PubMed Health⌕ Search

Biomedical subjects

S I Simon

Publications and source records attributed to S I Simon.

At least 37 records · Page 2Linked to original sources
PubMed1998

Beta2-integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells.

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and beta2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s(-1)) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s(-1) in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s(-1). The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed.

Animals↗
PubMed1998

The multistep process of homotypic neutrophil aggregation: a review of the molecules and effects of hydrodynamics.

Homotypic adhesion of neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes are supported by L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand the hydrodynamic stresses. Using cone and plate viscometry to apply a uniform linear shear field to suspensions of neutrophils and flow cytometry to quantitate the size distribution of aggregates formed over the time course of formyl peptide stimulation, we conducted a detailed examination of the affect of shear rate and shear stress on the kinetics of cell aggregation. The efficiency of aggregate formation was fit from a mathematical model based on Smoluchowski's two-body collision theory. Over a range of venular shear rates (400-800 s-1), approximately 90% of the single cells are recruited into aggregates ranging from doublets to grouping larger than sextuplets. Adhesion efficiency fit to the kinetics of aggregation increased with shear rate from approximately 20% at 100 s-1 to a maximum level of approximately 80% at 400 s-1. This increase to peak adhesion efficiency was dependent on L-selectin and beta 2-integrin, and was resistant to shear stress up to approximately 7 dyn/cm2. When L-selectin was blocked with antibody, beta 2-integrin (CD11a,b) supported adhesion at low shear rates (< 400 s-1). Aggregates formed over the rapid phase of aggregation remain intact and resistant to shear up to 120 s. At the end of this plateau phase of stability, aggregates spontaneously dissociate back to singlets. The rate of cell disaggregation is linearly proportional to the applied shear rate. The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to firm but reversible adhesion.

Animals↗
PubMed1998

[New approaches to tissue management in minimal invasive pediatric surgery].

Tissue management in minimally invasive surgery plays an important role in the indication for and feasibility of surgical interventions. Hemostasis and sealing of larger areas are possible with minimally invasive tissue gluing in liquid and bandage-bound form. For effective and efficient application of the precoated collagen fleece (TachoComb) a modularly constructed applicator (AMISA) was designed that is suitable for a wide range of indications in MIS and allows selective leak closure (SLC) in parenchymatous tissues with different lesions (Pneumothorax, Chylothorax, rupture of liver/spleen, biopsies, fistulae).

Aprotinin↗
PubMed1997

Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration.

L-selectin enables capture and rolling of neutrophils on inflamed endothelium. This may facilitate the binding of agonists such as IL-8 and platelet-activating factor (PAF), which signal CD18-mediated firm adhesion and transmigration. Recent studies demonstrate that L-selectin can mediate transmembrane signaling. However, the functional effects of costimulation through agonist and L-selectin require further study. Here, we quantify cell adhesion, motility, and transmigration in response to co-activation through L-selectin and agonist. The surface expression of CD11b/CD18 increased and L-selectin decreased in proportion to the extent of L-selectin cross-linking. A flow cytometric assay was used to measure CD11b/CD18-dependent adhesion to fluorescent beads adsorbed with albumin. Neutrophil adhesion was detected within seconds of adding PAF (20 pM), IL-8 (50 pM), or cross-linking L-selectin. Costimulation through agonist and L-selectin potentiated by up to threefold the rate and extent of bead capture. Stimulation through L-selectin induced membrane ruffling, whereas PAF or IL-8 induced bipolar shape change. L-selectin cross-linking sustained the transient shape change induced by low concentrations (10-50 pM) of agonist. Chemokinesis stimulated by IL-8 was inhibited in the presence of cross-linking L-selectin. This was attributed to enhanced cell spreading following costimulation. Migration across HUVEC monolayers stimulated with IL-1 was also potentiated in the presence of L-selectin cross-linking. We propose that cross-linking of L-selectin and binding of agonist receptors may act synergistically to amplify neutrophil activation and emigration in the inflamed vasculature.

Cell Adhesion↗
PubMed1997

Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners.

