There are not many reports about the contribution of IgA in MS patients, because its detection is cumbersome 12. It was reported that qualitative methods are the most sensitive methods to detect intrathecal immunoglobulin synthesis 7. For these reasons, we aimed to develop a new method based on IEF and immunodetection to detect OGIgAB in CSF and serum samples to analyze the presence of IAS in MS patients.
First, we developed a high sensitive quantitative ELISA to analyze the IgA concentration in CSF and serum samples. Once demonstrated the linearity, accuracy, sensibility and reproducibility of the ELISA, we analyzed the concentration of this immunoglobulin in samples from patients with MS and different neurological diseases.
In our cohort, IgA concentration in serum samples was found within normal ranges in most cases. The analysis of CSF showed an IgA concentration within the previously described normal range 12 in most case but a considerable number of the individuals (37.00%) had a low IgA concentration, which make difficult to detect IAS. We did not detect significant differences in the IgA concentration between patients with MS or different neurological diseases, neither in CSF nor in serum samples. Neither did we detect statistical differences when the different MS types were compared.
Then we analyzed the presence of IAS by a quantitative method, the Reiber diagram, and we observed that the percentage of MS patients showing IAS using this method was similar to described previously 7. Using this method the percentage of IAS were similar in MS patients and control group.
Then we developed a new high IEF and immunodetection to detect OGIgAB in order to study the presence of IAS in MS patients and control group. We overcame the difficulties associated with the analysis of this immunoglobulin by qualitative methods as follows. First, as it was described that the proportion of dimeric IgA increases from 5%, up to 53.9% when control and MS patients are compared 14, samples were reduced using DTT to obtain monomeric IgA. Second, to avoid the IgA precipitation we employed agarose instead of acrylamide gels 12. Third, to detect IgA at low concentration in CSF, biotin-streptavidin amplification method was used. Finally, we employed alkaline phosphatase to develop the staining, this enzyme improves 10 fold the sensitivity of the assay compared with peroxidase 1.
Once established the best conditions, we demonstrated the linearity, accuracy and reproducibility of the IEF and immunodetection. The sensitive of this new IEF technique, higher than methods used previously 8, was enough to identify OGIgAB in all samples in our cohort, despite of the high percentage of individuals with a low IgA concentration in CSF. This immunoassay showed four different patterns. Pattern I (polyclonal IgA in CSF and serum) indicates that all the B-lymphocytes release a limited amount of antibody because they are not stimulated, this is the normal situation. Pattern II (the same OGIgAB in CSF and serum) involves activated peripheral B-lymphocytes producing this immunoglobulin, which finally passes through the brain blood barrier to the CNS. Pattern III (presence of OGIgAB in CSF but not in serum) demonstrates the presence of B-lymphocytes releasing this immunoglobulin in the CNS. Pattern IV (two or more OGIgAB in CSF compared with serum) discloses the presence of activated B-lymphocytes in the CNS, but also indicates that some are also activated in the peripheral system.
According to these patterns, we could identify IAS in a higher percentage than described before for MS patients 14. This new assay is more sensitive than Reibergram to detect IAS, corroborating that qualitative techniques are more sensitive than quantitative methods 6,15. IAS was not related with IgA concentration in CSF, because we detected it in individuals with a low IgA concentration, but we did not in patients showing high levels.
IAS is highly specific of MS patients, because its incidence is higher in these individuals than in those form the control groups, despite of the inclusion of inflammatory diseases of the central nervous system as controls. Most of the MS patients with IAS showed Pattern IV, in contrast to the situation observed when IgG or IgM oligoclonal bands are analyzed 1,16. Therefore, the presence of additional oligoclonal bands in CSF is the most frequent pattern. Nevertheless, a considerable percentage of MS patients showed Pattern II. This was not expected, because this pattern is not usually observed when IgG or IgM oligoclonal bands are analyzed in MS patients 1,16. Interestingly, the higher percentage of IAS was observed in CIS patients. The incidence of Pattern II was particularly high in NonMSDND (75,00%) and NDND (71.43%) patients, indicating again a specific difference of IgA-bands distribution in MS patients. This also could indicate that origin of the lymphocyte activation is different depending on the disease.
All these data suggest that IgA, as the other immunoglobulins, could have a dual role in MS, protecting against the disease or promoting the cell damage.
Regarding the protective role of IgA in MS, it was recently described that recirculating intestinal IgA positive cells prevent the development of experimental autoimmune encephalomyelitis, the animal model of MS, because they release IL-10 17. We have described above the high incidence of OCIgAB in serum samples from the MS group, demonstrating the usual presence of peripheral activated B lymphocytes in these individuals.
Moreover, it is involved in the first line of defense against pathogens, and the presence of virus in MS patients has been broadly demonstrated, for example, our group has previously detected human herpesvirus 6, JC virus and Epstein Barr virus in the CSF and central nervous system of these MS patients 11,18,19. In addition, viruses were also found in the CSF at the first demyelinated event 19, and it was demonstrated that infections increase the risk of relapses 20. Regarding this, we observed a high incidence of IAS in CIS patients but a lower incidence in RRMS, SPMS and PPMS.
On the other hand, antibodies contribute to eliminate cells infected by viruses, because they promote antibody-dependent cellular cytotoxicity 21. Moreover, the activation of the immune system could trigger cross-reactions against self-antigens, one of the classical hypothesis in autoimmunity 22. IgA deposits observed in demyelinated areas of MS patients are related with axonal damage 9.
In summary, here we present a highly sensitive technique to detect OGIgAB in CSF and serum samples. Our findings draw attention to the high prevalence of IAS in MS patients, especially in CIS group. Further studies would be of great interest to clarify the role of this immunoglobulin in the physiopathology of MS.