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We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic α4β1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial α4β1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of β1integrins, particularly those associated with α5 integrins.Activation of β1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated β1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated β1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis. J. Clin. Invest. 103:613–625 (1999)

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activated protein (MAP) kinase is rapidly stimulated by flow in bovine aortic endothelial cells. Integrin receptors may act as mechanotransducers, as suggested by rapid remodeling of focal adhesion complexes in response to flow. To study the role of integrins in flow-mediated MAP kinase activation, we compared the effects of β1 integrin activation (with 8A2 antibody) and flow in cultured human umbilical vein endothelial cells (HUVECs). Both 8A2 (3 μg/mL) and flow (shear stress, 12 dynes/cm 2 ) stimulated MAP kinase, although the flow response was faster and greater. To characterize flow-activated tyrosine kinases, tyrosine-phosphorylated proteins were immunoprecipitated and identified by Western blot. There was a time-dependent increase in phosphotyrosine content in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD proteins. A 125-kD protein was identified as focal adhesion kinase (FAK), suggesting that flow activates integrins. In comparison with flow, 8A2 caused less tyrosine phosphorylation of fewer proteins, although FAK was tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 and flow caused additive increases in MAP kinase. Antibody 8A2 increased binding of the β1 affinity–sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a β1-activating antibody and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that “costimulatory” events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.

Abstract Avidity modulation and function of β 1 -integrin receptors in cultured human vascular smooth muscle cells (SMCs) were investigated using monoclonal antibody (mAb) 8A2, which binds to the β 1 subunit of integrin heterodimers and induces a high avidity state. The adhesion of SMCs to extracellular matrix proteins, but not to poly- l -lysine, was enhanced by pretreatment with mAb 8A2. A qualitative alteration of β 1 integrin was assessed with mAb 15/7, which binds to an activation-dependent epitope on the β 1 subunit. Binding of mAb 15/7 was enhanced by mAb 8A2 in a dose-dependent manner. Arg-Gly-Asp peptide and soluble fibronectin also enhanced expression of the 15/7 epitope, suggesting that the 15/7 epitope is closely related to the ligand-occupied state of β 1 integrin. Platelet-derived growth factor (PDGF)-AA and -BB increased SMC adhesion to type I collagen but did not augment mAb 15/7 binding, suggesting that PDGFs increase binding avidity by a postreceptor mechanism. In addition, mAb 8A2 inhibited PDGF-BB–induced SMC migration through Matrigel-coated filters. These results suggest that avidity modulation of β 1 integrin may play an important role in the function of SMCs.

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML. ispartof: Blood vol:87 issue:6 pages:2450-8 ispartof: location:United States status: published

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin αvβ3. Here we demonstrate that two other integrins, αvβ1 and αvβ5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin αvβ1. When the 293 cells are transfected with the β5 subunit, they can also adhere to GST-OPN using integrin αvβ5. Divalent cations regulate the binding of GST-OPN to both αvβ1 and αvβ5. Mg2+ and Mn2+ support the binding of GST-OPN to these integrins but Ca2+ does not. The highest affinity is observed in Mn2+. In the presence of this ion, the affinity of GST-OPN for αvβ1 is 18 nM and the affinity for αvβ5 is 48 nM. The antibody 8A2, which is an agonist for β1, promotes the adhesion of 293 cells to GST-OPN even when Ca2+ is present. This observation suggests that cellular events could modulate the affinity of αvβ1 for OPN. Collectively, these findings prove that integrins αvβ1, αvβ3, and αvβ5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.

