Interstitial leukocyte migration and immune function.

The trafficking of leukocytes into and within lymphoid and peripheral tissues is central to immune cell development, immunosurveillance and effector function. Interstitial leukocyte trafficking is the result of amoeboid polarization and migration, guided by soluble or tissue-bound chemoattractant signals for positioning and local arrest. In contrast to other migration modes, amoeboid movement is particularly suited for scanning cellular networks and tissues. Here, we review mechanisms of leukocyte migration and sensing involved in diapedesis, tissue-based interstitial migration and egress, immune cell positioning in inflammation, and emerging therapeutic interference strategies.

Catch bonds in adhesion.

One of the most exciting discoveries in biological adhesion is the recent and counter-intuitive observation that the lifetimes of some biological adhesive bonds, called catch bonds, are enhanced by tensile mechanical force. At least two types of adhesive proteins have been shown to form catch bonds--blood proteins called selectins and a bacterial protein called FimH. Both mediate shear-enhanced adhesion, in which cells bind more strongly at high shear than at low shear. Single-molecule experiments and cell-free assays have now clearly demonstrated that catch bonds exist and mediate shear-enhanced adhesion. However, the mechanics of cellular organelles also contribute to shear-enhanced adhesion by modulating the force applied to catch bonds. This review examines how individual catch bond behavior contributes to shear-enhanced cellular adhesion for the two best-understood examples. The lessons from these systems offer design principles for understanding other types of shear-enhanced adhesion and for engineering nanostructured force-dependent adhesives out of catch bonds.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.

Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement.

Cell movement within three-dimensional tissues is a cycling multistep process that requires the integration of complex biochemical and biophysical cell functions. Different cells solve this challenge differently, which leads to differences in migration strategies. Migration principles established for leukocytes share many characteristics with those described for ameba of the lower eukaryote Dictyostelium discoideum. The hallmarks of amoeboid movement include a simple polarized shape, dynamic pseudopod protrusion and retraction, flexible oscillatory shape changes, and rapid low-affinity crawling. Amoeboid crawling includes haptokinetic adhesion-dependent as well as biophysical migration mechanisms on or within many structurally and functionally different substrates. We describe central aspects of amoeboid movement in leukocytes and the implications for leukocyte crawling and positioning strategies within interstitial tissues.

[Adhesion molecules and their role in organ response after trauma]. / Czasteczki adhezyjne i ich rola w odpowiedzi ustroju na uraz.

In this paper, we presented general characterization of adhesion molecules and their role in organ response after injury. Severe trauma is frequently complicated by multiple organ dysfunction (MODS) or failure (MOF). The pathogenesis of these syndromes involves a complex interaction of humoral and cellular events. The recruitment of circulatory leukocytes into tissues is regulated in part by specific leukocyte-endothelial cell interactions with several families of cell adhesion molecules. In the adhesion cascade, the initial phase of inflammation, a transient slowing of neutrophils in postcapillary venules is mediated by selectins. Firm adhesion of neutrophils to the endothelium occurs by interaction of beta 2 integrins to ICAM-1. Diapedesis between endothelial cells requires the engagement of PECAM-1. Significance of the adhesion molecule in organ response after severe trauma permitted their diagnostic and prognostic use as well as new methods of treatment.

Integrinas: ¿qué están haciendo en los leucocitos in vivo? / Integrins: what are they doing on leukocytes in vivo?

Las integrinas constituyen una familia importante de receptores de adhesión que pueden reconocer tanto componentes de la matriz extracelular como ligandos celulares. Distintas aproximaciones experimentales in vitro han mostrado que las integrinas pueden jugar un papel importante en diversas funciones leucocitarias. Recientemente, la generación de ratones manipulados genéticamente ha proporcionado una herramienta muy útil para diseccionar las funciones que estos receptores de adhesión desempeñan in vivo en los leucocitos. De forma interesante, el análisis de ratones deficientes en alguna integrina ha revelado funciones insospechadas para estos receptores como es el caso del papel de las integrinas 1 en la migración de progenitores hematopoyéticos o del papel de la integrina M 2 en la apoptosis de neutrófilos. Sin embargo, otros hallazgos en estos ratones han confirmado las funciones que se habían sugerido previamente por otros abordajes como es el papel de algunas integrinas en el tráfico leucocitario en la migración leucocitaria durante la inflamación. Finalmente, los ratones deficientes en integrinas pueden servir en algunos casos como modelos animales de enfermedades humanas tales como la deficiencia de adhesión leucocitaria o el asma (AU)

MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions.

