Apcs Heparan Sulfates and Glycoprotein 96 in Encephalomyelitis in Vivo Interact with Experimental Autoimmune Amino Acid Copolymers That Alleviate Amino Acid Copolymers That Alleviate Experimental Autoimmune Encephalomyelitis in Vivo Interact with Heparan Sulfates and Glycoprotein 96 in Apcs

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS. One approved treatment for relapsing forms of MS is YEAK, a random copolymer of the amino acids tyrosine, glutamic acid, alanine, and lysine. YFAK, a second-generation copolymer composed of tyrosine, phenylalanine, alanine, and lysine, is more successful in treating experimental autoimmune encephalomyelitis, a mouse model of MS. Although originally designed and optimized based on the autoantigen myelin basic protein (MBP) and the MBP-derived peptide MBP85-99 presented to the MS-associated class II MHC molecule HLA-DR2, YEAK and YFAK also stimulate cytokine and chemokine production in APCs that lack class II MHC products. How YEAK and YFAK copolymers interact with APCs remains enigmatic. We used biotinylated YFAK to affinity-purify YFAK-interacting proteins from RAW264.7 cells and tested APCs from mice deficient in several of the newly identified interactors for their capacity to secrete CCL22 in response to YEAK and YFAK. We propose that initial contact of YFAK with cells is mediated mainly by electrostatic interactions, and find that interaction of YFAK with host proteins is strongly dependent on ionic strength. Cells deficient in enzymes involved in sulfation of proteins and proteoglycans showed strongly reduced binding of biotinylated YFAK. Lastly, cells stimulated with YFAK in the presence of heparin, structurally similar to heparan sulfates, failed to produce CCL22. We conclude that charge-dependent interactions of copolymers that alleviate MS/experimental autoimmune encephalomyelitis are critical for their effects exerted on APCs and may well be the main initial mediators of these therapeutically active copolymers. M ultiple sclerosis (MS) is a chronic inflammatory auto-immune disease that affects the CNS. CD4 + T cells that recognize Ags of the myelin sheath are held responsible for the inflammation and demyelination that characterize MS. A, K) n (hereafter referred to as YFAK) ameliorate experimental autoimmune encephalomyelitis (1–4) and other autoimmune diseases (5). Notwithstanding its heterogeneous composition, YEAK is one of the few compounds approved for clinical treatment of MS. YFAK and YEAK may suppress inflammation via regulatory and Th2-polarized CD4 + T cells that secrete IL-10 (1, 2, 6–8), via IL-10–producing B cells (9–11), or by modulating cytokine and chemokine secretion by dendritic cells and macrophages (12–14). Bone marrow–derived myeloid cells (15–17) and RAW264.7 cells exposed to YFAK and YEAK secrete CCL22, a chemoattractant for regulatory T and Th2 cells. Whereas the cellular targets cells have been defined especially for YEAK (15, 16), not much is known about the molecular …