Directional migration of leukocytes：their pathological roles in inflammation and strategies for development of anti－inflammatory therapies

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury also constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial presentation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.     

Endothelial activation drives lateral migration and diapedesis of leukocytes

To invade a tissue, leukocytes have to overcome the endothelial barrier. Prior to trans-endothelial migration, leukocytes move laterally on the endothelial surface-searching for an emigration site. It is still unclear, how the actual diapedesis step is initiated and whether the endothelium has a decisive role. Here, video-microscopy was employed to investigate, whether lateral migration of leukocytes is correlated to their diapedesis rate. To address the contribution of each cell type, selective stimulation of either leukocytes or endothelial cells with TNFα was performed. Stimulation of endothelial cells alone was sufficient for maximal effects, thereby underlining their decisive role for leukocyte diapedesis. Concomitant to the TNFα-enhanced diapedesis rate, leukocyte adhesion was intensified and, unexpectedly, the lateral leukocyte migration was accelerated.     

Neurotransmitters are regulators for the migration of tumor cells and leukocytes

Neurotransmitters are signal substances that have traditionally been regarded as mere mediators of signal states between cells in the nervous system. Whereas the mechanisms of this "classic" neurotransmitter regulation are well understood, only recently has new evidence come to light elucidating the modulatory role of neurotransmitters in immune function, and in the regulation of migration of leukocytes and tumor cells. The migration of leukocytes is, among other things, of primary importance for an anti-tumor immune response, whereas the migration of tumor cells is a prerequisite for invasion and the development of metastases. We here clarify and consolidate the latest tumor biological findings on the role of these neurotransmitters, which bind to serpentine receptors, and which are involved in leukocyte migration, tumor growth, invasion and metastasis. This review thus accentuates the complex, interactive involvement of neurotransmitters in the regulation of migration of both leukocytes and tumor cells.     

Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish,frogs, and birds during migration

Using the method of confocal laser scanning microscopy, changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration were studied. It has been shown that, during movement, in erythrocytes, like in leukocytes, reorganization of cytoskeleton microfilaments occurs. In the course of migration, in amphibians and birds, red blood cells form pseudopodia filled with bundles of actin filaments arranged in parallel, whereas no pseudopodia are formed in fish erythrocytes. The change in the structure of the actin cytoskeleton of nuclear erythrocytes, like in leukocytes, is responsible for the capability of red blood cells to undergo reactions of migration and phagocytosis.     

Genetic evidence for functional redundancy of Platelet/Endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.     

The reorganization of the actin cytoskeleton of nuclear erythrocytes and leukocytes in fish, frogs and birds during cell migration

Changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes during their migration in fish, frogs and birds have been studied by the method of confocal laser scanning microscopy. It has been shown that, during movement of cells, the reorganization of cytoskeleton microfilaments in erythrocytes is similar to that in leukocytes. During migration, red blood cells of amphibious and birds form pseudopodia filled with bunches in parallel laid actin filaments. Erythrocytes in fish do not form pseudopodia. Similar to leukocytes change in the structure of the actin cytoskeleton in nuclear erythrocytes determines the ability of red blood cells to reactions of migration and phagocytosis.     

Three or more routes for leukocyte migration into the central nervous system

Leukocyte migration into and through tissues is fundamental to normal physiology, immunopathology and host defence. Leukocyte entry into the central nervous system (CNS) is restricted, in part, because of the blood-brain barrier (BBB). During the past decade, crucial components that are involved in the process of leukocyte migration have been identified and progress has been made in understanding the mechanisms of neuroinflammatory reactions. In this review, present knowledge of the trafficking determinants that guide the migration of leukocytes is superimposed onto the vascular and compartmental anatomy of the CNS. We discuss three distinct routes for leukocytes to enter the CNS and consider how different populations of leukocytes use trafficking signals to gain entry.     

Restoration of antineoplastic reactivity of the leukocytes after preincubation]

Specific antitumor sensitization in patients with carcinoma of the stomach was revealed by means of the leukocyte migration inhibition test. After 24-hour preincubation at 4 degrees C in a serum-free medium the areactive leukocytes from patients with carcinoma of the stomach (stages III--IV) acquired the ability to react specifically to allogeneic antigens of the tumor of the same localization. Preincubation did not influence the inhibition of migration of leukocytes in non-tumor patients by stomach carcinoma antigens. The supernatant of preincubated leukocytes of tumor patients contained substances that inhibited migration of indicator leukocytes.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Separation of different sized particles by inertial migration

Inertial migration has been used to fractionate feed streams containing latex particles and sheep leukocytes. For latex particles, a purified stream of small particles, less than 4 m, was obtained. For sheep leukocytes, a stream containing almost no large leukocytes greater than 16 m was produced. The results obtained show that this method could be used to fractionate biological cells of different sizes. Inertial migration is most suitable for the removal of larger particles which contaminate a dilute suspension of smaller particles.     

