Effect of the serum from multiple sclerosis patients on an in vitro model of blood-brain barrier.

Abstract

Multiple sclerosis (MS) is characterized by focal inflammatory demyelination, largely due to autoimmune responses against different components of the myelin sheet. It is also generally accepted that the pathogenesis of MS consists of inflammatory and neurodegenerative phases, where demyelination should produce partially reversible clinical deficits that can remit, due to limited remyelination, while axonal degeneration produces permanent non-remitting clinical damage. It is also assumed that nervous system inflammation is initiated by autoreactive, myelin-specific T cells that permeate the blood-brain barrier and trigger a series of events leading to tissue destruction. In addition to antibodies, the analysis of CSF/serum of MS patient led to identification of soluble, CNS-derived proteins that could be considered biologic markers of disease and indicators of the ongoing pathologic process. Among these proteins, neurofilament components, as well as other proteins of the cytoskeleton have been found to be costantly modified in MS patients during the entire course of the disease, thus confirming continuous axonal damage. Actually, an hallmark of MS pathogenesis is blood-brain barrier disruption, possibly due to alterations of the tight junctions (TJs). Moreover, TJ disruption is accompanied by serum protein leakage across BBB. Starting from these observations, we looked for the presence in the sera from MS patients of factors able to induce both morphological and molecular modifications in brain cells. Moreover, by using a BBB in vitro model, set in our laboratory (1-4), we searched for a direct effect of MS sera on the permeability properties of the model BBB. We treated primary astrocytes (5) and neurons (6) with either control- or MS- serum and analyzed the treated cells by immunofluorescence and immunoblot, with antibodies against a variety of cytoskeletal- and angiogenic- proteins. Moreover we studied the effects of control and MS serum on the permeability of the BBB model, by using radiolabeled sucrose as a tracer of paracellular flux, and the measurement of the transendothelial resistance (TER) as a parameter of BBB alteration. We also looked at occludin localization as a marker of TJ formation. We found that cytoskeletal proteins, such as GFAP in astrocytes, and neurofilaments in neurons, respectively, are altered in cells treated with MS serum. In the three-cell type BBB model, treatment with MS serum induced a TER decrease and a loss of the normal peripheral localization of occludin, probably indicating a loss of TJs. Moreover, in treated cells, an increase of the paracellular flux of labeled sucrose was evident. Our results confirm the idea that MS serum contains factors able to alter the BBB and induce in brain cells alterations of the general organization and even cell death, as shown by viability tests in culture. 1. Savettieri et al (2000) NeuroReport 11: 1081-4 2. Cestelli et al (2001) J Controll Rel 76: 139-47. 3. Schiera et al (2003) J Cell Mol Med 7: 165-70 4. Schiera et al (2005) J Cell Mol Med 9 :373-9. 5. Proia et al (2008) Int J Mol Med 21:63-7. 6. Schiera et al (2007) J Cell Mol Med 11:1384-94.