Macrophage-Specific Chemokines Induced via Innate Immunity by Amino Acid Copolymers and Their Role in EAE

Abstract

The random amino acid copolymer poly(Y,E,A,K)n (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)n with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role. Here, both CCL22 (a Th2 cell chemoattractant) in large amounts and CXCL13 in much smaller amounts are shown to be secreted after administration of YFAK to mice and to a smaller extent by YEAK parallel to their serum concentrations. Moreover, bone marrow-derived macrophages secrete CCL22 in vitro in response to YFAK and to higher concentrations of YEAK. Strikingly, these chemokines are also secreted into serum of MHC Class II −/− mice, indicating that an innate immune receptor on these cells also has an important role. Thus, both the innate and the adaptive immune systems are involved in the mechanism of EAE amelioration by YFAK. The enhanced ability of YFAK to stimulate the innate immune system may account for its enhanced efficacy in EAE treatment.

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

“…Glutamic acid was replaced by phenylalanine to improve binding to Class II MHC molecules, based on the known binding motifs of the autoantigenic peptide MBP85-99 to HLA-DR2 (DRB1*1501) (3). Only recently attention was drawn to Class II MHC-independent effects of the YFAK and YEAK copolymers on innate immune cells, such as monocytes, macrophages and dendritic cells (12, 15-17). We confirmed the earlier observation that exposure to YFAK and YEAK stimulate RAW264.7 cells (Fig.…”

“…YFAK was synthesized as described in (12). The random copolymers were biotinylated with a twofold molar excess (assuming an average MW for the copolymers of 5500 Da (YFAK) and 7000 Da (YEAK)) of Sulfo ChromaLink Biotin reagent (Solulink, Inc., San Diego) in 50mM sodium phosphate pH=7.6, 75mM sodium chloride and 37.5% (v/v) acetonitrile for 14 hours at room temperature.…”

“…ELISA assays were done as described (12). In short, 500.000 cells were stimulated in 250μl medium containing different concentrations of stimulant.…”

“…The random amino acid copolymers Copaxone® (Copolymer-1, glatiramer acetate, poly(Y, E, A, K) n , called YEAK) and poly(Y, F, 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 T H 2-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 and T H 2 helper T-cells.…”

“…Whereas the cellular targets cells have been defined especially for YEAK (15, 16), not much is known about the molecular target(s) and mechanism(s) of action of these copolymers. They have been variously proposed to be presented via Class II MHC products or to engage TLRs, but Class II MHC-deficient antigen presenting cells still respond to copolymers (12, 16, 17). Here we sought to identify molecular target(s) by searching for proteins and other macromolecules that interact with biotinylated versions of YFAK and YEAK.…”