Abstract
In multiple sclerosis (MS), demyelinated CNS lesions fail to sufficiently remyelinate, despite the presence of oligodendrocyte precursor cells (OPCs) capable of differentiating into mature oligodendrocytes. MS lesions contain damaged myelin debris that can inhibit OPC maturation and hinder repair. rHIgM22 is an experimental human recombinant IgM antibody that promotes remyelination in animal models and is being examined in patients with MS. rHIgM22 binds to CNS myelin and partially rescues OPC process outgrowth on myelin. Since rHIgM22 does not affect OPC process outgrowth in vitro on permissive substrate, we examined the possibility that it acts by enhancing phagocytic clearance of myelin debris by microglia. In this study, we tested if rHIgM22 binding could tag myelin for microglial phagocytosis. A mouse microglial cell line and primary rat microglia were treated with myelin and rHIgM22 and assayed for myelin phagocytosis. We found that: 1) rHIgM22 stimulates myelin phagocytosis in a dose-dependent manner; 2) rHIgM22-mediated myelin phagocytosis requires actin polymerization; and 3) rHIgM22-stimulation of myelin phagocytosis requires activity of rHIgM22 Fc domain and activation of Complement Receptor 3. Since myelin inhibits OPC differentiation, stimulation of phagocytic clearance of damaged myelin may be an important means by which rHIgM22 promotes remyelination.
Highlights
Activation of the immune system is believed to be one of the main causes of many neurodegenerative disorders
To test if rHIgM22 can promote clearance of myelin debris, BV-2 cells were serum starved for two hours and treated with pHrodo-labeled myelin and rHIgM22 followed by monitoring pHrodo signal over the course of 24 hours (Fig. 1a,b)
In order to verify that increases in pHrodo signal and intracellular cyclic-nucleotide 3′-phosphodiesterase (CNPase) in response to rHIgM22 treatment can be attributed to phagocytic uptake of myelin, we demonstrated complete absence of both pHrodo and CNPase signal in the presence of Cytochalasin D (CytoD), regardless of IgM treatment, indicating a requirement for intact cytoskeletal function for all forms of uptake of extracellular myelin
Summary
Activation of the immune system is believed to be one of the main causes of many neurodegenerative disorders. While OPCs would appear to be good candidates for playing a central role in the remyelinating process(es) induced by rHIgM22, purified OPC in vitro cultures do not appear to respond to rHIgM22 treatment[5]. Instead, mixed glial cultures, which consist of astrocytes, OPCs, maturing oligodendrocytes (OLs) and microglia are required to detect cellular responses to rHIgM22 in vitro[5]. Under these conditions, OPCs show enhanced BrdU incorporation and Ki-67 expression after a 48-hour treatment. OPCs show enhanced BrdU incorporation and Ki-67 expression after a 48-hour treatment These findings suggest that other cell types may be required for rHIgM22 to exert its effect(s). Given the ability of rHIgM22 to bind CNS myelin, we decided to explore if rHIgM22 could act as a classical IgM and aid in clearance of myelin debris, potentially allowing for disinhibition of OPC differentiation
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