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 α₄ integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that α₄ integrin-dependent leukocyte rolling and adhesion was inhibited with a β₁ integrin, but not a β₇ integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for α₄β₁ integrin in the vasculature. However, α₄β₁ 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 α₄ 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 α₄ integrin. Another ligand for α₄β₁ 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 α₄β₁ integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.