Linomide (roquinimex) affects the balance between pro- and anti-inflammatory cytokines in vitro in multiple sclerosis

Objectives - Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro-inflammatory cytokines, e.g. interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and lymphotoxin (LT) that may make MS worse, and anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor-β (TGF-β) that may act beneficially. Substances that down-regulate cytokines such as TNF-α or promote IL-10 or TGF-β can be anticipated to affect MS beneficially. Material and methods - In situ hybridization to detect and enumerate IFN-γ, TNF-α, LT, IL-4, IL-10 and TGF-β mRNA expressing blood MNC after stimulation with myelin basic protein (MBP), control antigens and without antigen in presence and absence of Linomide (roquinimex, LS-2616) was employed. In parallel, ELISPOT assay to detect MBP- and PHA-reactive IFN-γ secreting blood MNCLinomide was used. Results - Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP-reactive TNF-a and increases MBP-reactive IL-10 and TGF-β mRNA expressing MNC from MS patients'blood when analysed in vitro. Compared to dexamethasone, Linomide up-regulated levels of blood MNC expressing mRNA of TGF-β after culture in presence of MBP. Conclusions - Changes of cytokine balance towards a production of anti-inflammatory cytokines could be a desirable effect to be evaluated in future drug studies of Linomide-like substances. At present, Linomide is not evaluable in MS clinical trials due to side-effects.     

Effect of high-dose methylprednisolone administration on immune functions in multiple sclerosis patients

Objectives – To investigate the in vivo effect of corticosteroid pulse therapy on immunocompetent cells in 18 patients given methylprednisolone to treat an acute episode of MS. Material and methods – Blood was sampled before and after 3 days of methylprednisolone administration at doses of 1 g/day. Lymphocyte subtyping was performed and whole blood cell cultures were used to measure the cytokine producing capacity for interleukin-1 (IL-1), interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-a (TNF-α) and interferon-α (IFN-α). In addition, serum levels of the immunoglobulin classes IgG, IgA and IgM were determined. Results – Before treatment, production of IL-1 was significantly increased in MS patients as compared to healthy controls. After therapy, production of all cytokines was significantly decreased, whereas there were significant increases in the numbers of monocytes, neutrophils and T and B lymphocytes. Treatment had no effect on serum immunoglobulin levels. Conclusion – An important mechanism for the antiinflammatory effect of corticosteroids in MS results from a suppression of the activation of the peripheral immune compartment through inhibition of cytokine production and lymphocyte endothelial adhesiveness.     

Interleukin-6 and Transforming Growth Factor-β1 mRNAs are Induced in Rat Facial Nucleus Following Motoneuron Axotomy

Transection of the rat facial nerve leads to a rapid activation of both astrocytes and microglia around axotomized motoneurons. The factors involved in glial activation in vivo are poorly defined but cytokines have been implicated as major regulators of glial activity in vitro. In the present study we have investigated the expression of cytokine mRNAs in the axotomized facial nucleus that might be involved in glial activation. Eight hours after axotomy unilateral transection of the facial nerve had already induced a rapid accumulation of interleukin (IL)-6-mRNA, with a peak at 24 hours. No IL-6 mRNA was detected on the unoperated control side. Transforming growth factor (TGF)-β1 mRNA was detected at low levels in the normal facial nucleus, increasing to three times the normal level 2 days after axotomy. After day 7 TGF-β1 mRNA levels gradually declined, with a second minor peak 21 days after axotomy. In situ hybridization experiments, 4 and 21 days after axotomy, localized TGF-β1 mRNA to activated microglial cells around regenerating motoneurons, as well as probably some astrocytes. Motoneurons did not express TGF-β1 mRNA. TGF-β3 was found to be normally expressed in the facial nucleus but was not regulated by axotomy. No mRNA for IL-1, tumour necrosis factor-α or interferon-γ was found in the regenerating facial nucleus at any point in time. Our data indicate that IL-6 might act as an early activating signal for glial cells in response to motoneuron axotomy, and that TGF-β1 expressed by activated glial cells might provide a long-lasting negative feedback signal to control glial activation.     

Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

Local expression of cytokines in idiopathic inflammatory myopathies

H. Lepidi, V. Frances, D. Figarella-Branger, C. Bartoli, A. Machado-Baeta & J-F. Pellissier (1998) Neuropathology and Applied Biology , 24, 73–79
Local expression of cytokines in idiopathic inflammatory myopathies
The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM), are regarded as autoimmune diseases. They are characterized by chronic lymphocytic and macrophagic infiltration in muscle tissue. Of particular importance in understanding the immune response to IIM is the specific pattern of locally produced cytokines. Frozen muscle tissues from IIM (5 DM, 3 PM, and 1 IBM) were used to investigate the cytokine responses. The RT-PCR technique was instrumental to determine the pattern of expression of pro-inflammatory (IL-1β, IL-6, TNF-α), Th1 (IFN-γ IL-2), and Th2 (IL-4) cytokines. Immunohistochemistry was also used to localize morphologically IFN-γ and IL-4. Our results show that pro-inflammatory cytokines and Th1 cytokines are mainly expressed in IIM. The accumulation of mononuclear inflammatory cells and the inflammatory syndrome in IIM are probably related in part to the production of pro-inflammatory cytokines. Moreover, the pattern of local cytokine expression is consistent with a Th1 immune response related to autoimmune diseases.     

High numbers of perforin mRNA expressing CSF cells in multiple sclerosis patients with gadolinium-enhancing brain MRI lesions

Enhanced expression of pro- and anti-inflammatory cytokines is a common finding in MS, but attempts to correlate cytokine expression with disease activity have produced conflicting results. In this paper, gadolinium-(Gd-)enhancing lesions on brain MRI were used as markers for active inflammation in patients with MS not treated with any immunomodulatory drugs. In parallel, in situ hybridization was used to detect blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) expressing cytokine mRNA. An association was observed between numbers of perforin mRNA expressing CSF MNC and numbers of Gd-enhancing brain MRI lesions. Perforin mRNA expressing CSF MNC were not detected in any of the patients lacking active lesions on brain MRI. The expression of tumor necrosis factor-alpha, interleukin-10 (IL-10) and IL-12 mRNA in CSF MNC did not differ between MS patients with and without active MRI lesions. Based on the present finding, a role for perforin in the disruption of the blood-brain barrier in MS can be hypothesized.     

Autoantigen-induced IL-13 mRNA expression is increased in blood mononuclear cells in myasthenia gravis and multiple sclerosis

Evidence has been presented for the involvement of immune mechanisms in the pathogenesis of myasthenia gravis (MG) and multiple sclerosis (MS). The production of autoantibodies in both diseases is regulated by T-cells by means of cytokines. Interleukin-13 (IL-13) is mainly produced by T-helper type 2 cells and induces B-cell proliferation and antibody class switch. The role of IL-13 in MG and MS is not known. We employed in situ hybridization with synthetic radiolabelled oligonucleotide probes to detect and enumerate blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) expressing IL-13 mRNA from patients with MG, MS, optic neuritis (ON), other inflammatory neurological diseases (OIND) and healthy controls. MG is associated with elevated levels of acetylcholine receptor (AChR) reactive IL-13 mRNA expressing blood MNC compared to control patients. In MS, numbers of MBP-reactive IL-13 mRNA expressing MNC were higher compared to cultures without antigen stimulation. The levels of MBP-reactive IL-13 mRNA positive MNC were higher in MS compared to MG, but not other controls. There were no differences in spontaneous IL-13 mRNA expressing blood MNC numbers between MG, MS, ON and control patients. The data suggest the involvement of IL-13 in both MG and MS.     

Immunocompetence of human microvascular brain endothelial cells: cytokine regulation of IL-1β, MCP-1, IL-10, sICAM-1 and sVCAM-1

Endothelia from the brains of four patients undergoing neurosurgery, including one multiple sclerosis (MS) patient, were studied in vitro to determine cytokine and chemokine production; the release of soluble adhesion molecules was also investigated. The same procedure was repeated on human umbilical vein endothelial cells (HUVECs) in order to detect possible district-specific differences. After isolation, the endothelium was cultured and stimulated with γ-interferon (IFN), tumour necrosis factor alpha (TNF-α) and LPS. The results showed that brain endothelium, in our experimental conditions, does not produce interleukin (IL)-10 and produces lower amounts of IL-1β and soluble intercellular adhesion molecule-1 (sICAM-1) than HUVECs do; no differences were detected in soluble vascular cell adhesion molecule-1 (sVCAM-1) production. MCP-1 mRNA was detected both without and after stimulation with TNF-α and γ-IFN in HUVECs and MS human brain endothelial cells (HBECs), while in non-MS-HBECs it was found only after γ-IFN stimulation. Received: 24 June 1997 Received in revised form: 20 February 1998 Accepted: 5 March 1988     

Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.

