Ligation of CD31 (PECAM-1) on endothelial cells increases adhesive function of alphavbeta3 integrin and enhances beta1 integrin-mediated adhesion of eosinophils to endothelial cells.

We determined the role of the heterophilic interaction of alphavbeta3 integrin on endothelial cells with CD31 on leukocytes in mediating leukocyte-endothelial cell interactions. Preincubation of interleukin-4 (IL-4)-stimulated human umbilical vein endothelial cells (HUVECs) with anti-CD31 monoclonal antibodies (MoAbs) enhanced eosinophil adhesion to the IL-4-stimulated HUVECs, and the endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs was prevented by anti-vascular cell adhesion molecule-1 (VCAM-1) MoAb and anti-very late activation antigen-4 (VLA-4) MoAb, but not by anti-intercellular adhesion molecule-1 (ICAM-1) MoAb, anti-lymphocyte function-associated antigen-1 (LFA-1) MoAb, anti-P-selectin MoAb, or anti-E-selectin MoAb. CD31 stimulation of HUVECs increased the adhesive function of alphavbeta3 integrin to its ligand RGD peptide, the binding of which reached a maximum at 10 minutes after the stimulation, and the CD31-induced alphavbeta3 integrin activation on HUVECs was inhibited by inhibitors of protein kinase C and phosphatidylinositol 3 kinase (PI3-kinase). Furthermore, anti-alphavbeta3 integrin MoAb and RGD peptide as well as soluble CD31 inhibited endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs. However, anti-alphavbeta3 integrin MoAb had no significant inhibitory effect on the eosinophil adhesion to IL-4-stimulated or unstimulated HUVECs without CD31 stimulation of HUVECs. Finally, CD31 stimulation of eosinophils increased the adhesive function of alpha4beta1 integrin (VLA-4) to its ligand fibronectin and their adhesion to IL-4-stimulated HUVECs in a VLA-4-dependent manner. These results indicate that CD31-mediated inside-out signaling activates alphavbeta3 integrin on endothelial cells, that the heterophilic alphavbeta3 integrin/CD31 interaction induces beta1 integrin-mediated adhesion of eosinophils to endothelial cells, and that the heterophilic interaction itself is not significantly involved in firm adhesion of eosinophils to endothelial cells.     

Effect of the interaction between fibronectin and VLA-4 on the proliferation of human B cells, especially a novel human B-cell line, OPM-3

Very late antigen (VLA)-4 integrin has been suggested to play an important role in haemopoiesis. However, little is known concerning the roles of the fibronectin (FN)/VLA-4 interaction in the proliferation of human B cells. In this study we investigated the effect of immobilized FN on the proliferation of various B-cell lines, including a newly-established B-cell line, OPM-3, and human tonsillar B cells, that primarily express VLA-4 but not VLA-5. Immobilized FN significantly promoted the proliferation of OPM-3 cells and normal B cells via VLA-4. The cross-linking of β1 integrins of OPM-3 cells resulted in the phosphorylation of the focal adhesion kinase (FAK) associated 90 kD protein, an increase in FAK-associated kinase activity, and the phosphorylation of Raf-1. Furthermore, the MEK1 inhibitor, PD98059, inhibited the FN-promoted proliferation of OPM-3 cells. These results demonstrate that the FN/VLA-4 interaction transmits the growth signal(s) which may be mediated by Ras pathway in OPM-3 cells, and suggest that OPM-3 cells may be of great value in studying the roles of the FN/VLA-4 interaction in human B-cell growth.     

