Expression of T-cell Receptor (TCR)α Chain on Normal Human Tonsils and T-cell Lymphomas

We analyzed immunohistologically the expression of T cell receptor (TCR)α chain on human tonsils and on T cell lymphoma (T ML) tissues using the avidin biotin peroxidase method. A murine monoclonal antibody αF1, specific for the constant region of the JCRα chain, was employed. On normal tonsil, αF1 positive cells were observed mainly in T zones and germinal centers. In T zones, the staining intensities varied markedly, with heavy staining evident in less than one fourth. In germinal centers, a proportion of stained cells showed a histiocytic pattern with small cytoplasmic projections. All the T ML tissues expressed TCRα, whereas the staining intensities varied among cases and among lymphoma cells. No correlations were observed in the expressions of TCRα chain and other T-cell markers including CD3, CD4 and CD8. Acta Pathol Jpn 40: 722-728, 1990.     

β1 Integrin induces adhesion and migration of human Th17 cells via Pyk2-dependent activation of P2X4 receptor

Integrin-mediated T-cell adhesion and migration is a crucial step in immune response and autoimmune diseases. However, the underlying signalling mechanisms are not fully elucidated. In this study, we examined the implication of purinergic signalling, which has been associated with T-cell activation, in the adhesion and migration of human Th17 cells across fibronectin, a major matrix protein associated with inflammatory diseases. We showed that the adhesion of human Th17 cells to fibronectin induces, via β1 integrin, a sustained release of adenosine triphosphate (ATP) from the mitochondria through the pannexin-1 hemichannels. Inhibition of ATP release or its degradation with apyrase impaired the capacity of the cells to attach and migrate across fibronectin. Inhibition studies identified a major role for the purinergic receptor P2X4 in T-cell adhesion and migration but not for P2X7 or P2Y₁₁ receptors. Blockade of P2X4 but not P2X7 or P2Y₁₁ receptors reduced cell adhesion and migration by inhibiting activation of β1 integrins, which is essential for ligand binding. Furthermore, we found that β1 integrin-induced ATP release, P2X4 receptor transactivation, cell adhesion and migration were dependent on the focal adhesion kinase Pyk2 but not FAK. Finally, P2X4 receptor inhibition also blocked fibronectin-induced Pyk2 activation suggesting the existence of a positive feedback loop of activation between β1 integrin/Pyk2 and P2X4 purinergic signalling pathways. Our findings uncovered an unrecognized link between β1 integrin and P2X4 receptor signalling pathways for promoting T-cell adhesion and migration across the extracellular matrix.     

Sphingosine‐1‐phosphate activates chemokine‐promoted myeloma cell adhesion and migration involving α4β1 integrin function

Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1‐dependent myeloma cell adhesion is up‐regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine‐1‐phosphate (S1P) regulates immune cell trafficking upon binding to G‐protein‐coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up‐regulated the α4β1‐mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high‐affinity α4β1 that efficiently bound the α4β1 ligand VCAM‐1, a finding that was associated with S1P‐triggered increase in talin‐β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1‐dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2‐Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1‐dependent adhesion and migration of myeloma cells by CXCL12‐S1P combined activities might have important consequences for myeloma disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CD44 and β1 integrins mediate ovarian carcinoma cell migration toward extracellular matrix proteins

