Leukocyte Locomotion Research Articles

Cooperative Activity of α4β1 and α4β7 Integrins in Mediating Human B-Cell Lymphoma Adhesion and Chemotaxis on Fibronectin Through Recognition of Multiple Synergizing Binding Sites Within the Central Cell-Binding Domain

AbstractWe have quantitated the relative contributions of the constitutively active α4β1 and α4β7 integrins and the domains embodying their cognate binding sites in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin. By cooperating, the central cell-binding and IIICS carboxy-terminal domains were entirely responsible for the adhesion activity displayed by fibronectin, and their relative contribution to this process was estimated to be 30% versus 70%. Assessment of the leukocyte-substrate binding strength (ie, dynes/cell) indicated a 10-fold higher avidity of the cell-IIICS domain interaction. The two integrins interchangeably recognized both domains, but differed quantitatively in their participation in the adhesive event, as well as in domain preference. The use of 3Fn (according to the nomenclature proposed by Bork and Koonin [Curr Opin Struct Biol 6:366, 1996] for the type III fibronectin modules) module-specific antibodies and recombinant polypeptides showed that α4 integrins recognized both the RGD sequence (3Fn10) and an apparently novel synergistic site located within the 3Fn8 module; even in this case, the integrins displayed a distinct binding site preference. Interleukin-1β (IL-1β)/IL-2–induced chemotaxis also involved cooperative function of the central cell-binding and IIICS domains, but the mechanisms regulating this phenomenon differed markedly from those controlling cell adhesion. First, the relative contribution of the individual domains was comparable, but neither of the individual domains promoted migration to the extent observed on intact fibronectin. Secondly, α4β1 and α4β7 integrins were both involved in the domain-binding necessary for initiation of migration, but the relative contribution of each receptor in the chemotactic process was less disparate than for initial cell adhesion. Thirdly, the mode by which chemotactic B-lymphoma movement was supported by the central cell-binding domain differed from that sustaining cell adhesion in that it involved independent recognition of either the 3Fn8 or the 3Fn9 module, which acted in synergy with the 3Fn10 module. Our data provide novel evidence concerning the relative importance of the constitutively active α4β1 and α4β7 integrins for the interaction of B-cell lymphoma cells with fibronectin, and they emphasize a multiple and diverse recognition of sites responsible for either anchorage or locomotion of tumor leukocytes on this matrix molecule.

Circulatory Levels of C-X-C Motif Chemokine Ligands 1, 9, and 10 Are Elevated in Patients with Ischemic Stroke.

Inflammation plays a significant role in the development of ischemic stroke. CXC chemokines play pleiotropic roles in prolonged leukocyte locomotion, astrocyte migration/activation, and neural attachment/sprouting in response to focal stroke. In this study, we aimed to explore the changes in serum levels of three chemokines, C-X-C motif chemokine ligand 1 (CXCL1), C-X-C motif chemokine ligand 9 (CXCL9), and C-X-C motif chemokine ligand 10 (CXCL10), in ischemic stroke patients at the time of admission and before discharge from the hospital ward. In this study, we recruited 43 unrelated ischemic stroke patients using an easy convenience method or accidental sampling which is a type of non-probability sampling that involves the sample being drawn from that part of the population that is close to hand. We also enrolled 50 genetically unrelated healthy controls showing no history of neurologic, cardiovascular, or inflammatory diseases. Serum levels of the considered chemokines were measured by enzyme-linked immunosorbent assay (ELISA) in patients and healthy controls. No significant difference was observed in ischemic stroke patients following hospitalization and prior discharging from the hospital; however, there was a significant difference in serum levels of CXCL9 and CXCL10 between patients and healthy controls. We also found that the level of the chemokine was not related to gender or medical therapy. It appears that CXCL9 and CXCL10 are more predisposing factors and play a direct role in stroke considering that they were higher in patients than in healthy controls. We believe that this study might be used as a basis for further studies on more effective medication regimens to prevent the onset and subsequent complications of stroke. However, these mediators are useful diagnostic and prognostic tools rather than therapeutic tools.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion ofleukocytes.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Michael Sixt: Love the way they move

Sixt studies the mechanisms of leukocyte locomotion.

