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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.01-1023fje. |
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2
Departments of
* Immunology and
Neurology, Mayo Medical and Graduate School, Rochester, Minnesota, USA
2Correspondence: Department of Immunology, Mayo Clinic, 428 Guggenheim Bldg., 200 1st Street SW, Rochester, MN 55905, USA. E-mail:rodriguez.moses{at}mayo.edu
SPECIFIC AIMS
Glatiramer acetate (GA) is an immunogenic mixture of synthetic peptides that is an effective treatment for multiple sclerosis (MS). Our objective was to the study the independent effects of GA-reactive lymphocytes and antibodies on demyelination and remyelination in the Theiler’s virus model of MS.
PRINCIPAL FINDINGS
1. Active immunization with glatiramer acetate does not alter CNS pathology in chronically diseased mice
SJL mice infected with TMEV develop lifelong viral persistence, inflammatory demyelination of the spinal cord, and progressive neurological deficits. We first tested whether active immunization with GA would affect neuropathology in mice with established disease by immunizing mice during the chronic stage of infection (27–52 wk postinfection), when the extent of white matter demyelination (lesion load) normally is stable and the extent of spontaneous remyelination is low. GA was administered subcutaneously either alone or with incomplete Freund’s adjuvant (IFA). Injections of 0.1 mg were repeated 4–8 times over treatment periods of 6–11 wk. Quantitative morphometry of spinal cord sections showed that lesion load was unaffected by immunization with GA. On average, 10% of the white matter was demyelinated after treatment with PBS, IFA, or GA. Lesions were characterized by extensive primary demyelination and infiltration with macrophages, lymphocytes, and activated glia (Fig. 2B
). Oligodendrocyte-mediated remyelination, characterized by abnormally thin myelin usually at the edge of lesions, was quantified in tissue sections and expressed as a percentage of lesion area. The extent of remyelination was similar among mice treated with PBS, IFA, or GA, averaging 
10% of the total lesion area. Thus, GA immunization did not quantitatively alter neuropathology in mice with established disease.
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2. Adoptive transfer of glatiramer acetate T cells does not alter CNS pathology
To address the possibility that GA-reactive T lymphocytes could affect disease independent of other effects of GA immunization, we adoptively transferred GA-reactive lymphocytes to chronically diseased mice. GA-reactive lymphocytes were generated in uninfected SJL mice by immunization with GA and IFA for 7 days. Lymph node-derived cells were stimulated in vitro with GA for another 5 days, then Ficoll-isolated lymphocytes were injected intravenously to mice infected for 24 wk (15 million cells/mouse). Chicken egg ovalbumin-reactive lymphocytes were generated similarly in other uninfected mice as adoptively transferred control cells. Thymidine incorporation assays performed on the freshly isolated lymph node-derived cells showed stimulation indices of 7.9 for GA lymphocytes and 8.7 for ovalbumin lymphocytes after 4 days of in vitro stimulation. Morphometric analyses performed 5 wk after adoptive transfer showed no statistically significant differences in lesion load or remyelination among the experimental groups. Lesions accounted for 10% of the sampled area and remyelination accounted for less than 10% of the lesion area in all groups. Thus, freshly activated GA-reactive lymphocytes by themselves neither worsened nor improved disease pathology.
3. Glatiramer acetate immunization generates polyreactive antibodies
All MS patients treated with GA develop GA antibodies, but the characteristics of these antibodies are almost completely unexplored. To study the specificity and role of GA antibodies in the Theiler’s model, we purified GA antibodies (GA Ig) by GA affinity chromatography from the serum of uninfected SJL mice immunized with GA plus IFA. Protein A/G-purified IgG from SJL mice (SJL Ig) or commercial pooled mouse serum (Pooled Ig) served as control immunoglobulin. Both naive GA Ig and biotinylated GA Ig strongly recognized GA by ELISA and Western blot. By ELISA, GA was detectable by antibody at concentrations as low as 8 ng/ml. Quantitative isotyping showed that IgG1 accounted for 70% of the purified GA Ig., followed by IgG2 (18%). GA IgM was nearly undetectable, and other isotypes accounted for 5% or less of the total Ig.
Cross-reactivity of purified GA Ig to CNS antigens was examined first by immunostaining rat brain-derived glia in culture. Two types of cells were found to bind GA IgG. Some were round, process-free, located at the top surface of the cultures, and belonged to the microglial lineage as suggested by consistent colabeling with isolectin B4. The other cells that bound GA IgG also were process-free but colabeled with A2B5, consistent with early lineage oligodendrocytes. Among the cells that did not bind GA IgG were activated microglia attached strongly to the culture substrate, process-bearing A2B5-positive oligodendrocytes, differentiated oligodendrocytes (01- O4-, or MBP-positive), and astrocytes (glial fibrillary acidic protein (GFAP)-positive). In peritoneal macrophage cultures, GA IgG bound the surface of a subset of cells, but also recognized intracellular antigens in all cells after fixation and permeabilization.
