Regulation of lymphocyte activation and apoptosis in the immune response in multiple sclerosis

Multiple sclerosis (MS) is a neurological inflammatory disease affecting the central nervous system (CNS) white matter. The patients usually develop the first symptoms between 20 and 40 years of age, translating into a huge social and economical impact in the western countries, where NIS has a high prevalence.

The main theory explaining MS pathology implies that autoreactive immune cells reactive to various myelin antigens enter the CNS to provoke demyelination and microglia activation. Therefore, both the process of activation and of elimination of immune cells are very important to the disease process and to the resolution of lesions.

Immune cells of MS patients seem to be somewhat resistant to apoptosis. The failure to adequately induce apoptosis of activated cells, especially those recognizing self-antigens, may contribute to the perpetuation of the inflammation and autoimmune attack. This reported resistance may be a consequence of regulation of different pathways, namely through triggering of so-called death-receptors, such as CD95, and of modulation of intracellular molecules like the Bcl-2 family of proteins.

In study I, competitive RT-PCR was used to quantify mRNA levels of CD95 and its ligand (CD95L) in peripheral blood (PB) mononuclear cells (MC) of NIS patients, HC and individuals suffering from myasthenia gravis, a disease of the neuromuscular junction. CD95L expression was found to be significantly elevated in MS patients, particularly in relapsing-remitting MS, suggesting a modulation of this pathway in the most inflammatory stage of MS.

Study II confirmed the previous results with real-time PCR, but also identified an elevation in gene expression of CD95, the upstream initiator of the apoptotic cascade caspase-8 and its negative regulator cFLIP. Again, RR MS patients exhibited higher levels of these molecules than patients in the secondary progressive (SP) phase of the disease. Different cell subsets (T cells, CD4+ and CD8+ T cells, B cells and monocytes) were isolated from PB MC in a small number of individuals. CD4+ T cells expressed the highest amounts of CD95 and cFLIP, while CD8+T cells showed the greatest expression of CD95L. These results indicate that the CD95-mediated pathway may be implicated in the survival of activated immune cells and promote their migration into the CNS, particularly in RR MS.

Study III aimed at integrating the gene expression pattern of the same molecules studied in the previous papers with magnetic resonance parameters of MS patients. Caspase- 10 was also included as it can be recruited upon CD95-triggering similarly to caspase-8, though with a different role. We found that PB MC of patients with gadolinium-enhancing lesions, an indicator of active inflammation within the CNS, had lower levels of CD95 and caspase-8. Lesion load and presence of atrophy did not correlate with any of the molecular markers studied. These results possibly suggest a depletion of sensitive CD95+ cells from the periphery or the migration of activated cells across the BBB upon active inflammation inside the CNS.

In study IV, gene expression of four members of the BcI-2 family of proteins was studied in PB MC in relation to MS. This family of proteins regulates the intrinsic pathway of apoptosis, which affects the mitochondria. Antiapoptotic Bcl-2 and Bcl-xL and pro-apoptotic Bax, but not Bak, were found to be significantly increased in RR MS compared to healthy controls (HC). Treatment with interferon (IFN)-beta, the immunomodulatory treatment most commonly used in MS, induced an increase in levels of Bax in RR MS patients and of Bcl-2 in SP MS. Furthermore, in vitro activation with anti-CD3 and -CD28 monoclonal antibodies (MAb) induced the upregulation of gene expression of Bcl-2, Bcl-xL and Bax in PB MC of MS patients and HC. Our observations implicate the regulation at the transcriptional levels of Bcl-2, BcI-xL and Bax in RR MS, possibly in connection with overactivation of the immune system in these patients.

Study V focused on the study of 4- 1 BB, a member of the tumour necrosis factor receptor (TNFR) superfamily like CD95. 4- 1 BB delivers a co-stimulatory signal mostly to T cells. MS patients had decreased mRNA levels of 4- 1 BB in PB MC compared to controls and there was a decreased number of CD4+CD25high regulatory T cells expressing it at the surface, as measured by flow cytometry. Serum soluble 4- 1 BB, on the other hand, associated with activated immune cells, was present in higher levels in MS patients. In vitro blockade of this pathway with anti-4-1BB ligand MAb failed to inhibit proliferation of antigen-specific cells. We speculate that 4- 1 BB costimulatory pathway might modulate the suppressive capacity of CD4+CD25high regulatory T cells in MS. Further studies detailing its role in MS are ebing considered.

List of scientific papers

I. Huang W-X, Huang MP, Gomes MA, Hillert J (2000). "Apoptosis mediators fasL and TRAIL are upregulated in peripheral blood mononuclear cells in MS. " Neurology 55(7): 928-34
https://doi.org/10.1212/wnl.55.7.928

II. Gomes AC, Jonsson G, Mjornheim S, Olsson T, Hillert J, Grandien A (2003). "Upregulation of the apoptosis regulators cFLIP, CD95 and CD95 ligand in peripheral blood mononuclear cells in relapsing-remitting multiple sclerosis. " J Neuroimmunol 135(1-2): 126-34
https://doi.org/10.1016/s0165-5728(02)00437-x

III. Gomes AC, Morris M, Stawiarz L, Jonsson G, Putheti P, Bronge L, Link H, Hillert J (2003). "Decreased levels of CD95 and caspase-8 mRNA in multiple sclerosis patients with gadolinium-enhancing lesions on MRI. " Neurosci Lett 352(2): 101-4
https://doi.org/10.1016/j.neulet.2003.08.030

IV. Gomes AC, Mjornheim S, Hillert J (2004). "Altered mRNA levels of Bcl-2, Bcl-xL and Bax in multiple sclerosis and modulation of Bcl-2 and Bax expression by IFN-beta treatment." (Manuscript)

V. Liu GZ, Gomes AC, Putheti P, Kostulas K, Press R, Hillert J, Hjelmstrom P (2004). "Dowregulated 4-1BB expression in multiple sclerosis." (Manuscript)