PHORBOL ESTER STIMULATION INCREASES SICKLE ERYTHROCYTE ADHERENCE TO ENDOTHELIUM - A NOVEL PATHWAY INVOLVING ALPHA(4)BETA(1) INTEGRIN RECEPTORS ON SICKLE RETICULOCYTES AND FIBRONECTIN

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and re ceptors on sickle erythrocytes. It has previously been reported that s ickle reticulocytes express the alpha(4) beta(1) integrin receptor and bind to cytokine-activated endothelium via an alpha(4) beta(1)/vascul ar-cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other r oles for alpha(4) beta(1) in sickle-cell adherence, the ability of act ivated alpha(4) beta(1) to promote adhesion to endothelium via a ligan d different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm(2). Preincubat ion of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increa sed adherence of sickle cells eightfold as compared with untreated sic kle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta(1) antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blo od cell (RBC) adhesion to endothelium. Anti-alpha(4) antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle -cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha(4) beta(1), was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PD Bu-treated sickle cells was completely kinhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endo thelium using an antifibronectin antibody in enzyme-linked immunosorbe nt assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/- 17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adheren ce 86% +/- 8%. Taken together, these data suggest that endothelial-bou nd fibronectin mediates adherence of PDBu-treated sickle cells. Interl eukin-8 (IL-8), a chemokine released in response to bacterial infectio n, viral infection, or other injurious agents, and known to activate i ntegrins, also increased adherence of sickle erythrocytes to endotheli al cells via fibronectin. This novel adherence pathway involving sickl e-cell alpha(4) beta(1) activated by PDBu or IL-8 may therefore be rel evant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observation s describe ways in which inflammation and immune responses cause vasoo cclusive complications in sickle-cell disease. (C) 1996 by The America n Society of Hematology.