Monocytes contribute to the development of atherosclerotic lesions in mouse models. The chemoattractant proteins (chemokines), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), are found in human atheroma, and mice lacking receptors for these chemokines are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions. Although MCP-1 has a powerful effect on monocytes, IL-8 is thought to act predominantly on neutrophils and it is unclear how it could recruit monocytes. Here we investigate the ability of chemokines to control the interaction of monocytes under flow conditions with vascular endothelium that has been transduced to express specific leukocyte-adherence receptors. We find that MCP-1 and IL-8 can each rapidly cause rolling monocytes to adhere firmly onto monolayers expressing E-selectin, whereas related chemokines do not. These effects do not correlate with either the induction of a calcium transient or chemotaxis. We conclude that chemokines are important modulators of monocyte-endothelial interactions under flow conditions. Moreover, our finding that IL-8 is a powerful trigger for firm adhesion of monocytes to vascular endothelium reveals an unexpected role for this chemokine in monocyte recruitment.

The leukocyte cell adhesion cascade and its role in myocardial ischemia-reperfusion injury.

Cell-cell and cell-matrix interactions are known to be mediated by specific cell adhesion receptors expressed on the cell surface. The characterization of these cell adhesion molecules has allowed researchers to examine their roles in a variety of physiologic and pathophysiologic conditions. Numerous studies have demonstrated that myocardial ischemia-reperfusion injury is an acute inflammatory process in which leukocytes are intimately involved. In this review, we summarize the current data on the leukocyte cell adhesion cascade, focus upon studies which have demonstrated specific cell adhesion molecule interactions which mediate the leukocyte involvement in myocardial ischemia-reperfusion injury and suggest future avenues of exploration and possible clinical implications of the studies reviewed.

Neutrophil adhesion to fibrinogen and fibrin under flow conditions is diminished by activation and L-selectin shedding.

The adhesion of neutrophils (polymorphonuclear leukocytes [PMNs]) to immobilized fibrinogen/fibrin is mediated by beta2-integrins. However, the influence of physiologic flow conditions on neutrophil adhesion to these surfaces is poorly defined. In this report, the effect of flow and neutrophil activation on adhesion to immobilized fibrinogen and fibrin was examined. For the evaluation of (the distribution of) neutrophil adhesion, real-time video-assisted microscopy and custom-made software were used. Under flow conditions, adherent neutrophils appeared to support the subsequent margination of other neutrophils, thereby enhancing the adherence of these cells to fibrin. Consequently, neutrophils adhered in clusters, especially at higher shear stresses (eg, cluster index 1.4 at shear 80 mPa). Preactivation of PMNs with fMLP (10(-7) mol/L) or 4beta-phorbol, 12-myristate, 13-acetate (PMA; 100 ng/mL) resulted in approximately 50% inhibition of adhesion to fibrin and a more random distribution (cluster index <0.5). L-selectin antibodies or neuraminidase treatment of PMNs also inhibited adhesion and clustering, indicating a role for L-selectin. Under static conditions, no clustering appeared and PMN activation with fMLP or PMA caused threefold and sevenfold increased adhesion, respectively. Under these conditions, anti-L-selectin antibodies or neuraminidase did not affect adhesion. These results indicate that, under flow conditions, adherent neutrophils support adhesion of flowing neutrophils by L-selectin-mediated cell-cell interactions. Preactivated neutrophils, with lowered L-selectin expression, are less susceptible for this interaction. By this mechanism, adhered leukocytes can modulate the recruitment of leukocytes to the vessel wall at sites of inflammation.

[Regulation of leukocyte migration by adhesion molecules]. / Régulation de la migration leucocytaire par les molécules d'adhésion.

The ability of leukocytes to leave the blood-stream and migrate into tissues is a critical feature of the immune system, essential in eliminating infectious pathogens and allowing leukocyte accumulation at sites of injury, infection or inflammation. Lymphocytes continuously recirculate between tissues, lymphoid organs and blood, whereas neutrophils or monocytes lack this capacity. Migration of various leukocyte subpopulations into tissues is regulated by specific combinations of adhesion receptors and chemoattractants which direct them into tissues. Selectins initiate leukocyte attachment along vascular endothelium by mediating leukocyte rolling along inflamed endothelium, whereas CD11/CD18 (alpha L, M, X/beta 2) integrins have a more important role in subsequent steps of leukocyte migration into tissues. alpha 4/beta 1 or alpha 4/beta 7 integrins play a role in mediating lymphocyte rolling and firm adhesion to vascular wall. Leukocyte migration is an important mechanism in the pathogenesis of inflammatory diseases, the regulation of hematopoiesis and hemostasis. This reaction is also involved in the pathogenesis of atherosclerosis, reperfusion injuries and malignant cell metastasis. Leukocyte migration inhibitors may have therapeutic potential against inflammation and associated diseases.

Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.

Peritoneal dialysis solution attenuates microvascular leukocyte adhesion induced by nitric oxide synthesis inhibition.

