Leukocyte Capture Research Articles

Cellular pathology of atherosclerosis: smooth muscle cells prime cocultured endothelial cells for enhanced leukocyte adhesion.

During the development of an atherosclerotic plaque, mononuclear leukocytes infiltrate the artery wall through vascular endothelial cells (ECs). At the same time, arterial smooth muscle cells (SMCs) change from the physiological contractile phenotype to the secretory phenotype and migrate into the plaque. We investigated whether secretory SMCs released cytokines that stimulated ECs in a manner leading to increased leukocyte recruitment and thus might accelerate atheroma formation. SMCs and ECs were established in coculture on the opposite sides of a porous membrane, and the cocultured cells were incorporated into a flow-based assay for studying leukocyte adhesion. We found that coculture primed ECs so that their response to the inflammatory cytokine tumor necrosis factor-alpha was amplified. ECs cocultured with SMCs supported greatly increased adhesion of flowing leukocytes and were sensitized to respond to tumor necrosis factor-alpha at concentrations 10 000 times lower than ECs cultured alone. In addition, coculture altered the endothelial selectin adhesion molecules used for leukocyte capture. EC priming was attributable to the cytokine transforming growth factor-beta(1), which was proteolytically activated to a biologically active form by the serine protease plasmin. These results suggest a new role for secretory SMCs in the development of atheromatous plaque. We propose that paracrine interaction between ECs and SMCs has the potential to amplify leukocyte recruitment to sites of atheroma and exacerbate the inflammatory processes believed to be at the heart of disease progression.

Correlating the Kinetics of Cytokine-Induced E-Selectin Adhesion and Expression on Endothelial Cells

Many human diseases are mediated through the immune system. In chronic inflammatory disorders, the processes ordinarily involved in tissue healing become destructive. Endothelial cells normally recruit leukocytes to inflamed tissue using cytokine-induced adhesion receptors on the surfaces of interacting cells. Leukocyte capture depends on specialized characteristics of these receptors, particularly the binding kinetics. This study is designed to clarify the relationship between cytokine-induced changes in cell properties and binding kinetics. Here, we measure the kinetics of expression and monoclonal antibody binding for E-selectin in interleukin-1 α-stimulated microvascular endothelium in vitro and incorporate the data into kinetic models. Quantitative flow cytometry is used to determine molecular density (expression), and micropipette assays are used to find the probability of adhesion (function). Within five hours of interleukin-1 α stimulation, E-selectin density increases from 0 to 742 sites/ μm 2, and antibody-E-selectin adhesion probability increases from a baseline of 6.3% to 64%. A kinetic model is applied to find an apparent association rate constant, k f , of 3.7 × 10 −14 cm 2/sec for antibody-E-selectin binding. Although the model successfully predicts experimental results, the rate constant is undervalued for a diffusion-limited process, suggesting that functional adhesion may be modified through cytokine-induced changes in microtopology and receptor localization.

Adhesion molecule cascades direct lymphocyte recirculation and leukocyte migration during inflammation.

Leukocyte interactions with vascular endothelium are highly orchestrated processes that include the capture of free-flowing leukocytes from the blood with subsequent leukocyte rolling, arrest, firm adhesion, and ensuing diapedesis. These interactions occur under high shear stresses within venules and depend on multiple families of adhesion molecules. Many of the adhesion molecules involved are now identified. In addition, precise mechanisms underlying their regulation and our understanding of how different families of adhesion molecules work together is becoming clearer. Specifically, leukocyte/endothelial cell interactions such as capture, rolling, and firm adhesion can no longer be viewed as occurring in discrete steps mediated by individual families of adhesion molecules, but rather as a series of overlapping synergistic interactions among adhesion molecules resulting in an adhesion cascade. Although long thought to be mediated by distinct adhesion pathways, overlapping adhesion cascades mediate normal lymphocyte recirculation to peripheral lymphoid tissues and inflammation-induced leukocyte migration. These cascades thereby direct leukocyte migration, which is essential for the generation of effective inflammatory responses and the development of rapid immune responses.