Pathophysiology of leukocyte-tissue interactions

Unlike most somatic cells, leukocytes are constitutively non-adherent. However, adhesive interactions are not only a required step in essentially all effector functions performed by leukocytes, but they also relay increasingly well-defined intracellular signals that affect the leukocyte as well as the surrounding tissues. Dissecting such signals in leukocytes has provided a wealth of information that contributes to our understanding of how adhesion controls higher-order biological responses, ranging from cell migration to proliferation, differentiation and survival.     

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.     

Meeting report: molecular mechanisms of inflammation: how leukocytes come,see and seize

Inflammation has developed in the course of evolution as a process to defend the body against invading microbes and to respond to injuries. Several mechanisms of interaction between endothelial cells and leukocytes have evolved to render inflammation an effective, tightly controlled, and self-limited process. Imperfect executions of this "game plan" lead to pathological abnormalities resulting in diseases. The meeting on Molecular Mechanisms of Inflammation held at Schloss Elmau, Germany in October 2002 has featured activation of endothelial cells, adhesion and migration of leukocytes, as well as receptor pathways for activation and deactivation of leukocytes and, concomitantly, of the inflammatory response. Thus, a review on some of the presented data casts interesting spotlights on different steps of the inflammatory cascade.     

Molecular control of neuronal migration

Our understanding of neuronal migration has been advanced by multidisciplinary approaches. At the cellular level, tangential and radial modes of neuronal migration contribute to different populations of neurons and have differential dependence on glial cells. At the molecular level, extracellular guidance cues have been identified and intracellular signal transduction pathways are beginning to be revealed. Interestingly, mechanisms guiding axon projection and neuronal migration appear to be conserved with those for chemotactic leukocytes.     

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.     

Vascular adhesion and transendothelial migration of eosinophil leukocytes

Tissues respond to injury with inflammation in an effort to protect and repair the damaged site. During inflammation, leukocytes typically accumulate in response to certain chemicals produced within the tissue itself. The passage of leukocytes through the vascular lumen into tissues occurs in several phases, including rolling, activation, firm adhesion, transendothelial migration, and subendothelial migration. Although infiltration of eosinophil leukocytes is one of the most important aspects of allergic inflammatory reactions, eosinophils also participate in nonallergic inflammation. Eosinophil accumulation is regulated not only by endothelial adhesion molecules, but also by interactions between eosinophil adhesion molecules and extracellular matrix elements. This review summarizes the regulation of eosinophil leukocyte adhesion and migration. A better understanding of eosinophil recruitment responses may lead to the development of novel therapeutics for chronic allergic diseases.     

Reverse leukocyte migration can be attractive or repulsive

The directional migration of cells within multicellular organisms is governed by gradients of both chemical attractants and repellents in diverse processes, including leukocyte trafficking and neuronal pathfinding in vivo. These complex extracellular environments direct the orchestrated bidirectional trafficking of leukocytes between the vasculature and tissues. Substantial progress has been made in dissecting the molecular mechanisms involved in orchestrating the directed movement of leukocytes into host tissues; however, less is known about the reverse migration of leukocytes from the tissues to the vasculature. In this article, we discuss the functional interplay between chemoattraction and chemorepulsion in the bidirectional movement of cells in complex in vivo environments, and we describe how these mechanisms influence both normal physiology and human disease.     

T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.     

Tumour-associated antigens in culture medium of malignant melanoma cell strains

Summary The presence of melanoma-associated antigens naturally shed from cultured melanoma cells in spent culture medium was investigated by means of a leukocyte migration test and culture medium from four melanoma and two control cell strains.Leukocytes from 29/64 melanoma patients showed a positive reaction with spent culture medium from at least one melanoma cell strain, whereas leukocytes from only 4/25 patients with other cancers and 1/30 normal donors reacted. On the other hand, leukocytes from only 8/51 melanoma patients reacted with control culture medium. Only melanoma patients' leukocytes reacted with two or more of the melanoma cell strains used. Culture media from two melanoma cell strains were more reactive (25.3% and 29.4% positive tests with melanoma patients' leukocytes) than others (12.5% and 17.2% positive tests); this may represent either a qualitative difference (i.e., different antigens) or a quantitative one (i.e., different levels of antigen expression according to tissue culture conditions). Both inhibition and stimulation of migration were observed, but with one exception, on a given occasion, leukocytes from the same donor always reacted in the same way (i.e., either inhibition or stimulation). Migration stimulation was observed mainly with melanoma patients' leukocytes, and more especially when leukocytes were sampled from patients within a few weeks from tumour removal; migration stimulation may thus reflect a particular state of sensitization in patients.From the evidence obtained in these studies, it is concluded that spent culture medium from melanoma cell strains contains melanoma-associated antigen (s) that is (are) reactive in the leukocyte migration test and that this may contribute to the study of specific antitumour reactivity in patients and to the study and purification of tumour-associated antigens by providing an homogeneous source of antigens spontaneously released from tumour cells in conditions close to natural ones.