In the mesenteric microcirculation, inhibition of nitric oxide (NO) synthesis results in an inflammatory response through increased leukocyte adherence to the microvascular postcapillary venular endothelium. Recent studies have demonstrated that elevated concentrations of endogenous NO synthesis inhibitors are present in renal failure. How peritoneal dialysis solutions may affect leukocyte-endothelial interactions during inflammation induced by NO synthesis inhibition has been previously unknown. Using in vivo intravital microscopy of the rat mesenteric postcapillary venules, microvascular leukocyte adherence was quantitated during baseline conditions in which the mesentery was superfused with a buffer solution, followed by the superfusion of a NO synthesis inhibitor NG-nitro-L-ARGININE methyl ester (L-NAME) added to the buffer, followed by 4.25% Dianeal (4.25% D). When compared to baseline, L-NAME increased the mean number of adherent leukocytes by fivefold (2.2 +/- 0.9 vs 11.6 +/- 3.6 leukocytes/100 microns venule/10 min, p < 0.05), while 4.25% D quickly reversed the L-NAME-induced inflammatory response, returning the number of adherent leukocytes back to baseline values (11.6 +/- 3.6 vs 2.4 +/- 1.3 leukocytes/100 microns venule/ 10 min, p < 0.05). These results confirm that NO synthesis inhibition induces inflammation in mesenteric postcapillary venules. Superfusion of 4.25% D reverses leukocyte adhesion induced by NO synthesis inhibition. Thus, a standard peritoneal dialysis solution (4.25% D) reverses the leukocyte-adhesive effects of NO synthesis inhibition in the mesenteric microcirculation.

Significance of the inflammatory response in brain ischemia.

Leukocytes appear to have a central role in the inflammatory response that develops during acute brain ischemia. This brief review adduces evidence that leukocytes accumulate in focal zones of acute brain ischemia at a sufficiently early stage to participate in the process of progressive ischemic brain damage and that partial inhibition of that accumulation, by various measures, can attenuate ischemic brain injury. Mechanisms of leukocyte adhesion are discussed in detail and an inference is put forward that leukocytes are an important factor in progressive ischemic injury, but almost certainly act in concert with a number of other similarly important factors. On this basis, leukocyte inhibition may have demonstrable benefit in acute stroke, but ultimately be found to only partially spare potentially salvageable tissue in the ischemic zone.

Neutrophil-derived adhesion molecules in human digital ischaemia and reperfusion.

In order to see whether leucocyte-derived adhesion molecules are involved in ischaemia and reperfusion, the total and differential leucocyte counts and expression of the LFA complex i.e. CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1) and CD11c/CD18 (p 150,95) were monitored before and after standard cold and heat tests in 8 females with Raynaud's Disease and 8 matched controls. All patients suffered from vasoconstriction during the cold test which, compared with controls, was associated with fewer granulocytes expressing significantly more CD11b/CD18 (Mac-1) integrin and a significant degree of neutropenia persisting during reperfusion. Leucocyte-endothelial adhesive interactions may therefore occur during ischaemia and reperfusion.

Cross-linking of CD18 in human neutrophils induces an increase of intracellular free Ca2+, exocytosis of azurophilic granules, quantitative up-regulation of CD18, shedding of L-selectin, and actin polymerization.

Polymorphonuclear leukocytes (PMNs) exert most of their physiological functions while adherent to surfaces rather than in suspension. PMN adhesion is largely dependent on the function of the beta 2 integrins, CD11a,b,c/CD18. We mimicked engagement of beta 2 integrins by antibody cross-linking of CD18 on isolated human PMNs using both intact monoclonal antibody and F(ab')2 fragments. Within seconds of CD18 cross-linking, we observed a significant, transient rise of intracellular free Ca2+ concentration by 200-300 nM, which was largely due to Ca2+ mobilization from intracellular stores. The Ca2+ signal was blocked after pretreatment with phorbol myristate acetate, an activator of protein kinase C, but not with herbimycin A, a potent inhibitor of tyrosine kinases. In addition to the rise of intracellular free Ca2+ concentration, CD18 cross-linking induced exocytosis of azurophilic granules (release of 26% of total PMN elastase), which was significantly inhibited by herbimycin A. Moreover, 2.2-fold up-regulation of CD18 antigen and significant down-regulation of surface expression of the granulocyte adhesion molecule L-selectin were induced. Granulocyte F-actin content as measured by nitrobenzoxadiazole-phallacidin increased significantly 1 min after CD18 cross-linking. By contrast, CD18 cross-linking by soluble antibodies did not induce superoxide production, but PMNs bound to immobilized monoclonal antibodies against CD18 released significant amounts of superoxide. Initial signaling through beta 2 integrins does not appear to be mediated by a phospholipase C isoform activated through tyrosine phosphorylation, because the Ca2+ signal was not altered by herbimycin A. However, more complex cellular responses including exocytosis were found to require tyrosine phosphorylation. We show that engagement of beta 2 integrins provides an important stimulatory signal to PMNs inducing degranulation, modulation of L-selectin, and cytoskeletal changes.