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial cells that line the peritoneal cavity. The aim of this study was to identify the cell–matrix interactions that mediate ovarian carcinoma cell migration toward components of the mesothelial cell-associated extracellular matrix. The human ovarian carcinoma cell lines NIH:OVCAR5 and SKOV3 were analyzed by flow cytometry for the expression of cell surface receptors. The ability of those receptors to mediate ovarian carcinoma cell migration toward fibronectin, type IV collagen, and laminin was determined. A monoclonal antibody against the β1 integrin subunit abrogated the migration of both cell lines toward the extracellular matrix proteins. Blocking antibodies against alpha integrin subunits suggest that ovarian carcinoma cell migration toward fibronectin is primarily mediated by the ∝5β1 integrin, type IV collagen by the ∝2β1 integrin, and laminin by the ∝6β1 integrin. These results suggest that ovarian carcinoma cell migration is regulated by multiple β1 integrin–matrix interactions. Significant reduction of cell migration was observed with a monoclonal antibody against CD44 that blocks the hyaluronan-binding site of CD44, but not with an antibody that binds at an alternate site on CD44. Intact hyaluronan and/or hyaluronan oligomers also inhibited cell migration, suggesting that the CD44–hyaluronan interaction provides an integrin-independent mechanism of control for ovarian carcinoma cell migration. These results suggest that ovarian carcinoma cell migration is regulated by both integrin-dependent mechanisms, involving the interaction of β1 integrins with extracellular matrix proteins, and an integrin-independent mechanism that involves the interaction of CD44 and hyaluronan.     

Integrin alpha chains exhibit distinct temporal and spatial localization patterns in epithelial cells of the Drosophila ovary

Integrins are heterodimeric transmembrane receptors that modulate cell adhesion, migration, and signaling. Multiple integrin chains contribute to development and morphogenesis of a given tissue. Here, we analyze the expression of Drosophila integrin alpha chains in the ovarian follicular epithelium, a model for tissue morphogenesis and cell migration. We find expression throughout development of the beta chain, βPS. Alpha chains, however, exhibit both spatial and temporal expression differences. αPS1 and αPS2 integrins are detected during early and mid‐oogenesis on apical, lateral, and basal membranes with the βPS chain, whereas αPS3‐family integrins (αPS3, αPS4, αPS5) are expressed in anterior cells late in oogenesis. Surprisingly, we find that αPS3‐family integrins are dispensable for dorsal appendage morphogenesis but play a role in the final length of the egg, suggesting redundant functions of integrins in a simple tissue. We also demonstrate roles for αPS3βPS integrin in border cell migration and in stretch cells. Developmental Dynamics 237:3927–3939, 2008. © 2008 Wiley‐Liss, Inc.     

The use of reactive polymer coatings to facilitate gene delivery from poly (-caprolactone) scaffolds

To functionalize biomaterials for bioconjugation, a chemical vapor deposition (CVD) polymerization technique was utilized to modify material surfaces. Poly [(4-amino-p-xylylene)-co-(p-xylylene)] (PPX–NH₂) was deposited on inert polycaprolactone (PCL) surfaces to provide a reactive amine layer on the substrate surfaces. The biocompatibility of PPX–NH₂ was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase (LDH) assays. The results demonstrated that cells continuously proliferated on CVD treated PCL surfaces with high survival rates. Biotin was conjugated on modified PCL surfaces to immobilize avidin for binding of biotinylated adenovirus. Scanning electron microscopy (SEM) examination illustrated that adenoviruses were evenly bound on both 2-D films and 3-D scaffolds, suggesting CVD was capable of modifying various substrates with different geometries. Using a wax masking technique, the biotin conjugation was controlled to immobilize avidin on specific sites. Due to the virus binding specificity on CVD-modified surfaces, cell transduction was restricted to the pattern of immobilized virus on biomaterials, by which transduced and non-transduced cells were controlled in different regions with a distinct interface. Because CVD was functional in different hierarchies, this surface modification should be able to custom-tailor bioconjugation for different applications.     

Active Targeting Behaviors of Biotinylated Pluronic/Poly(Lactic Acid) Nanoparticles In Vitro through Three-Step Biotin–Avidin Interaction