A Direct Comparison of Selectin-Mediated Transient, Adhesive Events Using High Temporal Resolution

Leukocyte capture and rolling on the vascular endothelium is mediated principally by the selectin family of cell adhesion receptors. In a parallel plate flow chamber, neutrophil rolling on purified selectins or a selectin-ligand substrate was resolved by high speed videomicroscopy as a series of ratchet-like steps with a characteristic time constant (Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.-M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Biophys. J. 64:1922–1933; Alon, R., D. A. Hammer, and T. A. Springer. 1995. Nature (Lond.). 374:539–542). Under shear, neutrophil arrests due to bond formation events were as brief as 4 ms. Pause time distributions for neutrophils tethering on P-, E-, L-selectin, or peripheral node addressin (PNAd) were compared at estimated single bond forces ranging from 37 to 250 pN. Distributions of selectin mediated pause times were fit to a first order exponential, resulting in a molecular dissociation constant ( k off) for the respective selectin as a function of force. At estimated single bond forces of 125 pN and below, all three selectin dissociation constants fit the Bell and Hookean spring models of force-driven bond breakage equivalently. Unstressed k off values based on the Bell model were 2.4, 2.6, 2.8, 3.8 s −1 for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Bond separation distances (reactive compliance) were 0.39, 0.18, 1.11, 0.59 Å for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Dissociation constants for L-selectin and P-selectin at single bond forces above 125 pN were considerably lower than either Bell or Hookean spring model predictions, suggesting the existence of two regimes of reactive compliance. Additionally, interactions between L-selectin and its leukocyte ligand(s) were more labile in the presence of flow than the L-selectin endothelial ligand, PNAd, suggesting that L-selectin ligands may have different molecular and mechanical properties. Both types of L-selectin bonds had a higher reactive compliance than P-selectin or E-selectin bonds.

Mice Lacking Two or All Three Selectins Demonstrate Overlapping and Distinct Functions for Each Selectin

Selectins support the capture and rolling of leukocytes in venules at sites of inflammation and in lymphocyte homing. Gene-targeted mice with null mutations at the L-, E-, or P-selectin locus develop normally and show mild (E-/-) to moderate (P-/-, L-/-) defects in inflammatory cell recruitment. Mice lacking both P- and E-selectin (E/P-/-) have severe neutrophilia and spontaneous skin infections that limit their life span. Other combinations of selectin deficiency have not been investigated. We have generated novel mice lacking L- and P-selectin (L/P-/-), L- and E-selectin (L/E-/-), or all three selectins (E/L/P-/-) by bone marrow transplantation. L/P-/- mice (only E-selectin present) show an absence of leukocyte rolling after trauma and severely reduced rolling (by approximately 90%) in inflammation induced by TNF-alpha. Residual rolling in L/P-/- mice was very slow (3.6 +/- 0.2 micrometers/s after TNF-alpha). L/E-/- mice (only P-selectin present) showed rolling similar to that of L-/- at increased velocities (15.1 +/- 0.3 micrometer/s). The number of adherent leukocytes after 2 or 6 h of TNF-alpha treatment was not significantly reduced in L/E-/- or L/P-/- mice. E/L/P-/- mice showed very little rolling after TNF-alpha, all of which was blocked by mAb to alpha4 integrin. Adherent and emigrated neutrophils were significantly reduced at 6 h after TNF-alpha. We conclude that any one of the selectins can support some neutrophil recruitment but eliminating all three selectins significantly impairs neutrophil recruitment.

Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.

