Targeting of IgMkappa antibodies to oligodendrocytes promotes CNS remyelination

Abstract

We previously identified the remyelinating activity of a natural IgMkappa oligodendrocyte-reactive autoantibody (SCH94.03), using a virus-induced murine model of multiple sclerosis. We now describe a second mouse IgMkappa monoclonal antibody (mAb) (SCH79.08) raised against normal mouse spinal cord homogenate, which reacts with myelin basic protein and also promotes remyelination. Because these two mAbs recognize different oligodendrocyte antigens, several previously identified oligodendrocyte-reactive IgMkappa mAbs (O1, O4, A2B5, and HNK-1), each with distinct antigen specificities, were evaluated and found to promote remyelination. In contrast, IgMkappa mAbs that did not bind to oligodendrocytes showed no remyelination. One of these, CH12 IgMkappa mAb, which shares variable region cDNA sequences with SCH94.03 except for amino acid differences in the complementarity-determining region 3 in both heavy and light chains, did not bind to oligodendrocytes and did not promote remyelination. The fact that multiple oligodendrocyte-reactive antibodies with distinct antigen reactivities induce remyelination argues against direct activation by a unique cell surface receptor. These findings are most consistent with the hypothesis that the binding of mAbs to oligodendrocytes in the lesions induces myelin repair via indirect immune effector mechanisms initiated by the mu-chain. Importantly, these studies indicate that oligodendrocyte-reactive natural autoantibodies may provide a powerful and novel therapeutic means to induce remyelination in multiple sclerosis patients.

Fig. 1.

Light micrograph demonstrating extensive CNS remyelination after treatment with SCH79.08, O1, O4, A2B5, and HNK-1. Sections are from the spinal cords of SJL/J mice chronically infected with TMEV. Note the CNS remyelination, characterized by abnormally thin myelin sheath as compared with axon diameter, in demyelinated lesions of mice treated with SCH79.08 (A), O1 (B), O4 (C), A2B5 (D), and HNK-1 (E). Note the demyelination without significant remyelination in mice treated with R24 (F), CH12 (G), and control IgMκ ABPC22 (H). Araldite-embedded sections were stained with 1%p-phenylenediamine (magnification, 875×).

Fig. 2.

Indirect immunofluorescence of cultured glial cells. Note the live surface staining of oligodendrocytes with SCH94.03 (A) and R24 (E) but the absence of surface staining with SCH79.08 (C). Also note intracellular staining of the cytoplasm of astrocytes with SCH94.03 (B) and SCH79.08 (D) but the absence of staining with R24 (F). Scale bar, 20 μm. Oligodendrocytes and astrocytes were isolated from telencephalon of newborn Sprague Dawley rats.

Fig. 3.

Protein antigen reactivity (A) and chemical hapten reactivity (C) of SCH79.08 are assessed by direct ELISA. Also shown are protein antigen reactivity (B) and chemical hapten reactivity (D) of control IgM (XMMEN-OE5). Abbreviations used in these panels: Ars, azophenylarsonate; BSA, bovine serum albumin;FL, fluorescein; HEL, hen egg lysozyme;KLH, keyhole limpet hemocyanin; MBP, myelin basic protein; NP, (4-hydroxy-3-nitrophenyl)acetyl; PC, azophenylphosphoryl-choline; PhoX, phenyloxazolone;RBC, red blood cells; TMA, azophenyltrimethyl ammonium; TNP, trinytrophenyl acetyl. No reactivity to these protein antigens or chemical haptens was detected with another control IgM mAb (TEPC 183) (data not shown).

Fig. 4.

Western blotting of TMEV proteins (A) and MBP (B). Proteins from purified TMEV and rabbit MBP (obtained from Sigma) were separated on 15% SDS polyacrylamide gels. Bound Ig was detected with alkaline phosphatase-conjugated secondary antibodies by using BCIP/NBT. Molecular weight markers are indicated in kDa at theright or left margin. A,Lane 1, Polyclonal rabbit anti-TMEV Ab; lane 2, SCH79.08; lane 3, O1; lane 4, O4; lane 5, A2B5; lane 6, HNK-1;lane 7, CH12; lane 8, R24; lane 9, control mouse IgM (MOPC 104E). Arrowsindicate TMEV capsid proteins. B, Lane 1, Control mouse IgM (MOPC 104E); lane 2, SCH94.03;lane 3, polyclonal rabbit anti-MBP Ab (obtained from Dako); lane 4, SCH79.08. Arrows indicate two MBP isoforms (21.5 and 18.5 kDa) recognized by SCH79.08.