Interactions between hematopoietic cells and bone marrow (BM) stroma, composed of extracellular matrix and stromal cells, are crucial for hematopoiesis. Integrins facilitate these interactions by mediating adherence of hematopoiesis. Integrins facilitate these interactions by mediating adherence of hematopoietic cells to both the extracellular matrix and stromal cells. Marrow stromal cells secrete a variety of growth factors, including stem cell factor (SCF). Because treatment with SCF in vivo mobilizes primitive hematopoietic cells from the BM, we investigated the effect of the growth factor SCF of hematopoietic cell adhesion. These studies show that SCF modulates adhesive function in a dose- and time-dependent manner, but does not modulate expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 in the SCF- responsive cell line MO7E. Treatment of MO7E cells with SCF (200 ng/mL) produced a transient increase in adherence to cytokine-activated human umbilical vein endothelial cells (HUVECs) or to vascular cell adhesion molecule 1 (VCAM-1)-transfected Chinese hamster ovary (CHO) cells with peak adhesion at 30 minutes and return to baseline by 60 to 90 minutes. This increase in adhesion was paralleled by increased binding of the beta 1 activation-dependent monoclonal antibody (MoAb) 15/7, as determined by flow cytometry. However, prolonged incubation of MO7E with SCF induced a marked decrease in integrin-mediated adherence, with maximal inhibition by 24 hours. No change in expression of integrins, as determined by flow cytometry, was observed with short- or long-term incubation with SCF. SCF-treated cells were still able to respond to phorbol esters and to the activating beta 1 MoAb 8A2 with increased adherence, but not to the level seen in control cells. This suggests that a subpopulation of expressed alpha 4 beta 1 and alpha 5 beta 1 integrins is disengaged by prolonged incubation with SCF.

Peripheral blood lymphocytes (PBLs) cultured in the presence of recombinant human interleukin-2 (rhIL-2) develop a natural killer (NK) cell phenotype (CD16+, CD56+, CD3-) and are referred to as lymphokine- activated killer cells (LAK). In developing the LAK phenotype, enhanced adherence to matrix components and endothelial cells have been described. In this report we investigated the functional behavior of adhesion receptors in rhIL-2-activated PBLs by in vitro adhesion assay and by flow cytometry. Compared to PBLs, IL-2-activated PBLs had increased integrin-mediated adherence to: (1) fibronectin (FN), (2) human umbilical vein endothelial (HUVE) cells, and (3) cultured melanoma and pancreatic tumor cell lines. This increase in adherence was mediated by increased surface expression of members of the beta 1 and beta 2 integrin subfamilies, as determined by flow cytometric analysis. No induction of an activation-dependent beta 1 (CD29) epitope was detected. We also investigated the effects of the methylxanthine derivative pentoxifylline (PTX) on PBLs and rhIL-2-activated PBL adhesion. PBLs co-cultivated in the presence of rhIL-2 (1,000 U/mL) and PTX exhibited reduced adherence to FN, HUVE and cultured tumor cell lines. This inhibition by PTX was concentration- and time-dependent. The increased expression of integrins induced by rhIL-2 was only in part inhibited by PTX, suggesting that PTX induced a subpopulation of integrins that are expressed but functionally inactive.

Extracellular matrix receptors of the integrin family have a dual role in the epidermis, regulating both adhesion and differentiation. Loss of contact with the extracellular matrix causes keratinocytes to become committed to terminal differentiation, and results in a decrease in the ability of the alpha 5 beta 1 integrin to bind fibronectin. We have investigated whether the decrease in ligand-binding ability is reversible and, if so, whether commitment to terminal differentiation can also be reversed. Keratinocytes that had been placed in suspension for 5 hours to induce commitment were compared with the starting population (0 hour cells) in the presence or absence of 8A2, an activating anti-beta 1 antibody. 8A2 IgG or FAb fragments increased the amount of alpha 5 beta 1 in cell extracts that bound to fibronectin-Sepharose and in the presence of 8A2 the amount of bound alpha 5 beta 1 in 0 hour and 5 hour extracts was equal. 8A2 also restored alpha 5 beta 1 function in adhesion assays of intact 5 hour cells. Ca2+, Mg2+ and Mn2+ alone, at concentrations of up to 1 mM, did not increase the adhesiveness of 5 hour cells relative to 0 hour cells; however, the effect of 8A2 on keratinocytes was dependent on Ca2+. Although 8A2 restored alpha 5 beta 1 ligand-binding ability it did not prevent committed cells from withdrawing from the cell cycle and expressing involucrin, a differentiation marker.(ABSTRACT TRUNCATED AT 250 WORDS)