Leukocyte migration into sites of inflammation involves multiple molecular interactions between leukocytes and vascular endothelial cells, mediating sequential leukocyte capture, rolling, and firm adhesion. In this study, we tested the role of molecular interactions between fractalkine (FKN), a transmembrane mucin-chemokine hybrid molecule expressed on activated endothelium, and its receptor (CX3CR1) in leukocyte capture, firm adhesion, and activation under physiologic flow conditions. Immobilized FKN fusion proteins captured resting peripheral blood mononuclear cells at physiologic wall shear stresses and induced firm adhesion of resting monocytes, resting and interleukin (IL)-2-activated CD8(+) T lymphocytes and IL-2-activated NK cells. FKN also induced cell shape change in firmly adherent monocytes and IL-2-activated lymphocytes. CX3CR1-transfected K562 cells, but not control K562 cells, firmly adhered to FKN-expressing ECV-304 cells (ECV-FKN) and tumor necrosis factor alpha-activated human umbilical vein endothelial cells. This firm adhesion was not inhibited by pertussis toxin, EDTA/EGTA, or antiintegrin antibodies, indicating that the firm adhesion was integrin independent. In summary, FKN mediated the rapid capture, integrin-independent firm adhesion, and activation of circulating leukocytes under flow. Thus, FKN and CX3CR1 mediate a novel pathway for leukocyte trafficking.

Adhesion Molecules Regulating Human Leukocyte Attachment to Porcine Vascular Endothelium

463 Xenograft rejection may partly be mediated by infiltration of the porcine organ by the recipient's leukocytes. In humans and mice, leukocyte recruitment to sites of inflammation involves a cascade of interactions between adhesion molecules and their ligands. We have therefore studied the role of adhesion molecules in human leukocyte binding to TNF-a stimulated porcine aortic endothelial cell (PAEC) monolayers. Activation of PAEC enhances surface expression of E-selectin, vascular cell adhesion molecule-1(VCAM-1), and L-selectin ligand as demonstrated by staining with human L-selectin/murine IgM fusion protein. In both binding assays and hydrodynamic flow chamber assays, human neutrophils and lymphocytes adhered to activated PAEC. E-selectin and L-selectin primarily mediated neutrophil adhesion under shear conditions, while E-selectin and the β2 integrins promoted firm arrest. Under flow conditions, combined blockade of E-selectin, L-selectin, P-selectin glycoprotein ligand-1 (a hematopoietic P-selectin ligand) and β2 integrins dramatically reduced neutrophil attachment to PAEC. L-selectin primarily mediated lymphocyte attachment under shear conditions, while β2 integrin and VCAM-1/VLA-4 interactions mediated static lymphocyte adhesion. Combined blockade of β2 integrins, VLA-4 (a β1 integrin), VCAM-1, and L-selectin essentially inhibited all lymphocyte binding to PAEC. These results demonstrate that interactions between porcine adhesion molecules and their human counterreceptors remain remarkably intact across species. Further, disrupting successive steps of the adhesion cascade, namely leukocyte capture, rolling, and stable arrest, could diminish infiltration of a transplanted porcine organ by the human recipient's leukocytes, thereby attenuating xenograft rejection.

Adhesion Molecules Regulating Human Leukocyte Attachment to Porcine Vascular Endothelium

463 Xenograft rejection may partly be mediated by infiltration of the porcine organ by the recipient's leukocytes. In humans and mice, leukocyte recruitment to sites of inflammation involves a cascade of interactions between adhesion molecules and their ligands. We have therefore studied the role of adhesion molecules in human leukocyte binding to TNF-a stimulated porcine aortic endothelial cell (PAEC) monolayers. Activation of PAEC enhances surface expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and L-selectin ligand as demonstrated by staining with human L-selectin/murine IgM fusion protein. In both binding assays and hydrodynamic flow chamber assays, human neutrophils and lymphocytes adhered to activated PAEC. E-selectin and L-selectin primarily mediated neutrophil adhesion under shear conditions, while E-selectin and the ß2 integrins promoted firm arrest. Under flow conditions, combined blockade of E-selectin, L-selectin, P-selectin glycoprotein ligand-1 (a hematopoietic P-selectin ligand) and ß2 integrins dramatically reduced neutrophil attachment to PAEC. L-selectin primarily mediated lymphocyte attachment under shear conditions, whileß2 integrin and VCAM-1/VLA-4 interactions mediated static lymphocyte adhesion. Combined blockade of ß2 integrins, VLA-4 (aß1 integrin), VCAM-1, and L-selectin essentially inhibited all lymphocyte binding to PAEC. These results demonstrate that interactions between porcine adhesion molecules and their human counterreceptors remain remarkably intact across species. Further, disrupting successive steps of the adhesion cascade, namely leukocyte capture, rolling, and stable arrest, could diminish infiltration of a transplanted porcine organ by the human recipient's leukocytes, thereby attenuating xenograft rejection.

