Use of 51Cr-labelled macrophages for the objective evaluation of macrophage migration inhibition]

A possibility of using 51Cr-labeld macrophages migrating from the capillaries and lysed with sodium dodecyl sulfate for objective and quantitative assessment of the results of macrophage migration inhibition (MMI) microassay was demonstrated on the basis of correlation analysis of the experimental data. Migration area and sensitivity of 51Cr-labeled macrophages to the action of the factor of migration inhibition were found not to differ from those of unlabeled macrophages. In the assessment of the absolute macrophage migration values and its reduction under the action of the factor of migration inhibition formed in the H-2 system, and also of the specificity of this reduction there was revealed a high correlation between the migration area and the value of the migrant cell label.     

Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes

Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.     

ERK1/2 and p38 MAP kinase control MMP-2, MT1-MMP, and TIMP action and affect cell migration: a comparison between mesothelioma and mesothelial cells

Pleural malignant mesothelioma is a locally aggressive tumor of mesothelial cell origin. In other tumor types high expression of matrix metalloproteinase (MMP)-2, together with membrane-type1-MMP (MT1-MMP), and low levels of the tissue inhibitor of MMP (TIMP)-2 have been correlated with aggressive tumor progression and low survival rates. Therefore, we compared the expression and activation of these three factors and their regulation by two mesothelioma associated growth factors, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)-beta1 in six human mesothelioma and one mesothelial cell line. Polymerase chain reaction (PCR), immunoblotting, zymography, and small inhibitory RNAs (siRNA) were used to study gene expression, protein activation, and signal transduction. To proof the relevance of our in vitro data immunohistochemistry was performed in tissue sections. PDGF-BB induced, while TGF-beta1 inhibited cell proliferation. PDGF-BB was a chemoattractant for mesothelial cells, and its effect was increased in the presence of TGF-beta1. TGF-beta1 stimulated the de novo synthesis of pro-MMP-2 in both cell types. Pro-MMP-2 synthesis involved p38 MAP kinase. In cell culture and tissue sections only mesothelial cells expressed MT1-MMP. Migration of mesothelioma cells was dependent on the presence of MT1-MMP. Migration, but not proliferation of mesothelioma cells was inhibited by oleoyl-N-hydroxylamide, TIMP-2, and siRNA for MT1-MMP. Our data suggest that in mesothelioma cells the phosphorylation of p38 MAP kinase is deregulated and is involved in pro-MMP-2 expression. Mesothelioma progression depends on an interaction with mesothelial cells that provide MT1-MMP necessary to activate pro-MMP-2 to facilitate migration through an extracellular matrix (ECM) layer.     

Migration of the ampicillin transposon in enteropathogenic Escherichia

Migration of Tn 1 from plasmid RP4 into the chromosome of enteropathogenic Escherichia (EPE) of serogroups O124 and O111 was studied. E. coli K 12 LC 411 (RP4) was used as the donor. It was shown that the transposition rate markedly differed depending on the period of ;the cell isolation from Tn 1 in the chromosome, i. e. during conjugation or after several subcultures of the transconjugants from the autonomic plasmid onto the selective media. During the conjugation process the migration rate of Tn 1 was equal to 0.5 and 0.8 per cell acquiring the plasmid. The transposition rate in the EPE carrying the R factor for a long period of time was 2.4 . 10(-2) and 1.4 . 10(-2) respectively for each serogroup. The above differences in the migration rate of Tn 1 were not concerned with changes in the environment.     

Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.     

In vitro migration of human large granular lymphocytes

Large granular lymphocyte (LGL)-enriched peripheral blood mononuclear cells were prepared on discontinuous gradients of Percoll. Migration into nitrocellulose filters was studied in a 2-hr assay with the use of modified Boyden chambers. LGL migrated into filters in response to casein or C5a. Migration depended on the presence of a concentration gradient of chemoattractant between the lower and upper compartment of the chambers. Percoll-purified high density small lymphocytes had little or no migratory capacity under these conditions, requiring a longer incubation time (4 hr) for consistent migration. Migratory capacity in response to stimuli correlated with the frequency of LGL in the various fractions of Percoll gradients. Fractionation of LGL-enriched Percoll preparations with monoclonal antibodies and immunoadsorbent columns or a cell sorter revealed that cells with migratory capacity in response to stimuli were B73.1+ and OKT3-, thus confirming that migrating cells were LGL. LGL preincubated with interferon (IFN) showed enhanced spontaneous motility but no increased responsiveness to chemoattractants. IFN did not modify the migratory capacity of small lymphocytes. The prompt migration of LGL in response to stimuli is consistent with the hypothesis that these cells may serve as one of the first easily mobilizable lines of resistance against infectious agents and tumors. The migration assay described here may offer a better insight into the functions and regulation of LGL.     