Functional Cooperation of Cyclin C and c-Myc in Mediating Homotypic Cell Adhesion Via Very Late Antigen-4 Activation and Vascular Cell Adhesion Molecule-1 Induction

Very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1(VCAM-1) are a pair of adhesion molecules mediating cell-cell interaction. The binding activity of each depends on its surface expression, yet integrin activity can also be modulated through inside-out signaling. However, the specific intracellular molecules involved in modulating integrin VLA-4 activation via inside-out signaling or in regulating VCAM-1 expression are poorly understood. Weshow here that constitutive coexpression of cyclin C and c-Myc inhematopoietic BAF-B03 cells induces homotypic cell adhesion, whichresults from enhanced VLA-4 ligand-binding activity and inducedexpression of VCAM-1. Furthermore, regulation of cell adhesion appearsto be a feature unique to cyclin C, but not other G1 cyclins, E and D3,and its regulatory function is independent of CDK8 kinase activity. Ourresults provide a novel role for cyclin C and c-Myc in the regulationof cell adhesion through distinct mechanisms.     

Characterization of adhesion molecules on human myeloma cell lines.

In multiple myeloma, malignant plasma cells are localized in marrow and rarely circulate in peripheral blood. To investigate the role of adhesion proteins in this process, we determined the expression and function of adhesion molecules on cell lines derived from patients with myeloma. The U266, ARH-77, IM-9, and HS-Sultan cell lines strongly expressed beta 1 and alpha 4 integrins (89% to 98% positive), confirming that VLA-4 is the principal integrin on these cell lines. The U266 and IM-9 cell lines also expressed alpha 3 integrin on 15% to 20% cells. In contrast, all lines lacked cell surface alpha 2, alpha 5, and alpha 6 integrin expression (< 5% positive). These cell lines adhered to fibronectin (20% to 40% specific binding), without significant binding to either collagen or laminin. Adhesion of these cell lines to fibronectin was partially blocked with either anti-beta 1 integrin monoclonal antibody (MoAb) (75% inhibition), anti-alpha 4 integrin MoAb (75% inhibition), or RGD peptide (50% inhibition), but was unaffected by anti-alpha v beta 3 or anti-alpha IIb beta 3 MoAbs. Moreover, the combination of anti-beta 1 plus RGD peptide or anti-alpha 4 plus RGD peptide inhibited binding to fibronectin by 80% and 95%, respectively. Finally, pretreatment and coculture of the IM-9 cell line with interleukin-6 (IL-6) resulted in a 52% decrease in specific binding to fibronectin (30% +/- 6% to 15% +/- 6%; P = .001), associated with a decrease in the number of cells expressing VLA-4 and a decrease in intensity of VLA-4 expression. These data suggest that myeloma cells adhere to fibronectin through VLA-4 as well as through RGD-dependent mechanisms, and that this binding can be downregulated by IL-6. Future studies of binding of both myeloma cell lines and freshly isolated tumor cells to extracellular matrix proteins and to marrow stroma may enhance our understanding of localization and trafficking of cells within the bone marrow microenvironment.     

Essential role for Rap1 GTPase and its guanine exchange factor CalDAG-GEFI in LFA-1 but not VLA-4 integrin mediated human T-cell adhesion

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3(+) human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1alpha (SDF-1alpha)-stimulated LFA-1-ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly, silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1alpha- and phorbol 12-myristate 13-acetate (PMA)-induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1, whereas adhesion to VCAM-1 was reduced. Thus, PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells, whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4.     

Characterization of VLA-4-dependent myeloma cell adhesion to fibronectin and VCAM-1

The integrin VLA-4 mediates attachment of myeloma cells to multiple myeloma (MM) bone marrow stroma. The alternatively-spliced CS-1 region of fibronectin (FN) and VCAM-1 are main ligands for VLA-4 and are both expressed on MM stroma. The H1 region is present in all FN isoforms and represents an additional binding site for VLA-4. We employed FN fragments FN-H89 and FN-H0, that contain either the CS-1 and H1, or only the H1 sites, respectively, as well as soluble VCAM-1 (sVCAM-1), to characterize VLA-4-mediated adhesion pathways used by myeloma cells to attach to MM stroma. CD38highCD45RA- cells from MM bone marrow, and the myeloma-derived cell lines NCI-H929, IM-9 and RPMI 8226, specifically adhered, by different degrees, to FN-H89, FN-H0 and sVCAM-1, and their VLA-4-dependent adhesion was substantially up-regulated by the anti-beta1 antibody TS2/16, which increases the affinity of VLA-beta1 integrins. Furthermore, VLA-4 function on NCI-H929 cells was enhanced by TS2/16 during adhesion to MM stroma. The alpha4beta7 integrin mediated a small portion of myeloma cell line adhesion to FN-H89, mainly upon integrin activation with Mn2+. These results indicate that myeloma cells use VLA-4 to interact with CS-1/FN, H1/FN and VCAM-1 on MM stroma, and that its function can be potentially up-regulated, enabling higher degrees of cell adhesion to these VLA-4 ligands, which might influence myeloma cell localization in the bone marrow.     

