Karin Mausner-Fainberg

Interferon-β therapy up-regulates BDNF secretion from PBMCs of MS patients through a CD40-dependent mechanism

We had reported that immune cells from relapsing remitting multiple sclerosis (RR-MS) patients secrete low levels of BDNF and that there is a defective regulation of its secretion via DC40. We now studied the effect of interferon-beta (IFN-beta1) on the secretion and regulation of BDNF from immune cells in patients with RR-MS. The PBMCs of IFN-beta1a treated RR-MS patients secreted higher BDNF levels vs. untreated patients. Anti CD40 mAb stimulation of PBMCs of IFN-beta1a treated patients upregulated the BDNF levels. There was no significant effect of CD40 stimulation on PBMCs of untreated patients. CD40(+) expression on CD14(+) cells was higher in IFN-beta treated patients vs. untreated patients. In vitro treatment with IFN-beta1a of PBMCs from healthy controls and untreated patients led to a significant increase in CD40 expression on CD14(+) cells in both groups. The addition of IFN-beta1a to CD40 stimulated PBMCs of untreated patients restored the up regulatory effect of CD40 stimulation on BDNF levels. Therefore, reduced BDNF secretion from PBMCs and defective regulation effect of CD40 stimulation on BDNF levels in untreated RR-MS are reversible by therapy with IFN-beta1a.

Dysregulated neurotrophin mRNA production by immune cells of patients with relapsing remitting multiple sclerosis

Recent studies have suggested a neuroprotective activity of the lesion-infiltrating immune cells in multiple sclerosis (MS) by secretion of neurotrophins. We had earlier reported that immune cells from relapsing remitting MS (RR-MS) patients secrete low levels of brain-derived neurotrophic factor (BDNF), and that its secretion is dysregulated after CD40 stimulation. Here, we measured mRNA levels for BDNF, NT3 and NGF-beta mRNA in unstimulated PBMCs and found levels lower in untreated RR-MS patients than in healthy controls (HC). T-cell stimulation with anti-CD3/CD28 mAb up-regulated neurotrophin mRNA expression in untreated RR-MS patients and not in HC, whereas stimulation of PBMCs with anti-CD40 mAb up-regulated neurotrophin mRNA expression in HC and not in RR-MS patients. Further cellular analyses of the production of the neurotrophin mRNA in individual cells revealed that T cells were the main producers of the neurotrophin mRNA in RR-MS patients, and that monocytes were the main producers of NT3 and NGF-beta mRNA in HC. BDNF mRNA was similarly produced in monocytes and T cells in the HC group. The cytokines TNF-alpha and IL-17 up-regulated the expression of neurotrophin mRNA in HC but not in RR-MS patients. The neuroprotective activity of PBMCs appeared to be dysregulated in untreated patients with RR-MS, while the differences between the IFN-beta-treated RR-MS patients and the HC were smaller.

Reduced production of noggin by immune cells of patients with relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) plaques are characterized by neurodegeneration, astrogliolis, the presence of immature oligodendrocytes and infiltrating immune cells. Recent studies revealed a putative role for noggin in both neurogenesis and oligodenrocytes development. In order to study the potential of peripheral immune cells to contribute to neurogenesis in MS, we studied the mRNA expression, protein secretion and regulation profile of noggin in peripheral blood mononuclear cells (PBMCs) of untreated patients with relapsing-remitting MS (RR-MS), interferon-β (IFN-β) treated RR-MS patients compared to matched healthy controls (HC). Basal levels of noggin mRNA expression, determined by quantitative real-time PCR were lower in untreated patients than in HC. No differences were found between untreated patients and IFN-β treated patients. Similarly, the secreted levels of noggin, detected in 24h PBMCs supernatants by ELISA, were decreased in untreated RR-MS patients than in HC. Again no significant differences were found between untreated patients and IFN-β treated patients. Stimulation with anti-CD3/CD28 mAbs increased noggin mRNA expression in untreated patients but not in HC. However, noggin mRNA levels in untreated patients PBMCs stimulated with anti-CD3/CD28 did not reach noggin levels in unstimulated PBMCs of HC. Purification of monocytes (CD14+) and T cells (CD3+ cells) by magnet-activated cell separation has demonstrated that noggin mRNA is predominantly expressed in CD3(+) cells in both HC and in RR-MS patients. This pattern also appeared in protein level of noggin, tested by Western blot. The incubation of the PBMCs with TNF-α increased the expression of noggin only in HC group. In conclusion, T cells possess the potential to participate in the induction of neurogeneration by the production of noggin. This potential seems to be defective in immune cells of RR-MS patients as there is reduced mRNA expression and protein secretion levels of noggin, insufficient stimulatory effect of CD3/CD28 stimulation and unresponsiveness to TNF-α in these patients PBMCs.

