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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 19, 2003 as doi:10.1096/fj.03-0606fje. |
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Servicio
* Bioquímica-Investigación and
Neurología, Hospital Ramón y Cajal, Madrid, Spain; and
Centro Nacional de Biotecnología, Madrid, Spain
2Correspondence: Servicio Bioquímica-Investigación Hospital Ramón y Cajal Ctra. Colmenar km 9.1, Madrid 28034, Spain. E-mail: alberto.alcazar{at}hrc.es
SPECIFIC AIMS
Oligodendrocyte precursor cells (OPCs) are extremely efficient at remyelination, persist in the adult human central nervous system (CNS) and can proliferate; however, the failure to remyelinate is a pathological characteristic of the human demyelinating disease multiple sclerosis (MS), which suggests that these cells are ineffective. In the present report we studied why does remyelination fail and an investigation was made to see whether OPCs present antigens that might act as targets for antibodies in the cerebrospinal fluid (CSF) from MS patients, preventing remyelination.
PRINCIPAL FINDINGS
Current evidence suggests that OPCs, known as O-2A progenitor cells are efficient at myelin repair, either spontaneously or after transplantation into the CNS. In culture, OPCs have a typical bipolar morphology, show immunoreactivity for A2B5 antibody (A2B5+) and most of them terminally differenciate into postmitotic oligodendrocytes. OPCs have been identified in perinatal and adult rodent brain and have also been described in adult human CNS. These human cells are in fact A2B5+ cells, immunophenotypically similar to their counterparts in cultured rodent cells.
1. Antibodies in CSF from MS patients recognize an antigen on OPCs
Cultures prepared from rat cerebral hemispheres contain substantial numbers of OPCs. Cell cultures were prepared from 17 to19-day-old rat embryos and OPCs were phenotypically identified by their characteristic bipolar morphology and A2B5+ immunoreactivity. Cells from 6-day cultures were fixed and incubated with individual CSF samples from control and MS patients and A2B5 antibody. CSF antibodies from 17 control patients did not label OPCs or weakly labeled a small number of them (range 0–6±1%). However, most of the OPCs (71±2%) were labeled with different fluorescence intensity by antibodies from 28 out of 33 MS patients. IgG from blood donors were also used as controls and they did not bind to OPCs. Cell extracts obtained from 6-day cultures were used for antigen detection by Western blot. Antibodies in CSF from 26 out of 33 MS patients detected a 82-kDa antigen, while CSF from control samples showed no reaction. In MS patients, the fluorescence intensity of bound antibodies per OPC and the quantification of 82-kDa antigen detected closely correlated (r=0.923, P<0.0001).
2. Identification of the antigen as heat shock protein 90 (Hsp90)
Antigens from cell extracts were immunoprecipitated with purified IgG from different MS CSF pools. Three proteins with apparent molecular masses of 82-, 70- and 42-kDa were detected (Fig. 1
A) and the 82-kDa spot was identified by MALDI-ToF-MS as heat shock protein 90 (Hsp90) ß (Fig. 1B
), a protein of 83,1 kDa and 5.1 pI according to the rat sequence. MS CSF antibodies reacted with the identified Hsp90 in the precise 82-kDa position on immunoblots of immunoprecipitates (Fig. 1C
). Monoclonal anti-Hsp90 antibody recognized OPCs on cell bodies in immunofluorescence assays (Fig. 1D
) and also detected the Hsp90 protein in 82-kDa position in immunoblots (Fig. 1E
), in a fashion similar to antibodies from MS patients. Competitive assays either co-labeling cultured cells in immunofluorescence assays or co-incubating immunoblots of cell extracts with both MS CSF antibodies and monoclonal anti-Hsp90 antibody, decreased the human IgG label on OPCs, (which remained only as a trace) and showed a lack of the 82-kDa antigen detection respectively. Antibodies in MS CSF against Hsp90 were purified by immunoaffinity. These immunopurified antibodies specifically detected the 82-kDa Hsp90 antigen on immunoblots (Fig. 1E
) and labeled OPCs in immunofluorescence assays (Fig. 1F
). MS CSF adsorbed against 82 kDa Hsp90 weakly detected the antigen in immunoblots and decreased the labeling on OPCs. Cell extracts from cell cultures were subjected to two-dimensional electrophoresis (2-DE) and immunoblot. Monoclonal anti-Hsp90 and polyclonal anti-Hsp90ß antibodies recognized five Hsp90 isoforms in 82-kDa position (Fig. 1G
) and antibodies from MS patients recognized two spots that precisely corresponded to two of the identified Hsp90ß isoforms (Fig. 1G
). Antibodies from MS patients also recognized human Hsp90 in immunoblots (Fig. 1H
).