Is MS an inflammatory or primary degenerative disease?

Multiple sclerosis (MS) is characterized by multiple areas of inflammation, demyelination and neurodegeneration. Multiple molecular and cellular components mediate neuroinflammation in MS. They involve: adhesion molecules, chemokines, cytokines, matalloproteases and the following cells: CD4+ T cells, CD8+ T cells, B cells, microglia and macrophages. Infiltrating Th1 CD4+ T cells secrete proinflammatory cytokines. They stimulate the release of chemokines, expression of adhesion molecules and can be factors that cause damage to the myelin sheath and axons. Chemokines stimulate integrin activation, mediate leukocyte locomotion on endothelial cells and participate in transendothelial migration. CD8+ cells can directly damage axons. B cells are involved in the production of antibodies which can participate in demyelination. B cells can also function as antigen presenting cells and contribute to T cell activation. Neuroinflammation is not only present in relapsing–remitting MS, but also in the secondary and primary progressive forms of the disease. The association between inflammation consisting of T cells, B cells, plasma cells and macrophages and axonal injury exists in MS patients including the progressive forms of the disease. The above association does not exclude the possibility that neurodegeneration can exist independently from inflammation. Very little inflammation is seen in cortical MS plaques. Anti-inflammatory therapies with different mode of action change the course of MS. Anti-inflammatory and immunomodulatory treatments are beneficial in the early relapsing stage of MS, but these treatments are ineffective in secondary progressive and primary progressive MS. In the stage of progressive MS, inflammation becomes trapped behind a closed or repaired blood–brain barrier. In such a situation current immunomodulatory, immunosuppressive or anti-inflammatory treatments might not reach this inflammatory process to exert a beneficial effect.

Neutrophils, reverse traffic and detrimental potential

Neutrophils, reverse traffic and detrimental potential

Modulation of leucocyte locomotion by interleukin-1

The influence of interleukin-1 (IL-1) upon leucocyte locomotion in vitro was studied, using either casein or zymosan-activated serum (ZAS) as chemotaxigens. A pre-incubation of polymorphonuclear cells (PMNC) with ultrapure (purity 99%) human IL-1 (1 U/ml) for 2 and 5 h followed by a washing step resulted in a significant decrease in leucocyte locomotion against casein (P less than 0.0005 and P less than 0.01, respectively), but not against ZAS (P greater than 0.1). Moreover, the direct addition of this IL-1 preparation to leucocyte locomotion assays without pre-incubation produced a similar and significant inhibition of leucocyte locomotion directed against casein (P less than 0.05). This inhibitory effect could not be augmented further by higher concentrations of ultrapure IL-1. In order to exclude the effect of possibly contaminating cytokines in the ultrapure IL-1 preparation used, additional assays with recombinant human IL-1 (rIL-1) alpha and rIL-1 beta (1 U/ml) were performed to investigate their influence upon leucocyte locomotion. It was found that both rIL-1 alpha and rIL-1 beta inhibited leucocyte locomotion directed against casein significantly (P less than 0.005). Similar to the previous experiments, leucocyte locomotion could not be further inhibited by higher concentrations (10 U/ml) or rIL-1. Thus, both ultrapure and rIL-1 were found to have the ability to inhibit leucocyte locomotion in vitro.

Distinct locomotive patterns of granulocytes, monocytes and lymphocytes in a stable concentration gradient of chemokines