Elevated and dysregulated bone morphogenic proteins in immune cells of patients with relapsing-remitting multiple sclerosis.

The abundance of neural stem cells (NSCs) in multiple sclerosis (MS) lesions with extensive astrogliosis suggests that fate factors of NSCs, such as the bone morphogenic protein (BMP) signaling maybe defective in MS. We found an elevated mRNA expression and protein secretion of BMP-2,4,5 but not of BMP-7. This was primarily in T cells. Cell stimulation with anti-CD3/CD28 antibodies or with IFN-γ induced expression of BMP-2,4,5 mRNA in untreated RR-MS patients, indicating that proinflammatory processes in MS may play a role in the BMP-2,4,5 productions in T cells. These results contribute to the understanding of the negligible extent of neurogenesis and oligodendrogenesis with extensive astrogliogenesis and the failure of adequate tissue repair in MS lesions.

Dysregulated production of leukemia inhibitory factor in immune cells of relapsing remitting multiple sclerosis patients

Leukemia inhibitory factor (LIF) is known to potentiate the differentiation and survival of neuronal and oligodendrocyte precursors. Systemic therapy with LIF reportedly ameliorated the severity of experimental autoimmune encephalomyelitis and prevented oligodendrocyte death. We studied the secreted LIF levels from immune cells of relapsing remitting multiple sclerosis (RR-MS) patients compared to age- and gender-matched healthy controls (HCs). LIF was barely detected in the supernatants when the cells were not stimulated. After stimulation with anti-CD3/CD28 monoclonal antibody, LIF levels were up-regulated in both patients and controls, although to a significantly lower extent in RR-MS patients compared to HC. There were no significant differences between untreated patients and interferon-β1a treated patients. This is a heretofore unreported aspect of immune dysregulation in patients with RR-MS that may be related to insufficient remyelination and neurogenesis in MS lesions.

Treatment with Anti-EGF Ab Ameliorates Experimental Autoimmune Encephalomyelitis via Induction of Neurogenesis and Oligodendrogenesis

Background. The neural stem cells (NSCs) migrate to the damaged sites in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the differentiation into neurons or oligodendrocytes is blocked. Epidermal growth factor (EGF) stimulates NSC proliferation and mobilization to demyelinated lesions but also induces astrogenesis and glial scar. Objective. To examine the clinical and histopathological effects of EGF neutralization on EAE. Methods. EAE-induced SJL mice were intravenously treated with either anti-EGF neutralizing antibody (Ab) or isotype control or PBS. On day 9 after immunization, 3 mice of each group were daily treated for 9 days with BrdU and then sacrificed for immunohistochemical analysis. Results. Treatment with anti-EGF Ab significantly ameliorated EAE symptoms during the second relapse. Anti-EGF Ab induced a shift from BrdU+GFAP+ NSCs to BrdU+DCX+ neuroblasts in the subventricular zone (SVZ), increased BrdU+NeuN+ neurons in the granular cell layer of the dentate gyrus, and increased BrdU+O4+ oligodendrocytes in the SVZ. There was no change in the inflammatory infiltrates in response to anti-EGF Ab. Conclusions. Therapy with anti-EGF Ab ameliorates EAE via induction of neurogenesis and oligodendrogenesis. No immunosuppressive effect was found. Further investigation is needed to support these notions of beneficial effect of anti-EGF Ab in MS.

