and intercellular adhesion molecule 1, but augmented PMN adherence and CD18 expression. The net effect of auranofin was a reduction of cytokine-driven adhesiveness and enhancement of formylpeptide-induced adhesion. Salazopyrin did not affect HUVEC or PMN adhesiveness or E-selectin and intercellular adhesion molecule 1 expression. Thus, the gold-containing drugs modulated HUVEC and PMN adhesiveness by different mechanisms but ones involving surface adhesion molecules.     

A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature

A two-step paradigm for leukocyte recruitment has been established in a number of tissues including the mesentery, skin, and muscle, and necessitates an initial rolling step via the selectins before firm leukocyte adhesion via the integrins. In view of the many inflammatory diseases that involve the liver, we investigated the importance of rolling and the selectins in the hepatic microvasculature and compared the responses to that of the commonly used mesentery or cremaster microvasculature. We visualized the liver microvasculature using intravital microscopy and we determined that within the liver the majority of leukocytes adhere within the sinusoids (80%) in response to a chemotactic stimulus such as FMLP (20% in postsinusoidal venules) whereas leukocytes adhere exclusively within postcapillary venules in tissue like the mouse cremaster. In the sinusoids, the adhesive response to FMLP is not dependent upon selectins inasmuch as adhesion was not reduced in the sinusoidal vessels of P-selectin-deficient mice or E-selectin/P-selectin- deficient animals in the presence or absence of L-selectin antibody. No rolling or adhesion was detected in response to FMLP in the selectin-deficient cremaster microvasculature. Immunoneutralization of selectins with fucoidan in wildtype mice eliminated rolling and adhesion in the cremaster but failed to affect adhesion in the liver sinusoids in response to FMLP. More long-term leukocyte recruitment with lipopolysaccharide (4 h) was also impaired in the cremaster but not the liver microvasculature in selectin-deficient animals. Leukocyte adhesion in the sinusoids was reduced in P-selectin-deficient mice also lacking intercellular adhesion molecule-1 (ICAM-1). This study for the first time demonstrates that selectins are not an essential step for leukocyte recruitment into the inflamed liver microvasculature.     

Activation of cultured vascular endothelial cells by antiphospholipid antibodies

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.     

IL-12 promotes the adhesion of NK cells to endothelial selectins under flow conditions

This study examined the adhesive interactions of peripheral blood NK cells with P- and E-selectin and analyzed the effect of IL-12 on the binding of NK cells to these selectins. P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on most resting and IL-12-activated NK cells. However, the percentage of resting NK cells bound to P-selectin-IgG was 15%, and that of activated NK cells bound to P-selectin-IgG was 65%. Furthermore, the number of IL-12-activated NK cells bound to P-selectin-transfected Chinese hamster ovary cells was significantly higher than that of resting NK cells under flow conditions. These interactions were abolished by the incubation of these NK cells with anti-PSGL-1 (PL-1) mAb. Thus, PSGL-1/P-selectin interaction is important in the binding of resting and activated NK cells to P-selectin. NK cells express sialyl-Lewis(x) (sLe(x)) structure recognized by anti-sLe(x) mAb (KM-93), and IL-12 activation of NK cells increased the mean fluorescence intensity of KM-93-reactive NK cells. Adhesion of IL-12-activated NK cells to E-selectin-transfected Chinese hamster ovary cells was stronger than that of resting NK cells under flow conditions. These interactions were reduced markedly by incubation with anti-sLe(x) mAb. Thus, sLe(x) is the major ligand of resting and activated NK cells for E-selectin. These findings indicate that IL-12 stimulation of NK cells promotes their adhesion activity to endothelial selectins.     

A juxtacrine mechanism for neutrophil adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process

The aim of this study was to identify the molecular mechanisms involved in neutrophil adhesion to immobilized platelets with particular focus on the possible existence of a juxtacrine system for neutrophil-platelet interactions. Platelets were immobilized onto collagen (type I)-coated coverslips that were placed in a flow chamber and neutrophils were perfused across these confluent monolayers at a shear stress of 1 to 4 dynes/cm2. Neutrophils rolled, and a significant proportion (25% to 50%) adhered to platelet monolayers. P-selectin was expressed in very large quantities on the surface of platelets and mediated all of the rolling, whereas the beta2-integrin mediated firm adhesion. An activation mechanism for adhesion was necessary inasmuch as fixed neutrophils continued to roll on immobilized platelets, but did not adhere. Platelets adherent to collagen produced significant levels of platelet-activating factor (PAF). Accordingly, the firm adhesion of neutrophils to platelets was significantly inhibited by a PAF receptor antagonist (WEB 2086). Treatment of only the platelets with acetylhydrolase, which converts membrane-associated PAF to lyso-PAF, prevented 60% of the adhesion. These data suggest that PAF, on the surface of platelets, mediated a significant portion of the adhesive interaction. Addition of some selectin-binding carbohydrates (fucoidan or soluble SLEx analogs but not dextran sulfate) to the platelets caused rolling neutrophils to immediately adhere, an event that was not observed on histamine or thrombin-treated endothelium or P-selectin transfectants. These data support the view that a juxtacrine activation process exists on immobilized platelets for neutrophils. This process can be greatly enhanced on platelets and may involve a signaling mechanism through P-selectin.     

Novel anti-inflammatory compounds induce shedding of L-selectin and block primary capture of neutrophils under flow conditions

Leumedins are small molecules that inhibit neutrophil movement into inflamed tissues. These compounds have been shown to inhibit the adherence of neutrophils in static adhesion assays mediated by beta2-integrins. We now report that leumedins, like activating agents, induce the loss of L-selectin from the neutrophil surface. The loss of L-selectin is unrelated to the inhibition of static adhesion, since neutrophils that have been pretreated with leumedins to cause shedding of L-selectin, followed by removal of drug, adhere normally in a static adhesion assay, and this adhesion is inhibited upon readdition of leumedin. In an assay of adhesion to endothelial cells under conditions of physiologic wall shear stress, leumedins prevent both primary capture of neutrophils mediated by L-selectin and firm adherence mediated by beta2-integrins.