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3. Hsp90 antigen localization on OPC surface
Cultured cells were fixed without a permeabilization procedure and thus, the Hsp90 antigen detected in immunofluorescence experiments was shown to have an extracellular membrane location. A subcellular fractionation was carried out and the immunoblotting analysis of subcellular fractions using antibodies from MS patients and monoclonal anti-Hsp90 antibody detected the antigen in the membrane fraction. Furthermore, living cultured cells were incubated with purified IgG from OND control and MS patients. The cells were then fixed and stained with A2B5 antibody. Antibodies from MS patients labeled 42 ± 8.3% of OPCs whereas antibodies from control patients labeled no cells. Polyclonal anti-Hsp90ß antibody also detected the antigen on 34 ± 6% of OPCs in living cells as did the IgG from MS patients.
4. Recognition of the Hsp90 antigen on OPC surface by antibodies from MS leads to complement activation and extinction of the OPC population
Living cells from 6-day cultures were incubated with purified IgG from MS patients in the presence of CSF for complement activation. The cells were then fixed and stained with A2B5 antibody and anti-human C5b9 antibody for detection of complement activation. Incubation of living cells with IgG and CSF from MS patients led to a significant extinction of the OPC population (516±215 OPCs/cm2). This did not occur when purified IgG and CSF from OND control patients or when IgG adsorbed against Hsp90 and CSF from MS patients were used (2021±230 OPCs/cm2, P<0.001 and 1830±149 OPCs/cm2, P<0.01, respectively). Three-dimensional projection of confocal images of MS CSF IgG/C5b9 co-positive OPCs located the human IgG binding site on the surface of these cells, co-localized with the complement label.
CONCLUSIONS AND SIGNIFICANCE
This report demonstrates that CSF from MS patients contain antibodies that specifically recognize an antigen—identified as Hsp90ß—on OPCs. The binding of antibodies from MS patients to the OPC surface led to complement activation and significant extinction of the OPC population. An immune attack on OPCs is one of the potential mechanisms that could compromise MS lesion remyelinating capacity. The role of Hsp in MS has emerged mostly from their high immunogenicity in mammals. Certain families of Hsp (e.g., Hsp60 and Hsp70) have been shown to elicit strong immunological reactions. Further, altered expression of these Hsp and Hsp90 in MS lesions has been described and very recently a microarray analysis has identified specific overexpression of the Hsp90 gene in active MS lesions. Hsp90 corresponds to a family of 90- to 82-kDa proteins highly conserved in mammals. Thus, Hsp90ß (Hsp84) is a protein with a 98% of homology between human and rat. Hsp90 has been located on cell surfaces of mouse tumor cell lines as well as microglial cells in Alzheimer’s disease. The expression of Hsp90 on cell surface could represent a target of the immune response, which has been reported in some diseases such as autoimmune forms of rheumatoid arthritis or in breast cancer. Antibodies against Hsp90 expressed (or overexpressed) on OPCs may play a pathogenic role leading to the exhaustion of these cells. This could contribute to the poor remyelination observed in MS lesions. These findings are in keeping with a recent report that describes a lower density of OPCs in MS lesions compared with non-lesion areas. In conclusion, this investigation demonstrates the potential biological roles of Hsp90 and anti-Hsp90 antibodies in the etiology of the human demyelinating disease MS (Fig. 2
). The development of strategies that enhance remyelination requires an understanding of the mechanisms that impair endogenous remyelination. The identification of anti-Hsp90 antibodies in CSF from MS patients that specifically damage OPCs is a step forward in this direction.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0606fje; 
This article has been cited by other articles:
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  K. Sidera, M. Samiotaki, E. Yfanti, G. Panayotou, and E. Patsavoudi Involvement of Cell Surface HSP90 in Cell Migration Reveals a Novel Role in the Developing Nervous System J. Biol. Chem., October 29, 2004; 279(44): 45379 - 45388. [Abstract] [Full Text] [PDF]
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