In order to prepare targeted drug carriers, previously a biotin group has been attached by our group to the end of Pluronic F87/poly(lactic acid) and Pluronic P85/poly(lactic acid) block co-polymers to obtain B-F87–PLA and B-P85–PLA, respectively. In this paper, the active targeting properties of B-F87–PLA and B-P85–PLA nanoparticles in vitro were investigated through a three-step biotin–avidin interaction by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) tests and fluorescence microscopy (FM). Two kinds of human ovarian cancer cells (OVCAR-3 and SKOV-3) and paclitaxel were chosen for the cytotoxicity tests. CA-125 antigen is over-expressed on OVCAR-3 cells but not on SKOV-3 cells. The loading and release behavior of paclitaxel loaded in B-Pluronic–PLA nanoparticles were also studied. Paclitaxel loaded in both B-F87–PLA and B-P85–PLA nanoparticles shows an initial rapid release followed by a slow release period. Compared with SKOV-3 cells, the cytotoxicity results implied that paclitaxel-loaded B-Pluronic–PLA nanoparticles were delivered more effectively to OVCAR-3 cells due to the specific interaction between the biotin groups on the surface of B-Pluronic–PLA nanoparticles and the avidin/biotinylated MAb X306/CA-125 antigen complexes on the surface of OVCAR-3 cells. The active targeting properties of B-F87–PLA nanoparticles were further confirmed by FM.     

Effect of specific antigen stimulation on intraepithelial lymphocyte migration to small intestinal mucosa

Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.     

Signal transduction events regulating integrin function and T cell migration

Integrin receptors facilitate T cell function by mediating adhesive events critical for T cell trafficking and recognition of foreign antigen, including interactions with vascular endothelium, extracellular matrix components, and antigen-presenting cells. Consequently, the functional activity of integrin receptors is acutely regulated by various intracellular signals delivered by other cell surface receptors, resulting in rapid changes in T cell adhesion and migration. This review highlights recent insights into our understanding of the signaling events by which the CD3/T cell receptor complex and chemokine receptors regulate integrin function and T cell migration. These studies highlight novel functions for several signaling molecules, including the tyrosine kinases Itk and ZAP-70, and the adapter protein SLAP-130/Fyb. In addition, analysis of the regulation of integrin function and chemokine-mediated migration has highlighted the critical role that spatial localization of signaling molecules plays in signal transduction, and the importance of the actin cytoskeleton in T cell function.     

Relative Rigidity of Cell–Substrate Effects on Hepatic and Hepatocellular Carcinoma Cell Migration

Abstract

Polyacrylamide gels with different stiffness and glass were employed as substrates to investigate how substrate stiffness affects the cellular stiffness of adherent hepatocellular carcinoma (HCCLM3) and hepatic (L02) cells. The interaction of how cell-substrate stiffness influences cell migration was also explored. An atom force microscope measured the stiffness of HCCLM3 and L02 cells on different substrates. Further, F-actin assembly was analyzed using immunofluorescence and Western blot. Finally, cell-surface expression of integrin β1 was quantified by flow cytometry. The results show that, while both HCCLM3 and L02 cells adjusted their cell stiffness to comply with the stiffness of the substrate they were adhered to, their tuning capabilities were different. HCCLM3 cell stiffness complied when substrate stiffness was between 1.1 and 33.7 kPa, whereas the analogous stiffness for L02 cells occurred at a higher substrate stiffness, 3.6 kPa up to glass. These ranges correlated with F-actin filament assembly and integrin β1 expression. In a migration assay, HCCLM3 cells migrated faster on a relatively soft substrate, while L02 cells migrated faster on substrates that were relatively rigid. These findings indicate that different tuning capabilities of HCCLM3 and L02 cells may influence cell migration velocity on substrates with different stiffness by regulating cy- toskeleton remodeling and integrin β1 expression.     

Immunocytochemical study of progesterone receptors in the human endometrium during the menstrual cycle

Specific mouse monoclonal antibody (alpha PR6) against progesterone receptor was used with an avidin biotin complex technique to localize progesterone receptors in frozen sections of 26 normal cyclic human endometria. Progesterone receptor was detected in the nuclei of epithelial and stromal cells in both the functionalis and basalis layers. In the functionalis, the receptor content increased from the early to the late proliferative phase in both cell components. It remained high in the early secretory phase and decreased in the mid- and late secretory phases, comparatively more rapidly in the epithelium than in the stroma. In the latter, the predecidual cell nuclei were receptor-positive. The menstrual phase endometrium lacked receptors. The basalis was rich in progesterone receptors during the proliferative, early and midsecretory phases in both components and receptor-free during the late secretory and menstrual phases of the cycle. Myometrial smooth muscle cell nuclei contained progesterone receptors, whereas they were absent in endometrial and myometrial vessels. Overall, the epithelial progesterone receptor content seemed to correlate with the endometrial tissue levels of estradiol, possibly reflecting its estrogen sensitivity, whereas the stromal progesterone receptor content during the secretory phase at least, in part, may be constitutively synthetized.     