LAD-III, a leukocyte adhesion deficiency syndrome associated with defective Rap1 activation and impaired stabilization of integrin bonds

Recently, we reported a rare leukocyte adhesion deficiency (LAD) associated with severe defects in integrin activation by chemokine signals, despite normal ligand binding of leukocyte integrins.(1) We now report that the small GTPase, Rap1, a key regulator of inside-out integrin activation is abnormally regulated in LAD Epstein-Barr virus (EBV) lymphocyte cells. Both constitutive and chemokine-triggered activation of Rap1 were abolished in LAD lymphocytes despite normal chemokine signaling. Nevertheless, Rap1 expression and activation by phorbol esters were intact, ruling out an LAD defect in Rap1 guanosine triphosphate (GTP) loading. The very late antigen 4 (VLA-4) integrin abnormally tethered LAD EBV lymphocytes to its ligand vascular cell adhesion molecule 1 (VCAM-1) under shear flow due to impaired generation of high-avidity contacts despite normal ligand binding and intact avidity to surface-bound anti-VLA-4 monoclonal antibody (mAb). Thus, a defect in constitutive Rap1 activation results in an inability of ligand-occupied integrins to generate high-avidity binding to ligand under shear flow. This is a first report of an inherited Rap1 activation defect associated with a pathologic disorder in leukocyte integrin function, we herein term it "LAD-III."     

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) and its G-protein-linked receptor CXCR4 are involved in hematopoietic progenitor cell and lymphocyte migration. The integrin VLA-4 is a cell adhesion receptor for CS-1/fibronectin and VCAM-1 and constitutes one of the main adhesion receptors mediating myeloma cell adhesion to bone marrow (BM) stroma in multiple myeloma (MM). It is shown here that MM CD38(hi)CD45RA(-) BM cells and myeloma-derived cell lines expressed CXCR4 and displayed a moderate chemotactic response to SDF-1alpha. Because cell migration in response to SDF-1alpha might require a dynamic regulation of integrin function, it was investigated whether SDF-1alpha can modulate VLA-4 function on myeloma cells. SDF-1alpha rapidly and transiently up-regulated VLA-4-mediated myeloma cell adhesion to both CS-1/fibronectin and VCAM-1, which was inhibited by pertussis toxin and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Modulation of VLA-4-dependent myeloma cell adhesion by SDF-1alpha could contribute to the trafficking and localization of these cells in the BM microenvironment.     

Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis

Adhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.     

VLA-4 (alpha(4)beta(1)) engagement defines a novel activation pathway for beta(2) integrin-dependent leukocyte adhesion involving the urokinase receptor