Caveolin-1 in cell polarization and directional migration

Migration is a complex process in which cells move in a given direction either in response to changes in the extracellular environment or as a consequence of an intrinsic propensity for directional movement. Migration plays key roles in many physiological and pathological processes, including development, angiogenesis, tissue regeneration and metastasis. An important role in migration is played by caveolin-1 and caveolae. Caveolae compartmentalize intracellular signalling pathways to orchestrate cell migration. Caveolin-1 presents a polarized distribution in migrating cells and is linked to the cytoskeleton, and changes in its expression modulate migration. Although there are some discrepancies regarding the regulatory effect of caveolin-1, most studies show that it promotes cell movement and polarity. The importance of caveolin-1 has recently been reinforced by studies with Cav1(-/-) cells, which indicate that it establishes polarity during directional migration by coordinating Src kinase and Rho GTPase signalling.     

Role of interstitial cell migration in generating position-dependent patterns of nerve cell differentiation in Hydra

The role of interstitial cell migration in the formation of newly differentiated nerve cells was examined during head regeneration in Hydra magnipapillata. When distal tissue was removed from the body of a wild-type strain (105), nerve cell differentiation occurred at a rapid rate during the first 48 hr of regeneration, slowing after this point. Rapid nerve cell differentiation was due primarily to migration of interstitial cells, some of which appeared to be nerve cell precursors, into the regenerating head. The migration decreased considerably after the first 48 hr of regeneration. In reg-16, a mutant strain deficient in head regeneration, no migration of interstitial cells and hence no new nerve cell differentiation were observed in the regenerating tip. However, the interstitial cells of reg-16 were observed to migrate into regenerating tissue of strain 105. These observations suggest that the migration of nerve cell precursors plays an important role when the new nerve net is being established during head regeneration.     

Spatiotemporal changes in HNK-1/L2 glycoconjugates on avian embryo somite and neural crest cells.

Neural crest cell migration was studied in trunks of quail and chick embryos using HNK-1 and L2 antibodies. At the intersegmental cleft, labeled crest cells were associated with the rostral wall of the somite rather than blood vessels. Migration into and through the rostral part of the sclerotomes was more rapid (40-70 microns/hr; quail) and the onset of localization was earlier (after 13-16 hr; quail) than previously supposed. Crest cells here were initially mono- to multipolar, scattered, and inconsistently oriented and formed numerous close (about 20 nm) homo- and heterotypic cell-cell contacts. In vitro models suggested that significant numbers of crest cells, however, could be unlabeled at this early phase. Somitic properties covarying with the hemisegmental pattern of crest cell immigration were investigated. Laminin distribution, although asymmetric in the somites, was not closely related to that of crest cells. Tenascin distribution matched that of crest cells, but only at the localization stage. Earlier, maximal tenascin expression occurred in the somite's caudal wall, a region avoided by crest cells. Chondroitin 6-sulfate proteoglycan expression was elevated in the caudal somite-half at the localization phase and also, at lumbar levels, at the immigration stage. This is consistent with tenascin and proteoglycan having a negative role in crest cell migration. The rostral somite-half was also labeled by HNK-1 and L2, but only in quails. This was associated with the cell surface, was transient, was stable to mild proteolysis, and was labile to cryoprocessing and lipophilic solvents. The spatial and temporal congruence with crest migration suggests that the HNK/L2 adhesion-related carbohydrate epitope on the somites indicates a molecule (possibly glycolipid) which acts via heterotypic cell-cell contacts to provide one cue in the patterned distribution of crest cells in the somites.     

α-1 antitrypsin promotes semimature, IL-10-producing and readily migrating tolerogenic dendritic cells

Tolerogenic IL-10-positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7-dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. α-1 antitrypsin (AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4(+) T cells, and AAT in vitro and in vivo. IL-1β/IFN-γ-mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT (0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and maintained inducible CCR7. Upon exposure of CD4(+) T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3(+) Treg cells were obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60 mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection, whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group. Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT.     

