Proteomic analysis of autoantibody reactivity to central nervous system antigens in sera and cerebrospinal fluid of multiple sclerosis patients

Multiple Sclerosis (MS) belongs to a large group of inflammatory demyelinating diseases of the central nervous system (CNS), in which the autoimmune response is directed to myelin and myelin-producing cells, the oligodendrocytes, and eventually leads to demyelination and oligodendrocyte loss. Heterogeneity in morphological alterations of the brain is detectable by magnetic resonance imaging (MRI), histopathological evaluation, as well as in clinica1 presentation. Three are the major forms of MS. It is not clear which factors are responsible for the different courses. Relapsing-remitting (M)-MS is the most frequent form (85%-90% of patients) characterized by unpredictable attacks (relapses) followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Most RR-MS patients later develop secondary progressive (SP)-MS, in which they begin to have neurologic decline between their acute attacks without any definite periods of remission. About 10%-15% of patients present with insidious disease onset and steady progression, termed primary progressive (PP)-MS. The autoimmune reaction has been shown to involve the activation of both T and B lymphocytes. The activation of CD4+ autoreactive T cells and their differentiation into a Thl phenotype are crucial events in the initial steps, and these cells are probably also important players in the long-term evolution of the disease. Damage of the target tissue, the central nervous system, is, however, most likely mediated by other components of the immune system, such as antibodies (Igs), complement, CD8+ T cells, and factors produced by innate immune cells. The observation that Igs are elevated in the cerebrospinal fluid (CSF) of MS patients has been the most important and earliest evidence suggesting a role for B cells and antibodies in the pathology of MS. In most of the studies the search for autoantigens has focused on myelin proteins and other CNS components. Although some studies emphasize the relevance of myelin-specific antibodies, others fail to confirm these data. We have overcome such restrictive approaches using a large pane1 of antigens derived from target tissue extracts. In the present study, we compared by bidimensional polyacrylamide gel electrophoresis (2D-PAGE) and immunoblotting the IgG repertoires from serum and CSF of contro1 and MS patients against antigens derived from CNS normal white matter. The reactive spots were then identified by mass spectrometry (MaS) andlor immunoblotting. This immunomic approach enabled the identification of a restricted number of neural protein isoforms specifically recognized by MS sera and CSF, which were mostly localized on oligodendrocytes andlor cytoskeleton. Almost al1 MS patients had CSF IgG directed to isoforms of one of the oligodendroglial molecules transketolase (TK), cyclic nucleotide phosphodiesterase type I (CNPase I), collapsin response mediator protein 2 and tubulin P4. Interestingly, 50% of MS CSF IgG recognized TK, which was mostly localized on oligodendrocytes in human white matter from normal and MS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2 and actin interacting protein 1) was prevalent in secondary progressive MS patients. Among the proteins recognized by serum IgG, almost al1 MS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, while CNPase I was the oligodendroglial antigen most frequently recognized (44%) by MS seric IgG. Our immunomic approach shed new light on the autoimmune repertoire present in MS patients revealing nove1 oligodendroglial andlor neuronal putative autoantigens, which may have potential important pathogenic implications and also serve as biomarkers of disease as well as useful diagnostic tools.