The Interaction of the RGD Sequence of Vitronectin with Integrin in Membrane Attack Complex (MAC) Mediated Synovial Damage in Juvenile Idiopathic Arthritis (JIA)

Purpose: Circulating immune complex (CIC) mediated activation of complement is a key pathologic event in synovial tissue of JIA patients. Complement receptors and regulatory proteins are not only crucial for the development of innate responses, but are also important in adaptive immunity. Ligation of CD46 by its ligand results in a T cell phenotype that produces high levels of Granzyme B (GrB) at the site of tissue inflammation. GrB cleaves vitronectin bound to soluble MAC, exposing the Arg-Gly-Asp (RGD) sequence that interacts with cell surface integrin, which is also up-regulated in tissue inflammation. We evaluated the role of the interaction of complement regulatory protein vitronectin with cell surface integrin in inflammatory synovium of JIA.
Methods: Synovial specimens were obtained from two patients with active JIA. Cryostat sections were made, and incubated with primary monoclonal antibodies to vitronectin, integrin, MAC, and GrB. Subsequently the specimens were incubated with either FITC or TRITC conjugated clone specific anti-Ig. The tissue sections were examined using a fluorescence microscope and images were captured with a camera attachment.
Results & Conclusion: H&E staining revealed inflammatory synovium pathology. The inflammatory regions revealed dense deposits of MAC. The other regulatory proteins i.e. vitronectin, integrin, and GrB were co-localized alongside of MAC. The presence of these molecules together in inflamed tissue points to their interaction in development of diseased pathology. The strongest staining was observed in the synovial lining-like layer and in vascular endothelial cells. Integrins act as cell adhesion receptors that are important for cell anchorage and polarity. Vitronectin, a serum plasma inhibitor of MAC, upon cleavage by Grb exposes the RGD sequence that interacts with integrin on the cell surface. T cells activated via CD46 release an abundance of GrB at the inflammatory site which cleaves Vitronectin. We propose that in inflamed synovium, a critical event is the interaction of the MAC-CIC-vitronectin (exposed RGD) with the over expressed cell surface integrin, which is due to the proinflammatory cytokine profile. This interaction may be necessary for the transfer of MAC to the cell membrane. In our previous studies, we have shown two to three fold higher amounts of vitronectin associated with MAC. The participation of vitronectin in MAC transfer could lead to cell damage, and sublytic levels of MAC will participate in matrix re-organization through signaling cascades, i.e. MAPK, PI3/Akt, S6kinase etc. We have studied the presence of these key molecules in synovial tissue of JIA patients, demonstrating their co-localization. In order to better understand the process of MAC mediated tissue damage, we are currently developing an in vitro model to study the interaction of MAC-CIC-vitronectin with integrin in synovial tissue.

 M.R. Reed, None; A.K. Chauhan, ProGen Biologics, L.L.C., 4; T.L. Moore, None.