Since macromolecular permeability between endothelial cells is regulated by tight junctions (zonula occludens), we wished to determine whether they also regulate neutrophil transendothelial migration. HUVEC monolayers, a commonly used model for studying leukocyte transmigration, were characterized using electric cell substrate impedance sensing and transmission electron microscopy. We show that culture medium containing endothelial cell growth supplement (50 microg/ml) was sufficient and necessary for the development of endothelial tight junctions. The frequency with which tight junctions were observed by transmission electron microscopy was further increased (twofold) by culturing HUVEC monolayers in a 1:1 mixture of endothelial medium and astrocyte-conditioned medium. These astrocyte-conditioned HUVEC monolayers showed a >1.5-fold increase in transcellular electrical resistance. The extent of neutrophil migration across IL-1-treated (10 U/ml for 4 h) HUVEC monolayers was the same whether tight junctions were present or absent, and the molecular requirements for neutrophil transmigration (CD18 and intercellular adhesion molecule-1) were unaffected by culturing in astrocyte-conditioned medium. Immunostaining for proteins associated with the intercellular junctional domain (occludin, ZO-1, cadherin, beta-catenin, gamma-catenin, and platelet-endothelial cell adhesion molecule-1) was localized to the endothelial borders, regardless of the culture conditions. Discontinuities were observed in the border staining for occludin, ZO-1, cadherin, and beta-catenin at the tricellular corner where the borders of three endothelial cells intersected. Significantly, 75% of neutrophil migration across IL-1-treated HUVEC monolayers occurred at tricellular corners. It appears that neutrophils preferentially migrate around endothelial tight junctions by crossing at tricellular corners rather than passing through the tight junctions that lie between two endothelial cells.

Astrocytes↗
PubMed1997

Regulation of human T lymphocyte coactivation with an alpha4 integrin antagonist peptide.

The cyclic hexapeptide CWLDVC (TBC 772) is an antagonist of alpha4 integrins and a potent inhibitor of lymphocyte interactions with fibronectin, vascular cell adhesion molecule-1, and muscosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). As such, peptide TBC 772 effectively inhibits the activation of freshly isolated human T lymphocytes stimulated with purified vascular cell adhesion molecule-1 coimmobilized with anti-CD3 mAb. The influence of peptide binding on distinct sites of the alpha4beta1 complex was determined by flow cytometry and cellular adhesion assays employing a panel of mAbs. Binding of the alpha4-specific mAb L25 and the beta1-specific mAb 33B6 was not altered by the peptide; however, binding of mAb 19H8, which is specific for a combinatorial epitope of alpha4beta1, was dramatically inhibited. Treatment of lymphocytes with the peptide caused an increase in a ligand-induced epitope on beta1 integrin defined by mAb 15/7. In T cell activation studies using coimmobilized anti-CD3 mAb and the anti-integrin mAbs, the peptide had broader inhibitory activity, suppressing costimulation induced by all the integrin mAbs. The peptide was not generally toxic and was integrin selective in its suppressive activity, as coactivation by ligation of CD3 in conjunction with CD28 or CD26 was not affected. These results suggest that the antagonist peptide CWLDVC can effectively neutralize integrin coactivation systems by a mechanism independent of competitive binding.

Antibodies, Monoclonal↗
PubMed1997

Neutrophil CD18-dependent arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow can be activated through L-selectin.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

Adult↗
PubMed1997

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.

Cell Adhesion↗
PubMed1996

GlyCAM-1, a physiologic ligand for L-selectin, activates beta 2 integrins on naive peripheral lymphocytes.

Naive T cells are selectively recruited from the blood into peripheral lymph nodes during lymphocyte recirculation. L-selectin, a lectin-like receptor, mediates the initial attachment of lymphocytes to high endothelial venules (HEV) in lymph nodes. A subsequent step involving the activation of beta 2 integrins has been proposed to facilitate firm adhesion, but the activating signals are poorly understood. We report here that either antibody-mediated cross-linking of L-selectin on human lymphocytes or treatment of the cells with GlyCAM-1, an HEV-derived, secreted ligand for L-selectin, stimulates their binding to ICAM-1 through the beta 2 integrin pathway. Furthermore, GlyCAM-1 causes the rapid expression of a neoepitope on beta 2 integrins associated with a high-avidity state. Naive (CD45RA+), but not memory (CD45R0+) lymphocytes, respond to L-selectin cross-linking or GlyCAM-1 treatment. Thus, the complexing of L-selectin by specific ligands may provide key signals to naive lymphocytes, contributing to their selective recruitment into peripheral lymphoid organs.

Antibodies, Monoclonal↗
PubMed1996

Chemotactic factors stimulate CD18-dependent canine neutrophil adherence and motility on lung fibroblasts.

The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown. We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts. Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry. Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts. However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1. Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts. PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent. Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA. In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h. Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils. These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium.

Animals↗
PubMed1996

Induction of homotypic lymphocyte aggregation: evidence for a novel activation state of the beta1 integrin.