Distinct phenotype of E-selectin-deficient mice. E-selectin is required for slow leukocyte rolling in vivo.

Leukocyte capture and rolling are mediated by calcium-dependent lectins expressed on most leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To study the role of the selectins during inflammation, we have investigated leukocyte rolling in venules of tumor necrosis factor-alpha (TNF-alpha)-treated mouse cremaster muscles in wild-type mice and gene-targeted mice with homozygous deficiency for L-, P-, or E-selectin (L-/-, P-/-, or E-/-, respectively). TNF-alpha treatment induces expression of E-selectin and increases expression of P-selectin on endothelial cells. Consistent with previous reports of redundant P- and E-selectin function, a combination of monoclonal antibodies (mAbs) against P- and E-selectin (RB40.34 and 9A9, respectively) was necessary to block rolling in wild-type mice. The rolling leukocyte flux fraction (percent rolling cells) in L-/- mice was similar to that in wild-type mice, but rolling in these mice was blocked by a P-selectin mAb. The velocity of rolling leukocytes in TNF-alpha-treated wild-type, P-/-, or L-/- mice was 5 to 10 times slower (3 to 7 microns/s) than during trauma-induced rolling (20 to 50 microns/s). In contrast, leukocytes in venules of TNF-alpha-treated E-/- mice rolled significantly faster (12 to 20 microns/s): the rolling leukocyte flux fraction was more than doubled compared with wild-type, L-/-, or P-/- mice; and the number of adherent leukocytes was reduced. Addition of an E-selectin mAb, but not a P-selectin mAb, increased rolling flux fraction and rolling velocity in wild-type mice. Histological analysis revealed that 90% to 95% of all leukocytes interacting (rolling and adherent) with the venular endothelium in TNF-alpha-treated wild-type, L-/-, P-/-, and E-/- mice were granulocytes. These results identify a previously unrecognized phenotype of E-/- mice by establishing that at the site densities prevailing in vivo, E-selectin in responsible for slow (approximately 5 microns/s) granulocyte rolling. E-selectin-dependent slow rolling drastically increases the transit time of leukocytes rolling through an inflamed tissue and thus aids in targeting leukocytes activated by chemoattractants to the inflammatory microenvironment.

Molecular mechanisms of leukocyte recruitment in the inflammatory process

The microcirculation constitutes the functional interface between the circulating blood and the interstitial space. To gain access to sites of inflammation, leukocytes must pass the endothelial barrier. The recruitment paradigm encompasses leukocyte margination, capture, rolling, activation, firm adhesion, and transmigration. Recent experimental work has shown that E-, L- and P-selectins and alpha 4 integrins can mediate leukocyte rolling. Upon activation by chemokines, complement peptides or lipid mediators, firm adhesion is afforded by beta 1 and beta 2 integrins, InterCellular Adhesion Molecules (ICAMs) and Vascular Cell Adhesion Molecule-1 (VCAM-1). Beta 1 and beta 2 integrins as well as Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1) have been shown to be involved in transmigration. Intravital microscopic techniques have been instrumental in applying the conceptual advances of cell and molecular biology to the in vivo situation. This review focuses on the current understanding of the leukocyte recruitment paradigm as suggested by in vivo observations and in vitro model systems. The paradigm of neutrophil recruitment is presented to serve as a model for the recruitment mechanisms of other inflammatory cells.

Leukocyte interactions with vascular endothelium. New insights into selectin-mediated attachment and rolling.

Among the earliest signs of inflammation is the capture of leukocytes from the blood stream and their subsequent rolling along the endothelium of postcapillary venules. This commentary summarizes recent insight into the molecular basis of leukocyte rolling gained from gene-targeted mice, Ab blocking studies, and in vitro and in vivo reconstitution assays. These data reveal how the selectins individually and collectively contribute to the process of leukocyte capture and subsequent rolling on vascular endothelium.