The pattern of leukocyte locomotion can be changed in many pathological situations, but its accurate analysis is difficult because of technological limitation. In the present study, by using a newly developed time-lapse videomicroscopic technique, we have analyzed the locomotive patterns of leukocytes in a stable concentration gradient of chemokines. Granulocytes, monocytes, and lymphocytes were purified from adult human peripheral blood. Locomotive behavior of the leukocytes was analyzed by an optical assay using a microchannel producing a stable concentration gradient of chemokines. Videomicroscopic analysis showed distinct locomotive patterns of granulocytes, monocytes, and lymphocytes. Granulocytes were intrinsically motile, vigorously moving in random direction without any chemokine stimulation. Upon stimulation with CXCL8/IL-8, the speed of migration was increased from 13.3 +/- 2.8 to 19.4 +/- 2.5 microm/min (P = 0.002, n = 100) and they moved toward the chemokine, although many cells still wandered very much. Stimulation with CCL5/RANTES or CXCL12/SDF-1alpha induced similar changes in locomotive pattern. On the other hand, most lymphocytes did not polarize or move spontaneously without chemokine stimulation. Stimulation with CXCL12 induced directional migration in 37% of the lymphocytes at a speed of 9.6 +/- 1.6 microm/min (n = 100). The movement pattern of monocytes was similar to that of granulocytes in that they tend to become polarized and move spontaneously, but they moved at a very slow speed ranging from 3.9 to 4.2 microm/min even with chemokine stimulation. The new optical assay may be useful for many diagnostic as well as basic studies.

Interplay between Shear Stress and Adhesion on Neutrophil Locomotion

Leukocyte locomotion over the lumen of inflamed endothelial cells is a critical step, following firm adhesion, in the inflammatory response. Once firmly adherent, the cell will spread and will either undergo diapedesis through individual vascular endothelial cells or will migrate to tight junctions before extravasating to the site of injury or infection. Little is known about the mechanisms of neutrophil spreading or locomotion, or how motility is affected by the physical environment. We performed a systematic study to investigate the effect of the type of adhesive ligand and shear stress on neutrophil motility by employing a parallel-plate flow chamber with reconstituted protein surfaces of E-selectin, E-selectin/PECAM-1, and E-selectin/ICAM-1. We find that the level and type of adhesive ligand and the shear rate are intertwined in affecting several metrics of migration, such as the migration velocity, random motility, index of migration, and the percentage of cells moving in the direction of flow. On surfaces with high levels of PECAM-1, there is a near doubling in random motility at a shear rate of 180 s −1 compared to the motility in the absence of flow. On surfaces with ICAM-1, neutrophil random motility exhibits a weaker response to shear rate, decreasing slightly when shear rate is increased from static conditions to 180 s −1, and is only slightly higher at 1000 s −1 than in the absence of flow. The random motility increases with increasing surface concentrations of E-selectin and PECAM-1 under static and flow conditions. Our findings illustrate that the endothelium may regulate neutrophil migration in postcapillary venules through the presentation of various adhesion ligands at sites of inflammation.

Regional rheological differences in locomoting neutrophils.

Intracellular rheology is a useful probe of the mechanisms underlying spontaneous or chemotactic locomotion and transcellular migration of leukocytes. We characterized regional rheological differences between the leading, body, and trailing regions of isolated, adherent, and spontaneously locomoting human neutrophils. We optically trapped intracellular granules and measured their displacement for 500 ms after a 100-nm step change in the trap position. Results were analyzed in terms of simple viscoelasticity and with the use of structural damping (stress relaxation follows a power law in time). Structural damping fit the data better than did viscoelasticity. Regional viscoelastic stiffness and viscosity or structural damping storage and loss moduli were all significantly lower in leading regions than in pooled body and/or trailing regions (the latter were not significantly different). Structural damping showed similar levels of elastic and dissipative stresses in body and/or trailing regions; leading regions were significantly more fluidlike (increased power law exponent). Cytoskeletal disruption with cytochalasin D or nocodazole made body and/or trailing regions approximately 50% less elastic and less viscous. Cytochalasin D completely suppressed pseudopodial formation and locomotion; nocodazole had no effect on leading regions. Neither drug changed the dissipation-storage energy ratio. These results differ from those of studies of neutrophils and other cell types probed at the cell membrane via beta(2)-integrin receptors, which suggests a distinct role for the cell cortex or focal adhesion complexes. We conclude that 1) structural damping well describes intracellular rheology, and 2) while not conclusive, the significantly more fluidlike behavior of the leading edge supports the idea that intracellular pressure may be the origin of motive force in neutrophil locomotion.