During acute inflammatory processes, beta(2) and beta(1) integrins sequentially mediate leukocyte recruitment into extravascular tissues. We studied the influence of VLA-4 (very late antigen-4) (alpha(4)beta(1)) engagement on beta(2) integrin activation-dependent cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion product vascular cell adhesion molecule-1 (VCAM-1)-Fc or by 2 anti-CD29 (beta(1) chain) monoclonal antibodies (mAb) rapidly induced adhesion of myelomonocytic cells (HL60, U937) to human umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated via beta(2) integrin (LFA-1 and Mac-1) activation: induced adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%). Adhesion to immobilized ligands of beta(2) integrins (intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole limpet hemocyanin) as well as to ICAM-1-transfected Chinese hamster ovary cells, but not to ligands of beta(1) integrins (VCAM-1, fibronectin, laminin, and collagen), was augmented. VCAM-1-Fc binding provoked the expression of the activation-dependent epitope CBRM1/5 of Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1-Fc was required for beta(2) integrin activation and induction of cell adhesion, whereas monovalent VCAM-1 or Fab fragments of anti-beta(1) integrin mAb were ineffective. Activation of beta(2) integrins by alpha(4)beta(1) integrin ligation (VCAM-1-Fc or anti-beta(1) mAb) required the presence of urokinase receptor (uPAR) on leukocytic cells, because the removal of uPAR from the cell surface by phosphatidylinositol-specific phospholipase C reduced cell adhesion to less than 40%. Adhesion was reconstituted when soluble recombinant uPAR was allowed to reassociate with the cells. Finally, VLA-4 engagement by VCAM-1-Fc or anti-beta(1) integrin mAb induced uPAR-dependent adhesion to immobilized vitronectin as well. These results elucidate a novel activation pathway of beta(2) integrin-dependent cell-to-cell adhesion that requires alpha(4)beta(1) integrin ligation for initiation and uPAR as activation transducer. (Blood. 2000;96:506-513)     

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion.Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.     

Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-gamma

Attachment of cells to extracellular matrix components is critical for the regulation of hematopoiesis. CD43 is a mucin-like transmembrane sialoglycoprotein expressed on the surface of almost all hematopoietic cells. A highly extended structure of extracellular mucin with negative charge may function as a repulsive barrier to hematopoietic cells. However, some investigators have shown that CD43 has proadhesive properties, and engagement of CD43 has been reported to upregulate integrin-mediated cell adhesion in T cells. We found that cross-linking of CD43 with monoclonal antibodies (MoAbs) enhanced integrin alpha4beta1 (very late antigen [VLA]-4) and alpha5 beta1 (VLA-5)-dependent adhesion of human cord blood CD34(+) cells to fibronectin. CD34(+) CD38(hi), but not CD34(+)CD38(-/low) cells responded significantly to the stimulus, suggesting that committed, but not stem and more immature progenitors are sensitive to CD43-mediated activation of integrin. To elucidate the molecular mechanism leading to integrin activation, we used the growth factor-dependent cell line MO7e. Cross-linking of CD43 induced tyrosine phosphorylation of several intracellular molecules including the protein tyrosine kinase Syk, the proto-oncogene product Cbl, and phospholipase C (PLC)-gamma2 in MO7e cells. Moreover, protein tyrosine kinase inhibitor herbimycin A and PLC inhibitor U73122 both blocked CD43-induced enhancement of adhesion to fibronectin. These results indicate that signals mediated through CD43 may increase integrin affinity to fibronectin via a pathway dependent on protein tyrosine kinase and PLC-gamma activation in hematopoietic progenitors.     

Integrin-dependent cell growth and survival are mediated by different signals in thyroid cells

Cell adhesion to extracellular matrix regulates proliferation and survival of several cell types including epithelial thyroid cells. Activation of integrin receptors by binding to extracellular matrix generates a complex cell type-dependent signaling. Adhesion to extracellular matrix induces proliferation and survival in primary cultures of thyroid cells and induces survival in immortalized human thyrocytes. In this study we demonstrate that in immortalized human thyrocyte cells, adhesion to immobilized fibronectin (FN) stimulates DNA synthesis and proliferation through the p21Ras/MAPK pathway, whereas cell survival is mediated by phosphatidylinositol 3-kinase (PI3K) signal pathway. Integrin activation by immobilized FN induced phosphorylation of pp125 focal adhesion kinase and paxillin and induced the formation of focal adhesion kinase/Grb-2/Sos complex. Western blot and in vitro kinase assay demonstrated the activation of Ras and the p44/p42 MAPK/ERK1/2. Inhibition of p21Ras activity and inhibition of MAPK enzymatic activity completely arrested cell growth but did not induce cell death. Integrin activation by cell adhesion to FN also induced activation of PI3K. Inhibition of PI3K enzymatic activity induced apoptosis demonstrated by annexin V-binding assay and loss of cellular DNA content. These results demonstrate that in thyroid cells adhesion to FN regulates proliferation through the p21Ras/MAPK signal pathway, whereas integrin-mediated cell survival is mediated by PI3K.     

Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.     

Signaling via beta1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.

Human epidermal stem cells express higher levels of beta1 integrins and are more adhesive than keratinocytes that are destined to differentiate. To investigate whether high beta1 integrin expression and adhesiveness are essential for maintaining keratinocytes in the stem cell compartment, we introduced a dominant-negative beta1 integrin mutant, CD8beta1, into cultured human keratinocytes, thereby interfering with beta1 integrin function. Surface beta1 integrin levels, adhesiveness, and mitogen-activated protein (MAP) kinase activation on fibronectin were reduced, and exit from the stem cell compartment was stimulated. Adhesiveness and proliferative potential were restored by overexpressing wild-type beta1 integrin or by constitutive MAP kinase activation. Conversely, a dominant-negative MAP kinase kinase 1 mutant decreased adhesiveness and stem cell number in the absence of CD8beta1. MAP kinase activation by alpha6beta4-mediated adhesion and mitogens was normal in CD8beta1 cells, and constitutive MAP kinase activation did not affect adhesion and proliferation of control keratinocytes. We conclude that beta1 integrins and MAP kinase cooperate to maintain the epidermal stem cell compartment in vitro.     

High frequency of adhesion defects in B-lineage acute lymphoblastic leukemia.

Aberrant proliferation, differentiation, and/or migration of progenitors observed in various hematological malignancies may be caused by defects in expression and/or function of integrins. In this study, we have developed a new fluorescent beads adhesion assay that facilitates flow cytometric investigation of lymphocyte function-associated antigen 1 (LFA-1)- and very late activation antigen-4 (VLA-4)-mediated functional adhesion in B-lineage acute lymphoblastic leukemia (ALL) of both the CD10(-) and CD10(+) (leukemic) cell population within one blood or bone marrow sample. Surprisingly, of the 20 B-lineage ALL patients investigated, 17 contained a leukemic cell population with LFA-1- and/or VLA-4-mediated adhesion defects. Five patients contained CD10(+) cells that did not exhibit any LFA-1-mediated adhesion due to the lack of LFA-1 surface expression. The CD10(+) cells from 10 ALL patients expressed LFA-1 that could not be activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA), whereas the CD10(-) cells expressed a functional LFA-1. Seven patients contained CD10(+) cells that expressed a PMA-unresponsive form of VLA-4. The PMA unresponsiveness of the integrins LFA-1 and VLA-4 expressed by the CD10(+) cells may be due to mutations in the integrins itself, in protein kinases, or in other intracellular molecules involved in integrin adhesion. These data clearly demonstrate the importance of investigating integrin function in addition to integrin surface expression. The strikingly high frequency (85%) of adhesion defects in ALL could suggest a causal relationship between integrin-mediated adhesion and B-lineage ALL.     

Ligation of lymphocyte function-associated antigen-1 on monocytes decreases very late antigen-4-mediated adhesion through a reactive oxygen species-dependent pathway

Monocyte-endothelial adhesion plays an important role in monocyte trafficking and hence is important for immune responses and pathogenesis of inflammatory diseases including atherosclerosis. The cross-talk between different integrins on monocytes may be crucial for a coordinated regulation of the cellular adhesion during the complex process of transendothelial migration. By using monoclonal antibodies and recombinant intercellular adhesion molecule 1 (ICAM-1) to engage lymphocyte function-associated antigen 1 (LFA-1) on monocytic cells, we found that the cellular adhesion to vascular cell adhesion molecule 1 (VCAM-1) mediated by very late antigen 4 (VLA-4) was suppressed after this treatment and the suppression depended on the presence of reactive oxygen species (ROSs). Inhibition of production of ROSs through the use of inhibitor of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, but not inhibitors of mitochondrial electron transport chain or xanthine oxidase, revealed that this suppression on VLA-4-mediated cellular binding was mediated by ROSs produced by phagocyte NADPH oxidase. Activation of phosphoinositol-3 kinase and Akt appears to mediate this NADPH oxidase activation through p47phox phosphorylation and Rac-1 activation. Our results provide a novel pathway in which ROSs play a critical role in integrin cross-talk in monocytes. This signaling pathway may be important for cellular transition from firm arrest to diapedesis during monocyte trafficking.     

