Maria Kvarnström

Longitudinal interferon-β effects in multiple sclerosis: differential regulation of IL-10 and IL-17A, while no sustained effects on IFN-γ, IL-4 or IL-13.

BACKGROUND Recent studies in experimental models and in vitro indicate lowering of IL-17/Th17 as an important mechanism of interferon-beta (IFN-β) treatment in multiple sclerosis (MS). MATERIAL AND METHODS In this longitudinal study of MS patients (n=25), spontaneous and myelin antigen-induced secretion of IL-4, IFN-γ and IL-10 (ELISPOT), mitogen stimulated secretion of IL-13 and IL-17A (ELISA) and circulating cytokine levels (Luminex) were recorded at inclusion and after 1.5, 3, 6 and 12months of IFN-β treatment. RESULTS Early changes were noted for IL-4, while after one year of treatment the only recorded significant effects were a decrease in secreted IL-17A levels and an increase in IL-10 secreting cells. While IL-17A levels tended to be higher in non-responders (n=8), the decrease in IL-17A levels seemed to be more pronounced in responders (n=17) showing significantly lower IL-17A levels after one year as compared with non-responders. CONCLUSION IFN-β treatment seems to mainly affect IL-17/IL-10-associated pathways rather than the IFN-γ/IL-4 axis.

Elispot assay detection of cytokine secretion in multiple sclerosis patients treated with interferon-β1a or glatiramer acetate compared with untreated patients

The mechanisms behind the beneficial effects of interferon-b1a (IFN-b1a) and glatiramer acetate (GA) in the treatment of multiple sclerosis (MS) are still uncertain. A ltered cytokine patterns have been suggested including inhibition of proinflammatory cytokines like interferon-g (IFN-g) and enhancement of anti-inflammato ry cytokines such as interleukin-4 (IL-4). Twenty-nine patients with MS (10 untreated, nine treated with IFN-b1a and 10 with GA) were investigated with elispot of peripheral blood mononuclear cells. Spontaneous and myelin induced (myelin basic protein (MBP), myelin oligodendro cyte glycoprotein (MO G)-14-39 and MO G 63-87) IFN-g, IL-4, IL-5 and IL-10 secretion was studied. We found a significant reduction of spontaneous IFN-g, IL-4 and IL-5, but no difference in IL-10 secreting cells in both groups of treated patients compared with the untreated patients. Myelin-specific responses showed a significant decrease of IFN-g and an increase of IL-5, but no change in IL-4 and IL-10 secreting cells in treated compared with untreated patients. Both treatment groups revealed similar cytokine secretion patterns except for a more pronounced decrease of both spontaneous and MO G 14-39 induced IL-4 secretion in the IFN-b1a treated group. Thus, immunological effects of IFN-b1a and G A were similar showing that disease promoting Th1 (IFN-g) cells were reduced while the potentially beneficial Th2 response (IL-4) was maintained.

Elevated number of cells secreting transforming growth factor β in Guillain-Barré syndrome

We used ELISPOT and cell ELISA to study secretion of IL‐4, IFN‐γ, TGF‐β, IL‐6, and TNF‐α by circulating mononuclear cells during the course of Guillain‐Barré syndrome (GBS). Compared to healthy controls, patients with GBS had higher numbers of TGF‐β‐secreting cells and the number of individuals with myelin‐peptide‐induced IL‐4 and TGF‐β secretion was higher in the GBS group. No significant differences were seen concerning the predominantly pro‐inflammatory cytokines IFN‐γ, IL‐6 or TNF‐α. Our findings indicate a down‐regulatory role for TGF‐β and IL‐4 in GBS.

Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)

Monoclonal expansion of B cells and plasma cells, producing antibodies against ‘self’ molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström’s macroglobulinaemia and B‐type of chronic lymphocytic leukaemia (B‐CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN‐MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin‐specific B cell clones from peripheral blood of patients with PN‐MGUS, by selection of cells bearing specific membrane Ig‐receptors for myelin protein P0, using beads coated with P0. P0‐coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein–Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti‐P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti‐P0 antibodies were of IgM‐λ type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin‐associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA‐DR, which is compatible with properties of antigen‐presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0‐specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen‐presentation by these cells followed by T cell activation.