Myelin-directed autoimmunity is considered to play a key role in the pathogenesis of multiple sclerosis (MS). Increased production of both pro- and anti-inflammatory cytokines is a common finding in MS. Interleukin-17 (IL-17) is a recently described cytokine produced in humans almost exclusively by activated memory T cells, which can induce the production of proinflammatory cytokines and chemokines from parenchymal cells and macrophages. In situ hybridisation with synthetic oligonucleotide probes was adopted to detect and enumerate IL-17 mRNA expressing mononuclear cells (MNC) in blood and cerebrospinal fluid (CSF) from patients with MS and control individuals. Numbers of IL-17 mRNA expressing blood MNC were higher in patients with MS and acute aseptic meningoencephalitis (AM) compared to healthy individuals. Higher numbers of IL-17 mRNA expressing blood MNC were detected in MS patients examined during clinical exacerbation compared to remission. Patients with MS had higher numbers of IL-17 mRNA expressing MNC in CSF compared to blood. This increase in numbers of IL-17 mRNA expressing MNC in CSF was not observed in patients with AM. Our results thus demonstrate increased numbers of IL-17 mRNA expressing MNC in MS with higher numbers in CSF than blood, and with the highest numbers in blood during clinical exacerbations.     

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.     

Multiple sclerosis: pro- and anti-inflammatory cytokines and metalloproteinases are affected differentially by treatment with IFN-beta

Interferon-beta (IFN-beta) has a beneficial influence on the course of multiple sclerosis (MS) and has become standard treatment of this disease, though its mechanisms of action are incompletely understood. This study examines the effect of IFN-beta treatment on the cytokines IL-6, TNF-alpha, IFN-gamma and IL-10; the metalloproteinases MMP-3, -7 and -9 and the tissue inhibitor of metalloproteinase-1 (TIMP-1). IFN-beta treatment resulted in decreased numbers of mononuclear cells (MNC) secreting IL-6 and TNF-alpha and expressing mRNA of MMP-3 and MMP-9 compared to pretreatment levels. On the contrary, numbers of IL-10 secreting MNC and TIMP-1 mRNA expressing were augmented during IFN-beta therapy. Whether the down-regulatory effects on pro-inflammatory and upregulatory effects on anti-inflammatory molecules are a direct result of IFN-beta on the immune system or secondary to clinical stabilization of MS pathology induced by IFN-beta remains to be evaluated.     

Increased transforming growth factor-β, interleukin-4, and interferon-γ in multiple sclerosis

The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell–associated interferon-σ (IFN-σ), the Th2 cell–related interleukin-4 (IL-4), and the immune response–downregulating cytokine transforming growth factor-β (TGF-β), but proof for their involvement in MS has been lacking. By adopting in situ hybridization with complementary DNA oligonucleotide probes for human IFN- IL-4, and TGF-β, the expression of mRNA for these cytokines was detected in mononuclear cells (MNC) from blood and cerebrospinal fluids. Strongly elevated levels of MNC expressing all three cytokines were found in peripheral blood and at even higher frequencies in cerebrospinal fluid from untreated patients with MS and optic neuritis, i.e., a common first manifestation of MS, compared with patients with other neurological diseases and healthy subjects. In MS and optic neuritis, IL-4 mRNA expressing cells predominated, followed by TGF-β– and IFN-σ–positive cells. Control patients with myasthenia gravis had similarly elevated levels of IFN-σ and TGF-β mRNA expressing blood MNC but lower numbers of IL-4–positive cells. No or slight disability of MS was associated with high levels of TGF-β mRNA expressing cells, while MS patients with moderate or severe disability had high levels of IFN-σ–positive cells. IFN-σ and TGF-β may have opposing effects in MS, and treatments inhibiting IFN-σ and/or promoting TGF-β might ameliorate MS.     

