Nicholas L. Kovach

A Biochemical Characterization of the Binding of Osteopontin to Integrins αvβ1 and αvβ5

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.

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating β1 integrin

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 alpha4beta1 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 alpha4beta1 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 beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 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.

Regulation of α4 integrin–mediated adhesion of human eosinophils to fibronectin and vascular cell adhesion molecule-1

Abstract Background: Eosinophils selectively accumulate at sites of allergic inflammation. Their recruitment is dependent on both the expression and functional activity of cell adhesion molecules. How the functional activity of cell adhesion molecules on eosinophils is regulated is poorly understood. Objective: Our objective was to examine the functional activity of α 4 integrins on human eosinophils and its regulation by various agents. Methods: Function of α 4 integrins on human eosinophils was examined by testing adhesion to immobilized fibronectin and vascular cell adhesion molecule-1 (VCAM-1) in the presence or absence of a monoclonal antibody (mAb) (8A2) that activates β 1 integrin function. Results: Spontaneous eosinophil adhesion to VCAM-1 was enhanced by 8A2, but adhesion to fibronectin could only be detected in the presence of 8A2. Concentrations of 8A2 that were approximately 100-fold less than saturating induced maximal eosinophil adhesion. Adhesion to VCAM-1 in the presence of 8A2 was effectively inhibited by α 4 and β 1 integrin mAbs; β 7 mAb had partial inhibitory activity. Connecting segment-1 peptide and α 4 mAb blocked 8A2-dependent fibronectin binding; β 1 , β 2 , and β 7 integrin mAbs had partial inhibitory activity. Eosinophils obtained from bronchoalveolar lavage fluids and blood eosinophils stimulated with IL-5, platelet-activating factor, or RANTES displayed increased β 2 integrin–dependent, not α 4 integrin–dependent, attachment. Spontaneous adhesion of eosinophils to VCAM-1 was significantly reduced by the tyrosine kinase inhibitor tyrphostin B46 (inhibitory concentration of 50% ≅ 20 μmol/L); this effect was reversed by 8A2. Conclusions: The functional activity of integrins on eosinophils can be positively and negatively regulated. Altered integrin avidity may influence eosinophil recruitment in vivo. (J Allergy Clin Immunol 1997;99:648-56.)

Activation of beta1 integrins on CML progenitors reveals cooperation between beta1 integrins and CD44 in the regulation of adhesion and proliferation.

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the α4β1 and α5β1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, β1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of α4, α5, and β1 integrins and CD44 are present on CML and normal CD34+ cells. We have previously demonstrated that β1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the β1 integrin activating antibody, 8A2, and demonstrated a role for the α5β1 integrin in this phenomenon. Since the integrin α4β1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between β1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN, but also to α4β1, α5β1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the α5, α4, or β1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34+ HLA-DR+ cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of β1 integrins on CML Ph+ CFC not only results in upregulation of β1 integrin-dependent adhesion and adhesion-mediated inhibition of proliferation, but also in the restoration of cooperation between β1 integrins and CD44. These studies suggest that decreased β1 integrin avidity may also affect the function of the proteoglycan adhesion receptor CD44, both of which may contribute to the abnormal circulation and expansion of malignant progenitors in CML.

Regulation of β1-Integrin Function in Cultured Human Vascular Smooth Muscle Cells

Abstract Avidity modulation and function of β1-integrin receptors in cultured human vascular smooth muscle cells (SMCs) were investigated using monoclonal antibody (mAb) 8A2, which binds to the β1 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 β1 integrin was assessed with mAb 15/7, which binds to an activation-dependent epitope on the β1 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 β1 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 PDG...

Role of Protein Synthesis and CD 11 /CD 18 Adhesion Complex in Neutrophil Emigration into the Lung

The mechanism of neutrophil (PMN) emigration into the lung may be stimulus-dependent. This study examined PMN emigration in the lung induced by intratracheal instillation of lipopolysaccharide (LPS), Streptococcus pneumoniae (S. pneu) organisms, supernatant from S. pneu incubated with alveolar macrophages (AM phi), Escherichia coli (E. coli) organisms, or phorbol myristate acetate (PMA). Rabbits were pretreated with either the CD18 monoclonal antibody (MAb) 60.3, the protein synthesis inhibitor cycloheximide (Cx), or, in one case, both. Animals were then given one of the above stimuli to elicit PMN emigration. Four hours after the stimulus was instilled, animals were killed and total and differential cell counts were performed on bronchoalveolar lavage (BAL) fluid. PMN emigration in response to PMA was virtually abolished by MAb 60.3, but was not significantly inhibited by Cx. Emigration induced by LPS was inhibited by 80% by either MAb 60.3 or Cx, and greater than 94% when MAb 60.3 and Cx were given simultaneously. Emigration in response to E. coli organisms was 80% inhibited by MAb 60.3. Emigration induced by S. pneu was approximately 50% inhibited by MAb 60.3, but was greater than 90% blocked by Cx. The MAb 60.3 had approximately the same effect on PMN emigration toward the supernatant from co-incubation of AM phi with S. pneu as it did toward live S. pneu. It is concluded that the mechanism of PMN emigration into the lung is stimulus-dependent. The CD18-dependent mechanism is responsible for the majority of the emigration in response to PMA, E. coli LPS, and E. coli organisms. S. pneu and supernatant from S. pneu + AM phi produce a CD18-independent pathway. These data suggest the requirement for de novo protein synthesis for PMN emigration in response to LPS and S. pneu, but not for PMA-induced emigration.