Intercellular adhesion of Jurkat lymphocytic cells was investigated by use of monoclonal antibodies 33B6 and 18D3, which bind to the beta1 integrin receptor. 33B6 induced homotypic aggregation of Jurkat cells, whereas 18D3 inhibited this aggregation. Jurkat cells could he induced to aggregate at low 33B6 concentrations corresponding to 5% beta1 integrin site occupancy, and the rate of aggregation was maximum at 30% occupancy. Simultaneous addition of mAb 18D3 and 33B6 demonstrated that the two antibodies mediate changes in the beta1 integrin activation state that are competitive in nature. Aggregation through beta1 integrin induced by 33B6 was reversed by subsequent addition of 18D3. To further examine the mechanism by which 33B6 and 18D3 affect cell adhesion function, we explored the binding of monoclonal antibody (mAb) 15/7. This mAb recognizes an activation epitope of the beta1 integrin and has been shown to sustain cell adhesion to vascular cell adhesion molecule 1 (VCAM-1) and fibronectin. Activation of Jurkat cells with Mn2+ caused a 2.5-fold increase in 15/7 binding but did not increase binding of 33B6. 33B6 partially blocked 15/7 binding to beta1 integrin on unstimulated and Mn2+-activated Jurkat cells. 18D3 did not affect mAb 15/7 binding. These results indicate that 33B6 and 18D3 modulated homotypic aggregation by inducing a novel activation state of the very late activation integrin distinct from the state recognized by 15/7, which supports cell binding to VCAM-1 and fibronectin.

Antibodies, Monoclonal↗
PubMed1996

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.

Antibodies, Monoclonal↗
PubMed1996

Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.

We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.

Animals↗
PubMed1995

L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin.

Emigration of leukocytes at sites of inflammation is initiated by the selectin family of carbohydrate-binding adhesion molecules. Molecular crossbridges initiate rolling of cells along the vascular endothelium where chemokines such as IL-8 and platelet activating factor (PAF) may be presented to their receptors on the leukocyte surface resulting in cell stimulation. Integrin activation appears to be a requirement for subsequent cell localization and diapedesis into the tissue. Several recent reports have demonstrated that ligation and cross-linking of neutrophil L-selectin results in neutrophil activation, including intracellular calcium release, superoxide production, and induction of mRNA for production of IL-8 and TNF-alpha. The purpose of this study was to examine whether ligation and cross-linking of L-selectin would specifically result in activation of beta 2-integrin-dependent adhesion. A fluorescence flow cytometric assay was developed that directly measures Mac-1-dependent cell adhesion. Fluorescent latex beads (2-microns diameter) were adsorbed with albumin or fibrinogen and added in excess to human neutrophils in a shear-stirred suspension. Following stimulation the kinetics of bead capture by neutrophils was continuously measured in real time on the flow cytometer. The onset of bead binding was detected in the presence of extremely low concentrations of PAF (10 pM) or formyl peptide (0.2 nM) stimulation. Ligation of L-selectin with whole IgG DREG200 or DREG56 Ab, but not controls (anti-CD44, -CD45, -CD11a), resulted in a significant potentiation of bead binding. Cross-linking F(ab')2 fragments of DREG200 with a goat anti-mouse F(ab')2 secondary Ab also stimulated beta 2-integrin-dependent adhesion in a dose-dependent fashion. A chimeric form of DREG200 expressing gamma 4 or gamma 1 isotypes of human Fc domain also stimulated cell adhesion when cross-linked. Surface expression of CD18 and an activation-dependent epitope, as detected with mAb24, also increased in response to L-selectin cross-linking. Cross-linking L-selectin induced significant adhesion and transmigration of neutrophils across human umbilical vein endothelial cells. We propose that cross-linking of L-selectin results in a cell signal that directly stimulates beta 2-integrin adhesive responses.

Adult↗
PubMed1995

P-selectin- and CD18-mediated recruitment of canine neutrophils under conditions of shear stress.