We report that a beta 1 integrin (alpha 5 beta 1) can exist in different affinity states for its soluble ligand, fibronectin. The alpha 5 beta 1 expressed by the erythroleukemic cell line K562 binds soluble fibronectin with low affinity (Kd > 1 microM), but is induced to bind it with 20-fold higher affinity (Kd-54 nM) in the presence of the anti-beta 1 mAb 8A2. This activation seems to be due to direct antibody-induced change in the receptor that does not require intracellular signaling, and is a plausible basis for the 8A2-induced enhancement of beta 1-dependent adhesion to fibronectin and other immobilized ligands (Kovach, N. L., T. M. Carlos, E. Yee, and J. M. Harlan. 1992. J. Cell Biol. 116: 499-509). Fab fragments of 8A2 bind with higher affinity to alpha 5 beta 1 receptor that is occupied by the GRG-DSP peptide ligand suggesting that the antibody functions by stabilizing a high affinity (occupied) conformer of the receptor. A functional consequence of the affinity modulation is that soluble fibronectin (at physiological concentrations) occupies the high affinity receptors, and so becomes an effective inhibitor of adhesion to immobilized fibronectin. In contrast, the majority of low affinity receptors remain unoccupied and are still to mediate cellular adhesion.

Vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) are adhesive proteins induced on endothelium by cytokines. We examined the contribution of these adhesive proteins to human peripheral blood monocyte adherence to endothelium using transfected Chinese hamster ovary (CHO) cells stably expressing these proteins and monoclonal antibodies (MoAbs) to ELAM-1, VCAM-1, or CD49d/CD29 (VLA-4), the leukocyte receptor for VCAM-1. Monocytes bound to CHO cells transfected with cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was inhibited by MoAb to VCAM-1 or the alpha-chain of very late activation antigen-4 (VLA-4) (CD49d). Additive inhibition of adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocytes were pretreated with both MoAb to CD49d and MoAb to CD18, the common beta-chain of the leukocyte beta 2 integrin receptors. Adherence of monocytes to HUVE stimulated by recombinant human tumor necrosis factor-alpha was not reduced by MoAbs to CD18, CD49d, or ELAM- 1 when used singly, but combinations of these MoAbs produced significant inhibition. We conclude that multiple receptor-ligand systems are involved in monocyte adherence to endothelium.

Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC(50) approximately 1-2 microM) alpha(4)beta(1)-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the beta(1) integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates beta(1) integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, alpha(6)beta(1)-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.

Parvovirus B19 (B19) DNA was detected by dot blot hybridization in sera from 5 (17%) of 30 human immunodeficiency virus (HIV)-infected patients with hematocrits (HCT) ofor =24 and 4 (31%) of 13 HRV-infected patients with HCT ofor =20, suggesting that B19 is a reasonably common cause of severe anemia in HIV infection. The anemia promptly remitted after immunoglobulin therapy in 3 of 4 treated patients. The presence of IgM to B19, the clinical circumstance in which anemia developed, and the marrow morphology were poor predictors of chronic B19 infection. DNA hybridization studies of sera from 191 HIV-infected and 117 HIV-seronegative homosexual males attending a clinic in the Seattle area revealed that 1 (0.5%) and 2 (2%) samples, respectively, from the 2 groups contained B19. However, when assayed by polymerase chain reaction (PCR), 5% of the serum samples from HIV-infected persons and 9% from uninfected persons contained B19, although each had an HCT ofor =40. The data argue that anemia results from chronic high-titer B19 infection. Although a negative PCR assay excludes this diagnosis, DNA hybridization may be the more specific serum test.

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.