α4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation

Rats immunized with Mycobacterium butyricum in Freund's adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an alpha(4) integrin Ab, Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that or, integrin-dependent leukocyte rolling and adhesion was inhibited with a beta(1) integrin, but not a beta(7) integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for alpha(4)beta(1) integrin in the vasculature. However, alpha(4)beta(1) integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature, To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient alpha(4) integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95-100%, Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via alpha(4) integrin. Another ligand for alpha(4)beta(1) integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that alpha(4)beta(1) integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.