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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)

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.

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.

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.

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.