Interaction of malignant MO4 cells with chick hypoblast in culture

Malignant MO4 mouse fibrosarcoma cells were confronted with fragments of hypoblast from stage 4 (Vakaet 1970) blastoderms in different dispositions either permitting or preventing contact of the hypoblast with the tissue culture plastic. Explantation of an MO4 cell aggregate on top of 24 h-old-hypoblast caused retraction of the hypoblast. Contact inhibition of ruffling in hypoblast cells at the inner margin, by MO4 cells migrating radially from the aggregate, prevented closure of the hole brought about by the initial retraction. Disintegration of hypoblast was not observed. Migration of MO4 cells during the first 24 h was faster from an aggregate explanted on top of hypoblast than from an aggregate explanted on tissue culture plastic. Hypoblast fragments explanted on top of confluent layers of MO4 cells attached and spread during the first 12 h. Later, the hypoblast progressively disintegrated. Here, MO4 cells accumulated underneath the hypoblast. We concluded 1) that the hypoblast attracted the MO4 cells by influencing their pattern of migration and 2) that contact with the artificial substrate allowed survival of hypoblast confronting malignant MO4 cells. Ultrastructural analysis suggested that formation of extracellular material played a major role in the interaction between the normal tissue and the malignant cells.     

Prostaglandin D2 regulates CD4+ memory T cell trafficking across blood vascular endothelium and primes these cells for clearance across lymphatic endothelium

Memory lymphocytes support inflammatory and immune responses. To do this, they enter tissue via blood vascular endothelial cells (BVEC) and leave tissue via lymphatic vascular endothelial cells (LVEC). In this study, we describe a hierarchy of signals, including novel regulatory steps, which direct the sequential migration of human T cells across the blood and the lymphatic EC. Cytokine-stimulated (TNF and IFN) human BVEC preferentially recruited memory T cells from purified PBL. Lymphocyte recruitment from flow could be blocked using a function-neutralizing Ab against CXCR3. However, a receptor antagonist directed against the PGD(2) receptor DP2 (formerly chemoattractant receptor-homologous molecule expressed on Th2 cells) inhibited transendothelial migration, demonstrating that the sequential delivery of the chemokine and prostanoid signals was required for efficient lymphocyte recruitment. CD4(+) T cells recruited by BVEC migrated with significantly greater efficiency across a second barrier of human LVEC, an effect reproduced by the addition of exogenous PGD(2) to nonmigrated cells. Migration across BVEC or exogenous PGD(2) modified the function, but not the expression, of CCR7, so that chemotaxis toward CCL21 was significantly enhanced. Thus, chemokines may not regulate all stages of lymphocyte migration during inflammation, and paradigms describing their trafficking may need to account for the role of PGD(2).     

Rabbit endometrial relaxin: immunohistochemical localization during preimplantation, pregnancy, and lactation

Relaxin was localized in rabbit endometrium (but not ovary) on Days 4-30 of pregnancy and Days 2-5 of lactation. The hormone was not observed on Days 2 and 3 post coitus. Relaxin was found in endometrial glands throughout the length of the uterus on Days 4-9 post coitus. Later, on Days 11-23, relaxin was localized in both uterine endometrial gland cells and luminal epithelial cells. At this time, staining was observed only in the endometrium directly associated with implantation sites. Areas between implantation sites were devoid of staining. On Days 25-30 of pregnancy, relaxin was found mainly in uterine luminal epithelial cells. Few glands were observed with relaxin. During the first week of lactation, the staining profile was the same as that observed on Days 25-30. Relaxin was not found in the endometrium of pseudopregnant rabbits (Days 1, 4, 8, 12, and 16). The early appearance of uterine relaxin at the time the blastocyst migrates into the uterine cavity coupled with the hormone's later confinement to implantation sites suggests that the blastocyst initiates and the conceptus maintains uterine relaxin.     

Canine mesenchymal stem cells are effectively labeled with silica nanoparticles and unambiguously visualized in highly autofluorescent tissues

ABSTRACT: BACKGROUND: Developing a long-term labeling method is critical and much needed to understand the fate, migration, and contribution in tissue regeneration. Silica nanoparticles have been developed recently and have been demonstrated to be biocompatible and to have high labeling capacity. Thus, this study was designed to assess the suitability of silica nanoparticles for canine MSCs and fluorescence efficiency in a highly autofluorescent tissue. RESULTS: Development of a method for long-term labeling of cells is critical to elucidate transplanted cell fate and migration as well as the contribution to tissue regeneration. Silica nanoparticles have been recently developed and demonstrated to be biocompatible with a high labeling capacity. Thus, our study was designed to assess the suitability of silica nanoparticles for labeling canine mesenchymal stem cells (MSCs) and the fluorescence efficiency in highly autofluorescent tissue.We examined the effect of silica nanoparticle labeling on stem cell morphology, viability and differentiation as compared with those of unlabeled control cells. After 4 h of incubation with silica nanoparticles, they were internalized by canine MSCs without a change in the morphology of cells compared with that of control cells. The viability and proliferation of MSCs labeled with silica nanoparticles were evaluated by a WST-1 assay and trypan blue exclusion. No effects on cell viability were observed, and the proliferation of canine MSCs was not inhibited during culture with silica nanoparticles. Furthermore, adipogenic and osteogenic differentiation of silica nanoparticle-labeled canine MSCs was at a similar level compared with that of unlabeled cells, indicating that silica nanoparticle labeling did not alter the differentiation capacity of canine MSCs. Silica nanoparticle-labeled canine MSCs were injected into the kidneys of BALB/c mice after celiotomy, and then the mice were sacrificed after 2 or 3 weeks. The localization of injected MSCs was closely examined in highly autofluorescent renal tissues. Histologically, canine MSCs were uniformly and completely labeled with silica nanoparticles, and were unambiguously imaged in histological sections. CONCLUSIONS: The results of the current study showed that silica nanoparticles are useful as an effective labeling marker for MSCs, which can elucidate the distribution and fate of transplanted MSCs.     