Transfer of myelin-specific cells deviated in vitro towards IL-4 production ameliorates ongoing experimental allergic neuritis

A causal role of IL‐4 (Th2) production for recovery in experimental allergic neuritis (EAN) was indicated by experiments where Th1‐like autoreactive cell populations, taken from the induction phase of the disease, were deviated to extensive secretion of IL‐4 in a selective fashion, by ex vivo stimulation with autoantigen in the presence of IL‐4. The deviated cells were adoptively transferred to EAN rats at a time just prior to the onset of clinical signs. This treatment ameliorated EAN compared with sham treatment. This therapeutic approach, with generation of autoreactive IL‐4‐secreting cells ex vivo followed by subsequent adoptive transfer, may become a new selective treatment of organ‐specific autoimmune diseases since, in contrast to previous attempts, it is done in a physiological and technically easy way.

Cytokine mapping in cerebrospinal fluid and blood in multiple sclerosis patients without oligoclonal bands

Objective: Since there are clinical and genetic differences between MS patients with intrathecal oligoclonal bands (OCB+) in the cerebrospinal fluid (CSF) compared with those without (OCB−), the aim was to find out if OCB− patients showed a different pattern of cytokine immune activation compared with OCB+ patients. Methods: The study included 25 MS patients (10 OCB− and 15 OCB+) and 13 controls. A panel of cytokines was measured; IL-1β, IL-6, IL-8/CXCL8, IL-10, TNF and GM-CSF in serum, CSF and in supernatants from polyclonally stimulated blood mononuclear cells, where also levels of IL-12p40, IL-13, IL-15, IL-17 and IFN-γ were measured. The concentrations of soluble (s) VCAM-1 and sCD14 were measured in serum and CSF. Results: In general, there were no extensive differences in cytokine concentrations between the OCB− and OCB+ groups. Conclusion: OCB− MS patients do not seem to constitute a separate entity concerning inflammatory parameters measured as cytokine concentrations in CSF and blood.

Myelin protein zero is naturally processed in the B cells of monoclonal gammopathy of undetermined significance of immunoglobulin M isotype: aberrant triggering of a patient’s T cells

Background Monoclonal gammopathy of undetermined significance of immunoglobulin M isotype is a condition with clonally expanded B cells, recently suggested to have an infectious origin. This monoclonal gammopathy is frequently associated with polyneuropathy and antibodies against myelin protein zero, whereas the role of the T cells remains largely unknown. We analyzed protein zero-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells. Design and Methods We used a well-characterized monoclonal gammopathy of undetermined significance-derived B-cell line, TJ2, expressing anti-protein zero immunoglobulin M. The ability of TJ2 cells to bind, endocytose, process, and present protein zero was investigated by receptor-clustering and immunofluorescence. The activation of protein zero-specific autologous T cells was studied by measuring interleukin-2 and interferon-γ with flow cytometry, immunobeads, and enzyme-linked immunospot assays. Results Surface-receptor clustering and endocytosis of receptor-ligand (immunoglobulin M/protein zero) complexes were pronounced after exposure to protein zero. Naturally processed or synthetic protein zero peptide (194–208)-pulsed TJ2 cells significantly induced interleukin-2 secretion from autologous T cells compared to control antigen-pulsed cells (P<0.001). The numbers of interferon-γ-producing T helper cells, including CD4+/CD8+ cells, were also significantly increased (P=0.0152). Affinity-isolated naturally processed myelin peptides were potent interferon-γ stimulators for autologous peripheral blood mononuclear cells, but not for control peripheral blood mononuclear cells. Conclusions We show for the first time that myelin protein zero is naturally processed in B cells from monoclonal gammopathy of undetermined significance of immunoglobulin M isotype, acting as aberrant antigen-presenting cells in activation of a patient’s T helper cells. Our findings cast new light on the important role of autoreactive protein zero-specific B cells in the induction of the pathogenic T-cell responses found in nerve lesions of patients with monoclonal gammopathy of undetermined significance with peripheral neuropathy.