Eosinophil trafficking: new answers to old questions

Eosinophil trafficking: new answers to old questions

L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.

L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.

Comparative Studies of the Colonic In Situ Expression of Intercellular Adhesion Molecules (ICAM-1, -2, and -3), β2 Integrins (LFA-1, Mac-1, and p150,95), and PECAM-1 in Ulcerative Colitis and Crohn's Disease

A dysregulated local immune defense with a constant influx of leukocytes provides a basis for continuous intestinal inflammation in ulcerative colitis (UC) and Crohn's disease (CD). Cell adhesion molecules are pivotal for the migration of leukocytes from the circulation toward the colonic epithelium. A study quantifying the cells expressing intercellular adhesion molecules (ICAMs), beta2 integrins, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the colon was performed to illustrate the leukocyte migration pathway in inflammatory bowel disease. Serial colonic sections (10 UC, 10 CD, and 10 controls) were stained immunohistochemically for ICAM-1, ICAM-2, ICAM-3, CD11a, CD11b, CD18, and PECAM-1. Cell adhesion molecule expression was evaluated quantitatively with reference to topographic localization. In UC, polymorphonuclear leukocytes (PMNs) in contact with the crypt epithelium and in crypt abscesses expressed CD11b. CD tissue was characterized by CD11a-, CD11c-, and ICAM-1-expressing cells. ICAM-1 was detected on endothelial cells, leukocytes, and apical parts of epithelial membranes, whereas ICAM-2 was expressed on basal epithelial membranes. Most infiltrating leukocytes expressed ICAM-3, whereas perivascular mononuclear cells expressed PECAM-1. Interestingly, the epithelial basement membrane in UC stained for CD18. In conclusion, CD11b, CD18, and ICAM-2 seem to be important for PMN transepithelial migration in UC, whereas CD11a, CD11c, ICAM-1, and ICAM-3 seem central in leukocyte locomotion and aggregation in CD. Differentiated upregulation of cell adhesion molecules is suggested to be essential for the diversities between UC and CD.

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with Leukocyte Adhesion Deficiency‐1: Normal displacement in close quarters via chimneying

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with Leukocyte Adhesion Deficiency‐1: Normal displacement in close quarters via chimneying

Does Entamoeba dispar Produce Monocyte Locomotion Inhibitory Factor (MLIF) Like Entamoeba histolytica?

Entamoeba histolytica (Schaudinn, 1903) and Entamoeba dispar (Brumpt, 1925) are two morphologically identical protozoan parasites of the human intestinal tract. The former is a potentially invasive microrganism capable of causing important intestinal damage and extraintestinal disease, while the latter is considered a noninvasive commensal. Indistinguishable under the microscope, E. histolytica and E. dispar can, however, be separated biochemically, immunologically, and genetically into two different protozoan species (1). Several strains of axenically grown E. histolytica have been shown to produce a heat-stable, small (582 Da) pentapeptide [monocyte locomotion inhibitory factor (MLIF)] with in vitro as well as in vivo anti-inflammatory properties (i.e., inhibition of leukocyte locomotion, respiratory burst, nitric oxide production, DNCB skin hypersensitivity, Rebuck window inflammation sequence, etc.) that selectively affect various types of leukocytes (2– 4).We now studied whether E. dispar produces MLIF.

Actin polymerization.

Locomotion of leukocytes is a complex event requiring coordinated activity of cytoskeletal, membrane, and adhesion systems (). Prior to the migration of cells into the inflamed tissue, they have to pass the endothelium. This process involves several types of adhesion molecule interactions between eosinophils and endothelial cells. Selectins mediate two steps, initial tethering to the vessel wall and their subsequent rolling. Integrins then bind to endothelial receptors, increasing adhesion which results in the arrest of the rolling leukocytes, which can then cross the endothelial layer of the blood vessel and enter the tissue using integrins for traction (, , , ).

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with leukocyte adhesion deficiency-1: normal displacement in close quarters via chimneying.

