Natalia Teleshova

CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms.

CTLA-4, expressed mainly on activated T cells, helps maintain, through its inhibitory function, immune-system homeostasis. Polymorphisms in the CTLA-4 gene (CTLA4) are known to be important in several autoimmune diseases, including multiple sclerosis (MS). Here, we have performed genotyping for CTLA4 polymorphisms, and investigated expression by peripheral blood mononuclear cells of CTLA-4 mRNA and protein, in patients with MS and myasthenia gravis and in healthy controls. Expression levels for mRNA and protein were similar in the patient and control groups; however, there was a clear relationship between genotype and CTLA-4 expression. Specifically, individuals carrying thymine at position −318 of the CTLA4 promoter (T−318) and homozygous for adenine at position 49 in exon 1 showed significantly increased expression both of cell-surface CTLA-4 after cellular stimulation and of CTLA-4 mRNA in non-stimulated cells. The association was seen most clearly for unsorted CD3+ cells and was absent in the CD8+ subset. The T−318 allele has been shown to be negatively associated with susceptibility to MS in an earlier study by our group. Thus, we propose that the susceptibility-influencing role of CTLA4 in MS may be related to genotypically conditioned promoter function, whereby high gene expression may decrease the risk of disease.

Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections

Dendritic cells (DC) accumulate in the CNS during inflammation and may contribute to local immune responses. Two DC subsets present in human cerebrospinal fluid (CSF) are probably recruited from myeloid (CD11c(+)CD123(dim)) and plasmacytoid (CD11c(-)CD123(high)) blood DC. In bacterial meningitis and especially in Lyme meningoencephalitis, numbers of myeloid and plasmacytoid DC in CSF were increased, compared to non-inflammatory neurological diseases, and correlated with chemotactic activity of CSF for immature monocyte-derived DC (moDC). Multiple DC chemoattractants, including macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-1, MCP-3, RANTES and stromal cell-derived factor (SDF)-1alpha were elevated in CSF in these two neuroinfections. Chemotaxis of immature moDC induced by these CSFs could be partially inhibited by mAbs against CXCR4, the receptor for SDF-1alpha, and CD88, the receptor for C5a. SDF-1alpha present in CSF also chemoattracted mature moDC, which in vivo could correspond to a diminished migration of antigen-bearing DC from the CSF to secondary lymphoid organs. Regulation of DC trafficking to and from the CSF may represent a mechanism of controlling the CNS inflammation.

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.

Multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines

Recent evidence emphasises a pivotal role for dendritic cells (DC) in the control of immunity by priming and tolerising T cells. DC capture and process antigens, express co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. In multiple sclerosis (MS), autoreactive T cells are proposed to play a pathogenic role by secreting pro-inflammatory cytokines, but studies on DC are lacking. To evaluate the involvement of DC in patients with MS, a modified procedure was used to prepare DC from blood of patients with MS and healthy subjects. DC were found to be potent stimulators of T cells in allogeneic and, to a lesser extent, in autologous mixed leukocyte reaction (MLR). Enzyme-linked immunospot (ELISPOT) assays were adopted to determine levels of IFN-gamma, TNF-alpha, IL-6 and IL-10 secreting DC vs. mononuclear cells (MNC). Proportionally more DC than MNC secreted IFN-gamma and IL-10 in both MS and healthy subjects. Patients with MS had higher levels of IFN-gamma, TNF-alpha and IL-6 secreting DC than healthy subjects. The differences for IFN-gamma and TNF-alpha secreting cells were confined to the subgroup of untreated MS patients and not observed in the subgroup examined during ongoing treatment with IFN-beta. Circulating DC secreting pro-inflammatory cytokines may represent another focus for the study of both immuno-pathogenesis and therapeutic interventions in MS.

Inflammation in the Central Nervous System: the Role for Dendritic Cells

Dendritic cells (DCs) are a subclass of antigen‐presenting cells critical in the initiation and regulation of adaptive immunity against pathogens and tumors, as well as in the triggering of autoimmunity. Recent studies have provided important knowledge regarding distribution of DCs in the central nervous system (CNS) and their role in intrathecal immune responses. DCs are present in normal meninges, choroid plexus, and cerebrospinal fluid, but absent from the normal brain parenchyma. Inflammation is accompanied by recruitment and/or development of DCs in the affected brain tissue. DCs present in different compartments of the CNS are likely to play a role in the defence against CNS infections, and also may contribute to relapses/chronicity of CNS inflammation and to break‐down of tolerance to CNS autoantigens. CNS DCs can therefore be viewed as a future therapeutic target in chronic inflammatory diseases such as multiple sclerosis.