In situ effector pathways of allograft destruction. 1. Generation of the "cellular" effector response in the graft and the graft recipient

Inflammatory leukocytes of DA-to-WF rat renal allografts displayed significant cytolytic activity to natural killer (NK) target cells on Day 2 after transplantation. The NK activity, which was associated with large granular lymphocytes in discontinuous Percoll gradients, peaked on Day 4 and disappeared rapidly thereafter. Coincident with the presence of NK activity in the graft, a decrease in NK activity in the recipient spleen was observed. Low NK activity was also recorded in WF-to-WF autografts. The cells displaying direct cytotoxic activity to donor (but not to recipient) strain peritoneal exudate target cells (PEC) were associated with the T suppressor/killer lymphocytes in affinity chromatography. They appeared in the graft between Days 2 and 4, peaked between Days 6 and 8 and disappeared slowly thereafter. In the spleen the cytotoxic T lymphocyte (CTL) activity appeared later and it reached a maximum between Days 16 and 20 before decreasing. In the blood distinct CTL activity was seen only from Days 16-20 onwards, after the graft had been rejected. No CTL activity was recorded in the graft, blood, or spleen of an autograft recipient. Addition of donor-directed post-transplantation antibody (antibody-dependent cellular cytotoxicity, ADCC) had a slight enhancing effect on the cytotoxic activity of inflammatory leukocytes up to Day 5. After this time, added antibody had a blocking effect on direct CTL activity. No ADCC activity was recorded in the inflammatory population of an autograft. On the contrary, high levels of ADCC activity to donor strain PEC were recorded in the spleens of both autograft and allograft recipients throughout the period of follow-up. The results demonstrate that at least three cellular effector pathways exist in an allograft: a strong natural killer cell component, a strong cytotoxic T lymphocyte component, and (possibly) a weak cell component participating in an ADCC type of cytotoxicity.     

The effect of proteases on endothelial cell migration in vitro

This communication reports on the role of proteases in the migration of endothelial cells in vitro. Endothelial cell (EC) migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Treatment with mitomycin C inhibited wound-induced proliferation of endothelial cells without affecting migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro in 10% serum was not affected by depletion of plasminogen, which inhibited plasmin production, or by various protease inhibitors: soybean trypsin inhibitor, Trasylol, E-amino caproic acid (EACA), ovalbumin, p-tpsyl-1-arginine-methyl ester (TAME), and benzamidine. However, migration and proliferation of endothelial cells in vitro was inhibited by acid-treated serum, a procedure commonly used to inactivate protease inhibitors. Migration of bovine smooth muscle cells, 3T3 cells and SV40-3T3 cells was inhibited by plasminogen-depleted serum; reconstitution with purified plasminogen reversed the depressed migration of only SV40-3T3. These results indicated that endothelial cell migration in vitro is not dependent on plasminogen, which may be another unique property of endothelial cells.     

壳聚糖薄膜成球培养对脐带间充质干细胞迁徙趋化的影响

基于细胞实验研究壳聚糖（chitosan,CS）薄膜成球培养技术对间充质干细胞(mesenchymal stem cells, MSCs)迁徙趋化特性的影响。从脐带组织中分离原代MSCs采取CS成球法培养,以常规贴壁培养MSCs作为对照,72 h后收集两组细胞分别进行划痕实验、Tranthwell迁徙实验观察并拍照记录,RT-PCR方法检测两种培养方式中MSCs迁徙相关基因表达水平的差异。研究结果显示,相较常规贴壁培养方式,CS培养组MSCs体外迁徙趋化能力增强,差异具有显著统计学意义（P<0.01）;CS成球培养组MSCs 中CXCR4、CXCR7、MCP-1、MMP-1、MMP-2、MMP-9、TIMP-2等迁徙相关基因表达均明显上调（P<0.01）。实验表明CS成球培养可显著促进MSCs的迁移趋化特性。     

Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force

Cell migration through 3D tissue depends on a physicochemical balance between cell deformability and physical tissue constraints. Migration rates are further governed by the capacity to degrade ECM by proteolytic enzymes, particularly matrix metalloproteinases (MMPs), and integrin- and actomyosin-mediated mechanocoupling. Yet, how these parameters cooperate when space is confined remains unclear. Using MMP-degradable collagen lattices or nondegradable substrates of varying porosity, we quantitatively identify the limits of cell migration by physical arrest. MMP-independent migration declined as linear function of pore size and with deformation of the nucleus, with arrest reached at 10% of the nuclear cross section (tumor cells, 7 µm²; T cells, 4 µm²; neutrophils, 2 µm²). Residual migration under space restriction strongly depended upon MMP-dependent ECM cleavage by enlarging matrix pore diameters, and integrin- and actomyosin-dependent force generation, which jointly propelled the nucleus. The limits of interstitial cell migration thus depend upon scaffold porosity and deformation of the nucleus, with pericellular collagenolysis and mechanocoupling as modulators.     

Lysophosphatidic acid receptor-selective effects on Jurkat T cell migration through a Matrigel model basement membrane

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and mononuclear phagocytes mediate T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) specific for LPA (Edg-2, -4, and -7) or S1P (Edg-1, -3, -5, -6, and -8). Jurkat leukemic T cells with the SV40 virus large T Ag (Jurkat-T cells) express Edg-3>-2>-4 Rs, as assessed by RT-semiquantitative PCR and Western blots with anti-Edg R mAbs. Jurkat-T cells expressing predominantly Edg-2 R (Jurkat-T-2 cells) and Edg-4 R (Jurkat-T-4 cells) were developed by cotransfection with the respective sense plasmids and a mixture of antisense plasmids for the other Edg Rs, and hygromycin selection. Migration of Jurkat-T-4 cells, but not Jurkat-T-2 cells, through a layer of Matrigel on a 5-um pore polycarbonate filter was stimulated up to 5-fold by 10(-9) to 10(-6) M LPA and by 30-300 ng/ml of anti-Edg-4 R Ab, but not anti-Edg-2 R Ab. LPA and anti-Edg-4 R Ab also enhanced by up to 4-fold the expression of matrix metalloproteinase by Jurkat-T-4 cells, but not Jurkat-T-2 cells, as assessed by cleavage of [(3)H]-type IV human collagen in the Matrigel. Enhancement of matrix metalloproteinase-dependent trans-Matrigel migration of Jurkat-T cells by the chemokine RANTES was suppressed by anti-Edg-2 R Abs, but was stimulated by anti-Edg-4 R Abs. The opposite effects of Edg-2 and Edg-4 LPA receptors on trans-Matrigel migration and some other T cell functions provide receptor-selective mechanisms for regulation of T cell recruitment and immune contributions.     

CCL11 and GM-CSF differentially use the Rho GTPase pathway to regulate motility of human eosinophils in a three-dimensional microenvironment

Asthma is a common disease that causes considerable morbidity. Increased numbers of airway eosinophils are a hallmark of asthma. Mechanisms controlling the entry of eosinophils into asthmatic lung have been intensively investigated, but factors regulating migration within the tissue microenvironment are less well understood. We modeled this by studying chemoattractant and growth factor-mediated human eosinophil migration within a three-dimensional collagen matrix. Stimulation with GM-CSF induced dose-dependent, random migration with a maximum of 77 +/- 4.7% of cells migrating. In contrast, CCL11 and C5a caused a more modest although significant degree of migration (19 +/- 1.8% and 20 +/- 2.6%, respectively). Migration to GM-CSF was partially dependent on Ca(2+) and alpha(M)beta(2) integrins. The Rho family of small GTPases regulates intracellular signaling of cell migration. GM-CSF-induced migration was only partially dependent on Rho kinase/Rho-associated kinase (ROCK) and was independent of RhoA activation. In contrast, CCL11-induced migration was fully dependent on both RhoA and ROCK. Activation of RhoA was therefore neither necessary nor sufficient to cause eosinophil migration in a three-dimensional collagen environment. This study suggests that eosinophil growth factors are likely to be required for eosinophil migration within the bronchial mucosa, and this involves signal transduction pathways distinct from those used by G protein-associated chemoattractants.