A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

Migration of keratinocytes through tunnels of digested fibrin

We report here a hitherto undescribed form of cell migration. When a suspension of human keratinocytes is plated on a fibrin matrix, single cells invade the matrix and progress through it as rounded cells by dissolving the fibrin and thereby creating tunnels. These tunnels are cylindrical or helical, the latter being the result of constant change in the path of cellular advance around the helical axis. Helical tunnel formation is strongly promoted by epidermal growth factor. The rate of migration of the cell through the track of a helical tunnel (up to 2.1 mm per day) is about 7-fold greater than through a cylindrical tunnel. Pericellular fibrinolysis leading to tunnel formation depends on the presence of plasminogen in the medium and its conversion to plasmin by a cellular activator. Formation of tunnels requires that plasminogen activator be localized on the advancing surface of the keratinocyte; we propose that the tunnel is cylindrical when the site of release of plasmin is located at a fixed point on the cell surface and helical when the site of release precesses.

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Transendothelial migration of neutrophils involves integrin-associated protein (CD47).

Inflammation is a primary pathological process. The development of an inflammatory reaction involves the movement of white blood cells through the endothelial lining of blood vessels into tissues. This process of transendothelial cell migration of neutrophils has been shown to involve neutrophil beta 2 integrins (CD18) and endothelial cell platelet-endothelium cell adhesion molecules (PECAM-1; CD31). We now show that F(ab')2 fragments of the monoclonal antibody B6H12 against integrin-associated protein (IAP) blocks the transendothelial migration of neutrophils stimulated by an exogenous gradient of the chemokine interleukin 8 (IL-8; 60% inhibition), by the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP; 76% inhibition), or by the activation of the endothelium by the cytokine tumor necrosis factor alpha (98% inhibition). The antibody has two mechanisms of action: on neutrophils it prevents the chemotactic response to IL-8 and FMLP, and on endothelium it prevents an unknown but IL-8-independent process. Blocking antibodies to IAP do not alter the expression of adhesion proteins or production of IL-8 by endothelial cells, and thus the inhibition of neutrophil transendothelial migration is selective. These data implicate IAP as the third molecule essential for neutrophil migration through endothelium into sites of inflammation.

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Gαq (the m3 muscarinic receptor), Gαi (the κ-opioid receptor), and Gαs (the β-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to Gαi, but not to Gαq or Gαs, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of Gα transducin, which binds to and inactivates free Gβγ dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of Gβγ dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of Gαi and the release of Gβγ dimers, and that Gαi-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.

Asian genotypes of JC virus in Native Americans and in a Pacific Island population: Markers of viral evolution and human migration

The human polyomavirus JC (JCV) causes the central nervous system demyelinating disease progressive multifocal leukoencephalopathy. Previously, we showed that 40% of Caucasians in the United States excrete JCV in the urine as detected by PCR. We have now studied 68 Navaho from New Mexico, 25 Flathead from Montana, and 29 Chamorro from Guam. By using PCR amplification of a fragment of the VP1 gene, JCV DNA was detected in the urine of 45 (66%) Navaho, 14 (56%) Flathead, and 20 (69%) Chamorro. Genotyping of viral DNAs in these cohorts by cycle sequencing showed predominantly type 2 (Asian), rather than type 1 (European). Type 1 is the major type in the United States and Hungary. Type 2 can be further subdivided into 2A, 2B, and 2C. Type 2A is found in China and Japan. Type 2B is a subtype related to the East Asian type, and is now found in Europe and the United States. The large majority (56–89%) of strains excreted by Native Americans and Pacific Islanders were the type 2A subtype, consistent with the origin of these strains in Asia. These findings indicate that JCV infection of Native Americans predates contact with Europeans, and likely predates migration of Amerind ancestors across the Bering land bridge around 12,000–30,000 years ago. If JCV had already differentiated into stable modern genotypes and subtypes prior to first settlement, the origin of JCV in humans may date from 50,000 to 100,000 years ago or more. We conclude that JCV may have coevolved with the human species, and that it provides a convenient marker for human migrations in both prehistoric and modern times.

Network of tangential pathways for neuronal migration in adult mammalian brain

Cells in the brains of adult mammals continue to proliferate in the subventricular zone (SVZ) throughout the lateral wall of the lateral ventricle. Here we show, using whole mount dissections of this wall from adult mice, that the SVZ is organized as an extensive network of chains of neuronal precursors. These chains are immunopositive to the polysialylated form of NCAM, a molecule present at sites of plasticity, and TuJ1, an early neuronal marker. The majority of the chains are oriented along the rostrocaudal axis and many join the rostral migratory stream that terminates in the olfactory bulb. Using focal microinjections of DiI and transplantation of SVZ cells carrying a neuron-specific reporter gene, we demonstrate that cells originating at different rostrocaudal levels of the SVZ migrate rostrally and reach the olfactory bulb where they differentiate into neurons. Our results reveal an extensive network of pathways for the tangential chain migration of neuronal precursors throughout the lateral wall of the lateral ventricle in the adult mammalian brain.

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible β-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.

Polarity of microtubule assemblies during neuronal cell migration.

The active migration of neurons from their sites of origin to their final destinations requires the unidirectional translocation of the nuclei and somatic cytoplasm within the growing leading processes. To explore the cellular machinery underlying this translocation, we determined the polarity of microtubules situated within the leading and trailing processes of migrating cerebellar granule cells in situ. Our analysis reveals that the newly assembled positive ends of the microtubules in the leading process uniformly face the growing tip, while their disintegrating negative ends face the nucleus. In the trailing process, by contrast, microtubule arrays are of mixed polarity. We suggest that the dynamics of slow polymerization in combination with fast disintegration of oriented microtubules create "push" and "pull" forces that contribute to the piston-like saltatory displacement of the nucleus and cytoplasm within the membrane cylinder of the leading process of the migrating neuron.

Potential roles of osteopontin and αVβ3 integrin in the development of coronary artery restenosis after angioplasty

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30–50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, αvβ3 integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with αvβ3 integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or αvβ3 integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-αvβ3 interaction was blocked by treatment of CASMCs with antibodies against OPN or αvβ3 integrin. Our results demonstrate that OPN and αvβ3 integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-αvβ3 interaction may provide a therapeutic approach to preventing restenosis.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.