Interferon-β1b leads to a short-term increase of soluble but long-term stabilisation of cell surface bound adhesion molecules in multiple sclerosis

Abstract. Adhesion molecules (AMs) are believed to regulate the transmigration of blood leukocytes across the blood-brain barrier (BBB), which is an essential step in the pathogenesis of multiple sclerosis (MS). Previous studies have investigated changes of the soluble forms of AM during interferon-_1b (IFN-_1b) treatment in MS patients. In this study, we analysed the influence of IFN-_1b treatment on the cell surface bound forms of the AMs cICAM-1 and cICAM-3 on blood mononuclear cells (MNC). Sixty-eight patients with relapsing-remitting MS were enrolled in this open study; thirty of them were treated with IFN-_1b. Blood samples were collected every three months over a period of 18 months. The expression levels of cell surface bound forms of AM on blood MNC were measured by two colour flow cytometry analysis. sVCAM-1, sICAM-1 and sICAM-3 were determined by ELISA. We found a short-term induction effect on the serum concentrations of sICAM-1 and sVCAM-1 after three months of IFN-_1b treatment. The expression levels of cell surface bound AMs on blood MNC remained stable during treatment. Untreated MS patients, however, showed a continuous decrease in the expression of cell surface bound AM expression over 18 months. Stabilisation of the expression of cell surface bound AMs on blood MNC may indicate the beneficial effects of IFN-_1b therapy in MS patients.     

Clues to the immunopathogenesis of multiple sclerosis by investigating untreated patients during the very early stage of disease

Plethora of abnormalities of the immune system has been described in multiple sclerosis (MS). They include a number of myelin antigens (e. g. MBP, MOG, PLP, MAG), the presence of reactive T cells in blood and, further enriched, in the cerebrospinal fluid (CSF), large numbers of B cells in the CSF secreting antibodies of multiple but unknown specificities, an increase of mononuclear cells (MNC) expressing and secreting both pro- and anti-inflammatory cytokines, including Th1 cytokines interferon-gamma (IFN-γ) and interleukin (IL)-6, the Th2 related IL-4 and IL-10, and the Th3-driven TGF-β, elevated numbers of MNC in both blood and CSF expressing a spectrum of metalloproteinases and their inhibitors, as well as many other aberrations. However, no consistent patterns have emerged that relate any of these findings to clinical variables such as exacerbations, during of disease, disability, or lesions in the central nervous system (CNS) detected at magnetic resonance imaging. In order to elucidate the relevance of these immunological abnormalities in the pathogenesis of MS, my colleagues and I studied patients with acute monosymptomatic optic neuritis (ON) and compared them with patients with clinically definite MS (CDMS). The patients have not been treated and have not received corticosteroids or interferon-β. When comparing these two groups, we were unable to identify any differences in any of the variables mentioned. Thus, very early MS, as represented by ON, shows the same full-blown pattern of immunological abnormalities seen in CDMS. Furthermore, a complete epitope spread affecting MBP, MOG, PLP, MAG and other myelin components is already present in ON. Whether any of these alterations play a pathogenetic role is still unsettled.     

MxA protein assay for optimal monitoring of IFN-beta bioactivity in the treatment of MS patients

Objectives –  Myxovirus resistance protein A (MxA) can be used as a marker of the bioactivity of interferon-beta (IFN-β) therapy. Two to forty per cent of IFN-β-treated multiple sclerosis (MS) patients develop IFN-β-neutralizing antibodies (NAb) with subsequent attenuation of MxA protein induction. The aim of this study was to set up a simple MxA enzyme immunoassay (EIA) for the measurement of MxA protein and to evaluate the EIA test by comparing the results with flow cytometric analysis and the measurement of NAb.
Methods –  total of 51 IFN-β-treated relapsing–remitting MS (RRMS) patients were tested for MxA protein expression by using both MxA EIA assay and flow cytometric analysis. Thirteen patients were confirmed to be NAb-positive.
Results –  The correlation between EIA and flow cytometric analysis was significant with a wider range of measured levels in the EIA. Patients with NAb had low MxA levels, but in some patients, remaining MxA induction could be detected despite NAb.
Conclusions –  The MxA EIA assay seems to be a practical method for large-scale analysis of the bioactivity of IFN-β treatment.     

