Integrin ␣ 4 ␤ 1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing ␣ 4 ␤ 1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670 -48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the ␣ 4 integrin.Cell adhesion to the vascular endothelium is important for inflammation, hemostasis, cancer cell metastasis, and hostparasite interaction. Integrins and their counterstructures, the cell adhesion molecules (VCAM-1 and ICAMs), 1 together with selectins and their counterstructures (cellular mucins) determine the cell adhesive properties in these processes (1). The adhesion between endothelial cells and leukocytes is regulated by changes in the number of interacting adhesion molecules due to the difference in the expression level, molecular trafficking, and/or internalization; changes in topographical distribution due to clustering, dimerization, and other forms of molecular assembly; and changes in affinity of the individual molecules to their counterstructures (2-7). ␣ 4 ␤ 1 integrin (very late antigen-4 (VLA-4), CD49d/CD29) is one of the integrins that can mediate initial capture, rolling, and firm cell attachment to the endothelial cells (8, 9). VLA-4 is expressed on several classes of blood cells. It mediates binding to the CS-1 domain of fibronectin and to the vascular cell adhesion molecule 1 (VCAM-1), an immunoglobulin superfamily member induced by cytokines on endothelium (10, 11). ␣ 4 ␤ 1 -Integrin adhesive properties can be modulated by cytokines and chemokines, but the mechanism controlling the regulation of integrin avidity is poorly understood. In particular, it has been suggested that the changes in VLA-4-dependent adhesion are due to either th...

Summary. The success of all-trans retinoic acid (ATRA) in the therapy of acute promyelocytic leukaemia (APL) has received increased attention. Unfortunately, life-threatening multiorgan failure commonly occurs, i.e. retinoic acid syndrome, and is thought to be the result of organ in®ltration by leukaemic cells. We hypothesized that ATRA-induced differentiation of APL cells leads to adhesion receptor alterations responsible for leucocyte extravasation from the blood into tissue. Changes in adhesive properties of the APL cell line NB-4 in response to ATRA were investigated using a parallel plate¯ow chamber under conditions that recapitulate physiologic¯ow conditions. Untreated NB-4 cells initially tether and roll on activated human umbilical vein endothelial cell monolayers using a combination of Eselectin, P-selectin and a4 integrin. After ATRA treatment, > 80% of initial NB-4 cell attachment to endothelial cells was E-selectin dependent. Stable arrest (®rm adherence) of NB-4 cells on activated endothelium was also altered by ATRA treatment. Untreated NB-4 cells used a4 integrin to arrest on endothelium, but b2 integrin dependent arrest was induced by ATRA. With the acquisition of b2 integrin function, ATRA-treated cells acquired the ability to transmigrate through activated endothelium. Thus, ATRA dramatically altered the adhesion phenotype on NB-4 cells: ATRA induced rolling largely attributable to E-selectin, abrogated a4 integrin dependent rolling, and promoted acquisition of b2 integrin dependent ®rm adherence and transmigration. These ®ndings represent novel cellular and differentiation effects of ATRA, and, to our knowledge, are the ®rst demonstration that a therapeutic agent differentially regulates a4 and b2 integrin on the same leucocyte.

The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail. We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer. We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils. Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below ∼20 sites/μm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia. The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear. Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates. These differences may account in part for the increase in eosinophil accumulation in allergic diseases.

While eosinophils rapidly and specifically leave the circulation after allergen challenge of atopic asthmatics, the remaining circulating eosinophils are not activated.