Tissue transglutaminase mediates the pro‐malignant effects of oncostatin M receptor over‐expression in cervical squamous cell carcinoma

Oncostatin M receptor (OSMR) is commonly over‐expressed in advanced cervical squamous cell carcinoma (SCC), producing a significantly worse clinical outcome. Cervical SCC cells that over‐express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro‐malignant effects, including increased cell migration and invasiveness. Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand‐dependent phenotypic effects of OSMR over‐expression in SCC cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of cervical and oral SCC. TGM2 depletion in cervical SCC cells abrogated OSM‐induced migration on fibronectin‐coated surfaces and invasiveness through extracellular matrix, while ectopic expression of TGM2 increased cell motility and invasiveness. Confocal microscopy and co‐immunoprecipitation assays showed that TGM2 interacted with integrin–α5β1 in the presence of fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin–α5β1 and fibronectin were also over‐expressed in cervical and oral SCC, where levels correlated with those of OSMR and TGM2. This combined tissue and in vitro study demonstrates for the first time that stimulation of over‐expressed OSMR in cervical SCC cells activates TGM2/integrin‐α5β1 interactions and induces pro‐malignant changes. We conclude that an OSMR/TGM2/integrin‐α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting. Copyright © 2013 Pathological Society of Great Britain and Ireland. © 2013 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

T‐cell regulation through a basic suppressive mechanism targeting low‐density lipoprotein receptor‐related protein 1

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T‐cell adhesion, and notably also T‐cell receptor (TCR) ‐induced activation, are subject to constant suppression through shedding of low‐density lipoprotein receptor‐related protein 1 (LRP1). The broad‐spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR‐induced activation and adhesion to β₁ and β₂ integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full‐length thrombospondin‐1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non‐adhering cells and expression of TSP170 correlated with T‐cell motility. The pro‐adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP‐1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP‐1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP‐1 in integrin‐dependent adhesion and TCR‐induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP‐1‐dependent pathway and ligation of CD28 antagonizes shedding independent of TSP‐1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T‐cell level.     

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.     

The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay

Ligands in the extracellular matrix (ECM) are known to mediate migration of normal as well as tumor cells via adhesion molecules such as the integrin receptor family. We developed a microliter scale (15-20 fu total volume) monolayer migration assay to investigate the ability of astrocytoma cells to disperse on surfaces coated with purified human ECM protein ligands. In this system the rate of radial migration of the cell population was constant over time. For human astrocytoma cell lines U-251 and SF-767, laminin and collagen type IV supported a migratory phenotype; fibronectin and vitronectin only minimally supported migration. The different ECM proteins also influenced growth rate: cells on laminin and collagen had a protracted lag phase. Furthermore, migrating cells seeded on laminin or collagen showed a lower labeling index than did stationary cells in the central, crowded region on the same substrate. This micro-scale migration assay should enable detailed molecular and biochemical studies of the determinants of migration.     

Functional blocking of specific integrins inhibit colonic cancer migration

For more effective oncological management of disseminated colorectal cancer, therapies must be devised that target the different individual stages of metastasis development. Recent work showed that integrin subunits α2, α6 and β4 are involved in the colorectal cancer cell extravasation process. By means of Immunocytochemistry and Western blotting, it was shown that all three integrins are expressed not only in human colorectal cancer cells (HT29) but also in rat colonic cancer cells (DHDK12). Using in vivo models and intravital video microscopy techniques, it was shown that functional blocking of these integrin subunits by specific antibodies produced a significant reduction in cancer cell extravasation and migration. In conclusion, integrin subunits α2, α6 and β4 are expressed in unrelated colorectal cancer cell strains and appear to play a key role in cancer cell migration.     