Humoral and cellular immune responses to Copolymer 1 in multiple sclerosis patients treated with Copaxone

Little is known about the involvement of cytokines in the pathogenesis of primary progressive (PP) multiple sclerosis (MS). We evaluated in this cross-sectional study whether IL-18, IL-12p35, IL-12p40, TNF-, IFN-γ, IL-10, IL-4, TGF-β, IL-12Rβ1, and IL-12Rβ2 mRNA expression in unstimulated white blood cells showed significant differences between relapsing–remitting (RR), secondary progressive (SP) and PP MS patients, and healthy controls. All clinical subtypes showed unique mRNA expression patterns as compared to the controls. Both RR and SP patients displayed increased levels of IL-12p40, IL-18, and TGF-β mRNA compared to controls, whereas PP patients showed only increased IL-18 mRNA levels. Both in PP and SP patients, IFN-γ and IL-10 mRNA were decreased compared to RR patients and controls. PP patients were unique in that they showed decreased IL-12Rβ1 mRNA. In conclusion, our data show that the assessment of cytokine (receptor) mRNA profiles is useful to discriminate between the different clinical subtypes and suggest that different cytokines are involved in the pathogenesis of PP MS as compared to RR and SP MS.     

Subunit- and brain region-specific reduction of GABAA receptor subunit mRNAs during chronic treatment of rats with diazepam

The mRNA levels for several GABA_A receptor subunits were measured by Northern blot analysis. Rats were treated for 3 wk by continuous release of diazepam (DZP) from subcutaneous reservoirs, and then sacrificed immediately or 48 h after removing the reservoirs. Poly(A)⁺ RNAs, isolated from cerebral cortex, cerebellum, and hippocampus, were hybridized with oligonucleotide probes for GABA_A receptor subunits and a cDNA probe for β-actin. Subunit mRNAs were expressed relative to the corresponding β-actin mRNA. DZP treatment decreased the α₁ subunit mRNA level 40% in hippocampus, but it was not changed in cortex or cerebellum. The α₅ subunit mRNA level was decreased in cerebral cortex (28%) and hippocampus (15%). The γ₂ subunit mRNA level was decreased (40%) only in cortex. DZP treatment did not affect α₂, α₃, α₄, β₂, or β₃ subunit mRNA levels. Decreases in mRNA levels had reversed within 48 h after stopping chronic treatment. Acute DZP did not change α₁, α₅, or γ₂ subunit mRNA levels. The decreases in GABA_A receptor subunit mRNA levels were specific to subunit and brain region. These results, coupled with those after chronic flurazepam treatment, also indicated that the effects on GABA_A receptor subunit mRNA levels are specific to the benzodiazepine (BZ) used for chronic treatment.     

Induction of serum soluble tumor necrosis factor receptor II (sTNF-RII) and interleukin-1 receptor antagonist (IL-1ra) by interferon beta-1b in patients with progressive multiple sclerosis

Background   Cytokine inhibitors, such as soluble tumor necrosis factor receptor II (sTNFRII) and interleukin-1 receptor antagonist (IL-1ra) are possible regulators of proinflammatory cytokine activity. Although previous studies have shown induction of sTNF-RII and IL-1ra by interferon-beta (IFNβ) in patients with relapse-onset forms of multiple sclerosis (MS), to date no studies of these cytokine inhibitors have been performed in patients with essentially progressive forms of MS. Objective   To address the effects of IFNβ on serum levels of sTNF-RII and IL-1ra in a cohort of progressive MS patients and to assess the relationship between levels and changes of sTNF-RII and IL-1ra and clinical and radiological variables. Methods Serial blood samples were obtained from a cohort of 73 patients with progressive MS who participated in a placebo-controlled clinical trial with IFNβ-1b. Serum levels of sTNF-RII and IL-1ra were measured by multiplex enzyme-linked immunosorbent assay at baseline and after 3, 6, 12, and 24 months. EDSS and MSFC scores were recorded at the time of blood sampling, and MR scans were obtained at baseline and after 12 and 24 months. Results   Treatment with IFNβ was associated with significant increases of sTNF-RII and IL-1ra serum levels during the followup period. A strong correlation at 24 months was observed between levels of sTNF-RII and EDSS scores in the placebo group. Finally, a trend for negative association was found between changes in sTNFRII and percentage change in T2-weighted lesion load at 24 months in the IFNβ treated group. Conclusions   sTNF-RII and IL-1ra levels are increased in the serum of progressive MS patients during IFNβ therapy and may be one mechanism by which IFNβ mediates its effects in the treatment of MS.