Multiple sclerosis and optic neuritis: CCR5 and CXCR3 expressing T cells are augmented in blood and cerebrospinal fluid

Abstract. A role for chemokines as mediators of Th1 cell recruitment to the central nervous system (CNS) is probable in MS. Therefore we studied expression of Th1-related CCR5 and CXCR3 chemokine receptors in patients with MS and controls. Patients with untreated MS had elevated percentages of CCR5 and CXCR3 expressing T cells vs. healthy controls (HC) in blood, and vs. other non-inflammatory neurological diseases (OND) patients in CSF. Such elevation was not found in MS patients examined during ongoing treatment with IFN-β. Patients with optic neuritis (ON), a common first manifestation of MS, had elevated percentages of CXCR3 expressing T cells in blood compared with HC, and of CCR5 expressing T cells in CSF compared with OND patients. High chemokine receptor expression may be one prerequisite for Th1 cells to migrate to the CNS. Inhibition of chemokine receptor expression may constitute a potentially important therapeutic effect of IFN-β.

Elevated expression of CCR5 by myeloid (CD11c+) blood dendritic cells in multiple sclerosis and acute optic neuritis

Myeloid and plasmacytoid dendritic cells (DC) are present in cerebrospinal fluid (CSF) in non‐inflammatory neurological diseases (NIND) and elevated in clinically definite multiple sclerosis (MS) and in early MS – acute monosymptomatic optic neuritis (ON). Here, we show that expression of CCR5, a chemokine receptor for regulated on activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)‐1α/β, is elevated on blood myeloid (CD11c+) DC in MS and ON compared to non‐inflammatory controls. In contrast, expression of CXCR4, a receptor for stromal cell‐derived factor (SDF)‐1α, is similar in all groups. Blood myeloid DC from MS patients respond chemotactically to RANTES and MIP‐1β, which are expessed in MS lesions. In active MS and ON, expression of CCR5 by myeloid DC in blood correlates with numbers of these cells in CSF. Thus, elevation of CCR5 may contribute to recruitment of myeloid DC to CSF in MS and ON. Recruitment of plasmacytoid DC to CSF appears to be CCR5‐independent.

Multiple sclerosis: a study of chemokine receptors and regulatory T cells in relation to MRI variables

Magnetic resonance imaging (MRI) remains the most valuable tool for monitoring disease activity and progression in patients with multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system (CNS) with presumably autoimmune etiology. Chemokine receptors have been implicated in MS as key molecules directing inflammatory cells into the CNS. Regulatory (CD4+CD25+) T cells (Tr cells) are important in suppressing autoimmunity, and their absolute or functional deficit could be expected in MS. In the present study, venous blood was obtained from MS patients concurrent with MRI examination of the brain, and expression of chemokine receptors CCR1, CCR2, CCR5, CXCR3 and CXCR4 by CD4 T cells and monocytes, proportions of Tr cells, as well as expression of CD45RO, CD95, CTLA‐4, HLA‐DR and interleukin (IL)‐10 by Tr cells and non‐Tr (CD25−) CD4 T cells was analyzed by flow cytometry. Surface expression of CXCR3 by CD4 T cells was downregulated in the group of patients with high lesion load (LL) on T2‐weighted images and gadolinium (Gd)‐enhancing lesions on T1‐weighted images, compared to the group with high LL and no Gd‐enhancing lesions, and to the group with low LL, suggesting internalization of CXCR3 due to the release of its chemokine ligand (IP‐10/CXCL10) from active MS lesions. Proportions of Tr cells amongst all CD4 T cells, and expression of IL‐10 by Tr cells were increased in the patients with high LL and Gd‐enhancing lesions. These results suggest that there is correlation between MRI parameters, chemokine receptor expression and the status of circulating Tr cells in MS, but further studies need to discriminate between pathogenetically relevant and bystander phenomena.

No evidence for elevated numbers of mononuclear cells expressing MCP-1 and RANTES mRNA in blood and CSF in multiple sclerosis.

The perivascular accumulation of mononuclear cells (MNC) in brain white matter is critical in the development of active lesions in multiple sclerosis (MS). Chemokines contribute to leukocyte recruitment by increasing the adhesiveness of integrins expressed on leukocytes and by promoting migration through endothelium and extracellular matrix. By using an in situ hybridization technique, it was possible to enumerate blood and CSF MNC expressing mRNA for the two CC chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cells, expressed and secreted) in MS patients and controls. No differences in numbers of blood MNC expressing MCP-1 or RANTES could be found in MS patients compared to healthy individuals or patients with acute aseptic meningoencephalitis (AM). High numbers of CSF MNC expressing MCP-1 and RANTES were found in some MS patients, but also in patients with AM. This shows that elevated numbers of MCP-1 and RANTES mRNA expressing CSF MNC are not specific for the inflammatory process in MS. We conclude that there is no evidence for a systemic dysregulation of the CC chemokines MCP-1 and RANTES in MS.