Neutrophil mobilization at sites of inflammation or thrombosis involves the participation of several adhesion molecules expressed on neutrophils and vascular endothelial cells. Local vascular damage with disruption of the endothelium results in adhesion of platelets to the exposed subendothelium, and these platelets could also participate in neutrophil recruitment. This initial phase of mobilization could be followed by heterotypic aggregation to recruit more leukocytes in the area. The present study first examined the interactions of adherent canine platelets and flowing canine neutrophils using an in vitro system that simulates vascular flow conditions. Results showed that collagen-adherent platelets express the adhesion molecule P-selectin on their surface and can support neutrophil arrest (612 +/- 43 neutrophils/mm2) at shear stresses of approximately 2.5 dynes/cm2. Both transient adhesion (manifested by a rolling-type behavior) and complete arrest were observed. These interactions could be totally inhibited by a monoclonal antibody directed against platelet P-selectin (24 +/- 18 neutrophils/mm2) but not by a monoclonal antibody against neutrophil CD18 (625 +/- 46 neutrophils/mm2). Additionally, under shear mixing conditions (700 RPM), canine blood leukocytes exhibited aggregation (> 80% singlets recruited into aggregates after 5 minutes), and this process does not involve P-selectin but is dependent on the neutrophil integrin CD18. However, stimulation of the blood with platelet-activating factor (5-20 ng/ml) induced a rapid aggregation with a significantly greater number of aggregates when compared with stirring alone (68.3% +/- 3.2% versus 35.2% +/- 6.3% at 1 minute, P < 0.05), and this aggregation was both P-selectin and CD18 dependent. Overall, these two mechanisms of leukocyte recruitment (neutrophil arrest on adherent platelets and aggregation) could act sequentially and in a cooperative manner to bring into close contact platelets and neutrophils at sites of inflammation and thrombosis in pathologic conditions in the dog.

Animals↗
PubMed1994

Lipopolysaccharide enhances CD11b/CD18 function but inhibits neutrophil aggregation.

Human neutrophils are primed in the presence of complexes of lipopolysaccharide (LPS) with its serum binding protein (LBP) in a manner dependent on CD14. Cellular consequences of priming include increased responsiveness, the upregulation of surface proteins including the adhesive integrin CD11b/CD18 (Mac-1), the increased binding of certain ligands to CD11b/CD18, and the concurrent shedding of the L-selectin homing receptor. Because expression of both CD11b/CD18 and L-selectin is obligatory for formyl peptide-stimulated neutrophil aggregation in vitro (Simon et al, Blood 82:1097, 1993), we have examined the consequences of bacterial endotoxin on the expression of neutrophil adhesive molecules. We observed that the exposure of neutrophils to LPS/LBP, while enhancing the surface numbers and adhesive function of CD11b/CD18 for latex particles, did not induce aggregation. In contrast, as the LPS/LBP concentration increased (ED50 = 30 ng/mL LPS/LBP), the ability of neutrophils to aggregate decreased in parallel with the shedding of L-selectin. Moreover, when L-selectin adhesive activity was blocked by treatment with Fab fragments of Dreg-200, aggregation was inhibited to an extent roughly proportional to the available L-selection. Blocking of LPS/LBP with CD14-specific monoclonal antibodies suppressed L-selectin shedding and preserved formyl peptide-stimulated aggregation. Taken together, the data suggest that inhibition of neutrophil aggregation by LPS/LBP is related to the expression of L-selectin via CD14 rather than LPS inhibition of CD11b/CD18 function during cellular stimulation.

Acute-Phase Proteins↗
PubMed1994

A role for lectin interactions during human neutrophil aggregation.

We have recently reported that neutrophil aggregation is dependent on both L-selectin and the beta 2-integrin Mac-1, raising the possibility that carbohydrate interactions play a role in aggregation. We used mono- and polysaccharides known to inhibit L-selectin-dependent adhesion of lymphocytes to high endothelial venules to test whether these carbohydrates could inhibit neutrophil aggregation. Similar types and concentrations of carbohydrates found by others to inhibit lymphocyte adhesion were effective in blocking neutrophil aggregation. Thus, nanomolar concentrations of the polysaccharides dextran sulfate (m.w. 500,000) and fucoidan inhibited aggregation, whereas dermatan sulfate, alpha-carrageenan, and dextran sulfate (m.w. 5,000) showed no inhibition. All of the phosphorylated monosaccharides tested inhibited aggregation with ED50 values between 8 and 17 mM, the most potent being mannose-6-phosphate and fucose-1-phosphate. The nonphosphorylated monosaccharides glucose and fucose were noninhibitory. The inhibitory effects of fucoidan or dextran sulfate (m.w. 500,000) did not appear to be due to altered regulation of L-selectin after stimulation because fucoidan reduced the rate of L-selectin shedding, whereas dextran sulfate had no effect compared with control. Neither carbohydrate inhibited the binding of formyl peptide to its receptor. However, carbohydrates were able to compete with mAb binding to a number of known leukocyte adhesion proteins. We used endotoxin pretreatment to create L-selectin-deficient neutrophils to study the minimum adhesive requirements for aggregation using two-color fluorescence flow cytometry. Our results implicate a lectinlike contribution to neutrophil aggregation, and suggest that L-selectin is the molecule that mediates the carbohydrate-dependent adhesive event.

Antibodies, Monoclonal↗