Inhibition of Very Late Antigen–4 (VLA–4) Prevents Endotoxin–Induced Uveitis

Abstract

Abstract: : Purpose: Leukocyte adhesion to the vascular wall is a critical early step in the pathogenesis of inflammation and is mediated, in part, by VLA–4, an integrin expressed in leukocytes that binds to endothelial VCAM–1. In this study, we investigated the efficacy of anti–VLA–4 inhibition via a blocking antibody (Ab) in prevention of Endotoxin Induced Uveitis (EIU) in rats. Methods: EIU was induced in Lewis rats via footpad injection of lipopolysacharide (LPS). Immediately after the LPS administration, the animals were randomized in two groups and received a single intraperitoneal injection of 5mg/kg anti–VLA–4 Ab (n=11) or an isotype–matched IgG (n=13). Twenty–four hours after the EIU induction, severity of uveitis was evaluated in vivo using slit–lamp biomicroscopy and cell and protein measurement by aspiration of the aqueous fluid. Retinal leukocyte adhesion was quantified with FITC– lectin labeling and by counting of vitreous leukocytes in H&E–stained sections of paraffin–embedded eyes. Retinal VCAM–1 levels were evaluated by Western Blotting. Leukocytes from LPS–injected and control rats were isolated with a density gradient and leukocyte adhesion to rat endothelial cells was quantified using a static in vitro adhesion assay. Results: In vivo, VLA–4 Ab prevented all uveitis–induced inflammatory parameters. Clinical score and protein content in the aqueous fluid of VLA–4 treated animals were decreased by 45 % (p<0.001) and 21% (p<0.01) respectively compared to the control rats. Retinal leukostasis decreased by 68% (p<0.001), and leukocyte accumulation in the vitreous body decreased by 75% (p<0.01) in VLA–4 treated versus control eyes. Leukocytes derived from EIU animals showed increased adhesion to endothelial monolayers in vitro, which was effectively reduced by 70% with a VLA–4 blocking Ab (n=6, p<0.001).VCAM–1 expression in the retinas of control EIU animals was upregulated compared to normal controls. Conclusions: VLA–4 blockade prevents the development of intraocular inflammation in an animal model of EIU. These findings suggest that anti–VLA–4 might be a promising therapeutic agent for the treatment of inflammatory eye diseases.

Keywords: uveitis–clinical/animal model • retina • inflammation