Increased neutralization capacity of TNF-α in sera of relapsing remitting multiple sclerosis patients is not related to soluble TNF-α receptors or anti-TNF-α autoantibody levels☆

The increased TNF-α levels in active lesions and CSF of multiple sclerosis (MS) patients and the beneficial effect of TNF-α blockade in animal models of MS led to the therapeutic usage of TNF-α antagonists. However, systemic TNF-α blockade exacerbated MS activity and was associated with new-onset MS when implemented for treating other autoimmune disorders. We employed a TNF-α neutralization bioassay and demonstrated that the capacity of sera from untreated and IFN-β-treated relapsing remitting MS patients to neutralize TNF-α is significantly higher than that of matched healthy controls. This finding was not related to sTNFRI, sTNFRII, or anti-TNF-α Abs levels.

High serum levels of BMP-2 correlate with BMP-4 and BMP-5 levels and induce reduced neuronal phenotype in patients with relapsing-remitting multiple sclerosis

Blockage of bone morphogenetic protein (BMP) signaling is required for differentiation of neurons and oligodendrocytes from neural stem cells (NSCs). Sera of untreated relapsing-remitting multiple sclerosis (RR-MS) patients expressed significantly higher levels of BMP-2 compared to sera of healthy controls. BMP-2 levels correlated with BMP-4 and -5 levels only in sera of untreated MS patients. Furthermore, sera of untreated patients inhibited the neuronal differentiation of RA-treated P19 cells, which was associated with induction of phospho-SMAD signaling pathway. These results suggest that BMP-2 sera levels may play a role in the failure of remyelination and neuro-regeneration in RR-MS.

Natural and induced immunization against CCL20 ameliorate experimental autoimmune encephalitis and may confer protection against multiple sclerosis

Th-17 type immune response that occurs in multiple sclerosis (MS) is linked to CCR6-CCL20 interaction. We confirmed the dependency on CCR6 in EAE development. Vaccination of mice with hCCL20, but not mCCL20, produced anti-murine CCL20 and ameliorated EAE. The EAE clinical score negatively correlated with anti CCL20 levels. A beneficial effect was transferred by sera from hCCL20-immunized mice. Immunized mice with cyclic peptide that include a bacterial outer membrane protein A (ompA), that share homology sequence with hCCL20 produced anti CCL20, anti ompA and anti-cyclic peptide. Immunization of mice with ompA or the cyclic peptide ameliorated EAE. The cyclic peptide inhibited CCL20 activity in an adhesion assay. A significantly higher level of anti CCL20 were found in healthy individuals compared to RR-MS patients. There was no similar difference for anti-CXCL10. Natural or induced immunization against CCL20 confer protection against EAE and may be beneficial in MS.

Differential screening-selected gene aberrative in neuroblastoma (DAN) is increased in the CSF of patients with MS and may be induced by therapy with interferon-β

Bone morphogenic proteins (BMPs) signaling blockade induce neurogenesis and oligodendrogenesis. Differential screening-selected gene aberrative in neuroblastoma (DAN) is a glycoprotein that antagonizes BMPs. We found that DAN levels were higher in CSF compared to serum in all participants. CSF-DAN levels were elevated in RR-and progresssive MS patients compared to controls. Moreover, serum-DAN levels were reduced in those patients, but elevated in IFN-β1a treated patients. The main source of DAN is apparently CNS- resident cells. The enhanced levels of CSF-DAN in MS patients suggest a tendency to induce neurogenesis/oligodendrogenesis in the patients CNS. Our results suggest an unreported mode of action of IFN-β1a.