Neuregulin‐1/glial growth factor stimulates Schwann cell migration by inducing α5 β1 integrin–ErbB2–focal adhesion kinase complex formation

After peripheral nerve injury, Schwann cells gain a migratory phenotype and remodel their extracellular matrix to provide a supportive environment for axonal regeneration. The soluble neuregulin‐1 isoform, that is, glial growth factor (GGF), is expressed in regenerating axons of injured peripheral nerves and regulates Schwann cell motility by activating the ErbB family of tyrosine kinase receptors, but how GGF/ErbB signaling contributes to Schwann cell motility remains unclear. Here, we show that GGF stimulates Schwann cell migration by inducing the formation of a protein complex containing the fibronectin receptor α5β1 integrin, ErbB2, and focal adhesion kinase (FAK). ErbB2 co‐localizes and co‐immunoprecipitates with the focal complex members including α5β1 integrin and FAK after GGF treatment. These effects of GGF appear to involve FAK activation, which occurs downstream of ErbB2 stimulation. RNAi‐mediated down‐regulation of α5 integrin expression in primary cultured Schwann cells resulted in significantly decreased interaction between FAK and ErbB2, as well as decreased GGF‐induced migration. An increase in the α5β1 integrin–ErbB2–FAK complex formation was observed in injured nerve Schwann cells, but not uninjured control. Taken together, these data suggest that GGF plays an important modulatory role in Schwann cell migration after nerve crush by inducing α5β1 integrin–ErbB2–FAK complex formation.     

移植物抗宿主病小鼠肝脏及异体CD8+ T细胞趋化因子和趋化因子受体表达分析

为探讨趋化因子及其受体介导的效应性细胞迁移在移植物抗宿主病(graft-versus-host disease,GVHD)发生发展中的重要作用,采用次要组织相容性抗原异配的GVHD小鼠模型,应用流式细胞分选术(FACS)、实时荧光定量聚合酶链反应等方法,分析GVHD靶器官和异体CD8+T细胞趋化因子及受体的表达。肝脏高表达干扰素诱导蛋白-10(IP-10)、干扰素诱导的T细胞趋化因子(ITAC)、γ干扰素诱生单核因子(MIG)、巨噬细胞炎症蛋白(MIP)-1α/β等趋化因子。异体CD8+T细胞上则高表达CXC趋化因子受体(CXCR)3和CC趋化因子受体(CCR)5。随着肝脏趋化因子的表达增加,异体CD8+T细胞迁移浸润的数量也增高。因而趋化因子及异体CD8+T细胞上趋化因子受体的特异对应关系,促使大量异体CD8+T细胞迁移浸润并造成肝脏损伤。     

Functional cloning of ARM-1, an adhesion-regulating molecule upregulated in metastatic tumor cells

Interactions of tumor cells with the endothelium and tissue stroma are considered to be critical steps in metastasis formation and progression of cancer. To identify cellular receptors that mediate the binding of tumor cells to endothelium, a murine T cell lymphoma-derived expression library was screened for adhesion-inducing cDNA clones. We identified a novel cell adhesion-promoting molecule, termed ARM-1 (adhesion regulating molecule-1), which is homologous to a human M _r 110.000 tumor-associated antigen. The ARM-1 cDNA codes for a type I transmembrane protein of 407 amino acids with potential O- and N-glycosylation sites that does not belong to any of the known families of cell adhesion molecules. Overexpression of ARM-1 in 293T human embryonic kidney cells significantly increased adhesion to different endothelial cells. ARM-1 expression in 293T cells did not alter integrin expression or β1-integrin-mediated cell adhesion. Northern blot analysis of human breast cancer cell lines revealed 3- to 5-fold elevated ARM-1 mRNA levels in metastatic as compared to non-metastatic cells. In conclusion, we have identified ARM-1 as a novel cell adhesion-promoting receptor that is upregulated in metastatic cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.