β1 integrin–mediated signaling induces intercellular adhesion molecule 1 and Fas on rheumatoid synovial cells and Fas‐mediated apoptosis

Objective

Rheumatoid arthritis (RA) synovial cells interact with inflammatory cells, as well as extracellular matrices, through integrins. However, the relevance of β1 integrin to inflammatory processes in RA remains unclear. We examined the role of β1 integrin–mediated signaling in RA.

Methods

Expression of cell‐surface molecules was assessed by FACScan. Engagement of β1 integrins was performed by crosslinking using a specific monoclonal antibody (mAb) and ligand matrices such as fibronectin or collagen. To determine the involvement of tyrosine kinases in β1 integrin–mediated signaling, the cells were pretreated with various inhibitors of intracytoplasmic signaling or were transfected with a wild‐type focal adhesion kinase (FAK) or a dominant negative truncation of the FAK expression plasmid via cationic liposome‐mediated transfection. Apoptosis of synovial cells was detected by double staining with propidium iodide and annexin V.

Results

β1 integrin was highly expressed on RA synovial cells. Engagement of β1 integrins by crosslinking as well as by ligand matrices markedly up‐regulated expression of intercellular adhesion molecule 1 (ICAM‐1) and Fas. Up‐regulation of ICAM‐1 and Fas induced by β1 integrin was mediated by the tyrosine kinase signaling pathway, especially involving FAK. Fas‐mediated early apoptotic change in the cells was amplified by β1 crosslinking.

Conclusion

Our results suggest that interaction of β1 integrins with extracellular matrix augments expression of ICAM‐1 and Fas on RA synovial cells, as well as Fas‐mediated apoptosis of synovial cells. This might lead to the spontaneous growth arrest through the Fas/Fas ligand pathway observed in RA synovitis.

     

beta1 integrin-mediated signaling induces intercellular adhesion molecule 1 and Fas on rheumatoid synovial cells and Fas-mediated apoptosis

OBJECTIVE: Rheumatoid arthritis (RA) synovial cells interact with inflammatory cells, as well as extracellular matrices, through integrins. However, the relevance of beta1 integrin to inflammatory processes in RA remains unclear. We examined the role of beta1 integrin-mediated signaling in RA. METHODS: Expression of cell-surface molecules was assessed by FACScan. Engagement of beta1 integrins was performed by crosslinking using a specific monoclonal antibody (mAb) and ligand matrices such as fibronectin or collagen. To determine the involvement of tyrosine kinases in beta1 integrin-mediated signaling, the cells were pretreated with various inhibitors of intracytoplasmic signaling or were transfected with a wild-type focal adhesion kinase (FAK) or a dominant negative truncation of the FAK expression plasmid via cationic liposome-mediated transfection. Apoptosis of synovial cells was detected by double staining with propidium iodide and annexin V. RESULTS: beta1 integrin was highly expressed on RA synovial cells. Engagement of beta1 integrins by crosslinking as well as by ligand matrices markedly up-regulated expression of intercellular adhesion molecule 1 (ICAM-1) and Fas. Up-regulation of ICAM-1 and Fas induced by beta1 integrin was mediated by the tyrosine kinase signaling pathway, especially involving FAK. Fas-mediated early apoptotic change in the cells was amplified by beta1 crosslinking. CONCLUSION: Our results suggest that interaction of beta1 integrins with extracellular matrix augments expression of ICAM-1 and Fas on RA synovial cells, as well as Fas-mediated apoptosis of synovial cells. This might lead to the spontaneous growth arrest through the Fas/Fas ligand pathway observed in RA synovitis.