The beta2 integrins are known to be important in the motile function of leukocytes in general and in the adhesive response to inflammatory stimuli in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the locomotion of human blood PMN from a patient with Leukocyte Adhesion Deficiency-1 (LAD), a disorder in which beta2 integrins on the cell surface are markedly deficient in number or function. In thin slide preparations such that the leukocytes were somewhat compressed between slide and cover slip, PMNLAD exhibited normal random locomotion and chemotaxis, apparently by using the opposing surfaces to generate the force for locomotion (chimneying). In thicker preparations, an adherence deficit was evident, but chemotaxis still occurred, even by PMNLAD anticoagulated in EDTA. Consistent with the paucity of beta2 integrins on the surface of the PMNLAD was their failure to aggregate in the presence of antibodies to beta2 integrins, even when they had been brought together by chemotaxis. We relate these findings to the reported independence from integrins of PMN in the lung vasculature in LAD, as well as in certain experimental conditions.

The effect of chemotaxis and chemokinesis on leukocyte locomotion: a new interpretation of experimental results

A mathematical model is developed to describe the motion of leukocytes through a Boyden chamber. The model is based on the Keller–Segel model of cell motion and comprises three partial differential equations which describe the evolution of the neutrophils, the chemoat-tractant, and a neutrophil-derived chemokinetic factor. Where other authors have concentrated on chemotaxis, here attention is focused on the manner in which the chemokinetic factor influences neutrophil locomotion. Numerical simulations show how the number of neutrophils initially placed on top of the chamber affects cellular motion through the system and reproduce the qualitative behaviour observed by Takeuchi & Persellin (Am. J. Physiol. 236, C22–C29). In particular, the simulations show how dense packing of the neutrophils increases the levels of the chemokinetic factor. This enhances random cell motion and increases the speed with which the neutrophils reach the source of chemoattractant. For a particular asymptotic limit of the system parameters, the model reduces to a nonlinear partial differential equation for the neutrophils. Similarity solutions of this caricature model yield algebraic expressions relating the speed with which the neutrophil front penetrates into the chamber to the number of neutrophils initially placed on top of it. The implications of the results are also discussed.

The effect of chemotaxis and chemokinesis on leukocyte locomotion: a new interpretation of experimental results

A mathematical model is developed to describe the motion of leukocytes through a Boyden chamber. The model is based on the Keller-Segel model of cell motion and comprises three partial differential equations which describe the evolution of the neutrophils, the chemoattractant, and a neutrophil-derived chemokinetic factor. Where other authors have concentrated on chemotaxis, here attention is focused on the manner in which the chemokinetic factor influences neutrophil locomotion. Numerical simulations show how the number of neutrophils initially placed on top of the chamber affects cellular motion through the system and reproduce the qualitative behaviour observed by Takeuchi & Persellin (Am. J. Physiol. 236, C22-C29). In particular, the simulations show how dense packing of the neutrophils increases the levels of the chemokinetic factor. This enhances random cell motion and increases the speed with which the neutrophils reach the source of chemoattractant. For a particular asymptotic limit of the system parameters, the model reduces to a nonlinear partial differential equation for the neutrophils. Similarity solutions of this caricature model yield algebraic expressions relating the speed with which the neutrophil front penetrates into the chamber to the number of neutrophils initially placed on top of it. The implications of the results are also discussed.

Assays of leukocyte locomotion and chemotaxis

This review discusses the range of methods which are currently available for measuring locomotion and chemotaxis of leukocytes in vitro, their history, and some definitions of terms. Assays of the net migration of large cell populations, such as the filter assay are the most popular and are useful for identifying chemoattractant molecules, but give no direct information about how these molecules influence the speed and direction of cell movement (chemokinesis and chemotaxis). Visual assays including measures of orientation in gradients and time-lapse filming give detailed information about cell paths and direct evidence for chemotaxis and chemokinesis. The polarization assay is a useful visual screening assay. Assays which simulate the situation in living tissues are becoming more popular and include migration through collagen or fibrin gels or through monolayers of vascular endothelium. Locomotion is a complex process, no single assay gives full information and the use of more than one assay is recommended.