CNS reactivity also was examined by immunostaining tissue sections from frozen spinal cords. Biotinylated GA IgG cross-reacted extensively in sections compared to low background staining with pooled mouse Ig or SJL Ig. Notable staining by GA IgG in lesioned white matter included perivascular-infiltrating cells, which consist of macrophages, microglia, and lymphocytes. GA IgG also recognized a subset of glia that had an abundance of thin, randomly oriented processes and often colabeled with the astrocyte marker GFAP. More noticeable in unlesioned white matter were round cellular profiles with the morphology of oligodendrocytes. The absence of myelin staining is consistent with binding to early oligodendrocyte progenitors. Other prominent reactivities included neuronal cell bodies in the gray matter and peripheral nerve, which were also present in sections from uninfected mice. Thus, in CNS tissue sections, GA Ig exhibited extensive cross-reactivity with endogenous antigens.
4. Glatiramer acetate antibodies promote remyelination
The role of GA antibodies in vivo, independent of other effects of immunization, was tested by adoptive transfer of GA Ig. Chronically diseased mice were treated for 5–6 wk with GA Ig or normal Ig. Each mouse received 5 weekly injections for total antibody doses of 0.5 or 1.5 mg (0.1–0.3 mg/injection). None of the antibody treatments affected lesion load compared with PBS-treated mice (Fig. 1
), indicating that GA antibodies were not pathogenic. In contrast, adoptively transferred GA Ig had a positive effect on oligodendrocyte-mediated remyelination (Fig. 1B
). At a total dose of 1.5 mg, the mean area of remyelination was increased 2.4-fold by GA Ig, which was significant by t test (P=0.02 vs. PBS) and one-way ANOVA (P=0.04 comparing PBS, SJL Ig, pooled Ig, and 1.5 mg GA Ig). Based on axon densities within remyelinated regions, the 2.4-fold increase in remyelination was equivalent to the repair of 7000 to 21,000 internodes in the sections we analyzed. In some lesions, the GA Ig-promoted repair was nearly complete (Fig. 2
D). Remyelination tended to increase with 0.5 mg GA Ig treatment but without statistical significance. Normal mouse IgG administered at identical doses did not enhance remyelination. Thus, polyclonal GA antibodies stimulated lesion repair independent of the full effects of immunization.
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CONCLUSIONS
In this study, we introduce a novel mechanism by which GA might exert its therapeutic effect in multiple sclerosis. The rationale for this study was the recently published observation that all patients treated with GA (Copaxone®) develop antibodies to the compound. Patients who remain relapse-free after 18–24 months of treatment have statistically higher GA antibody titers than those who develop relapses, consistent with a protective role for the antibodies. Our findings suggest that demyelinated lesion repair is a possible mechanism as to how GA antibodies could be protective.
GA antibodies join a list of immunoglobulins that enhance remyelination in Theiler’s virus model. The list includes polyclonal antibodies to MBP, pooled human IgG (IVIg) and IgM, and several monoclonal murine and human IgMs. GA Ig has two characteristics in common with other remyelination-promoting antibodies: extensive cross-reactivity to both cell surface and intracellular antigens, and binding to oligodendrocyte lineage cells. The multireactivity makes it difficult to determine how the antibodies promote myelin repair, but several mechanisms might be important. GA antibodies may stimulate glia or immune cells to produce factors that promote lesion repair. They might deactivate cytokines, complement, or other pathogenic factors. They may opsonize cellular debris, thereby creating a better environment for oligodendrocyte redifferentiation. Finally, direct binding to early oligodendrocytes may trigger expansion of oligodendrocytes or myelinogenesis. Thus, GA Ig may resemble IVIg in being a heterogeneous mixture of immunoglobulins with a multitude of therapeutic effects in vivo.
We propose that generation of polyreactive antibodies is one of many mechanisms rather than the sole mechanism by which GA is therapeutic in MS (Fig. 3
). Down-regulation of pathogenic lymphocytes is another likely mechanism. GA blocks activation of Type 1 helper T lymphocytes, most notably through displacement of stimulatory peptides from class II MHC and through generation of suppressor lymphocytes. Changes in macrophage and dendritic cell function and lymphocyte migration have also been described. Such a diversity of effects is at least partly attributable to the heterogeneous nature of GA—a mixture of potentially millions of different, randomly synthesized peptides averaging 70 amino acids in length. Sorting out what effects of GA are important at different stages or in different types of MS is an important future goal. Different mechanisms may complement each other, although it is also possible that different mechanisms might counteract each other. The latter possibility could explain our finding that adoptively transferred antibodies but not complete immunization promoted remyelination. MS is a complex disease, and tailoring treatment regimens or peptide formulations to emphasize one effect of GA over another may be useful.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1023fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.01-1023fje 
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R. R. Kreitman and F. Blanchette On the horizon: possible neuroprotective role for glatiramer acetate Multiple Sclerosis, June 1, 2004; 10(1_suppl): S81 - S89. [Abstract] [PDF]
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