Studies on co-stimulatory molecules, chemokines and chemokine receptors in neuroimmunological diseases

Abstract

Background: Multiple sclerosis (MS) and myasthenia gravis (MG) are characterised by activation of T cells directed against self antigens. Such autoreactive T cells are considered to play a central role in destruction of myelin in MS and in damage of neuromuscular junctions in MG. Two groups of molecules are involved in function of autoreactive T cells: (1) co-stimulatory molecules provide essential signals for T cell activation; (2) once activated, T cells are directed to target organs by chemokine and chemokine receptor interactions. Antigen presenting cells, such as dendritic cells (DC), can also be attracted by chemokines to inflammatory foci and contribute to T cell activation in situ.

The aim of this thesis was to study expression of co-stimulatory molecules, chemokines and chemokine receptors in MS and MG, and to evaluate the functional importance of chemokine receptors for migration of DC in MS. Specific aims of the study: (1) To study expression of CD40/CD40L co-stimulatory molecules in MS; (2) To evaluate expression of co-stimulatory molecules (CD40/CD40L, CD28/CD80-CD86) in MG; (3) To analyse expression of inflammatory chemokines (MCP-1, RANTES, IP-10) and chemokine receptors (CCR5, CXCR3) in MS; (4) To study expression of inflammatory chemokine receptors (CCR1, CCR5, CXCR1, CXCR4) by immature monocyte-derived dendritic cells (moDC) in MS and its functional importance; and (5) To evaluate effects of IFN-beta, a cytokine that beneficially modulates the course of MS, on these immunological variables.

Materials: Blood and CSF MNC were obtained from patients with untreated MS, IFN-beta treated MS, optic neuritis (ON), other inflammatory neurological diseases (OIND), other non-inflammatory neurological diseases (OND), MG and healthy controls (HQ).

Methods: (1) Flow cytometry to detect MNC expressing co-stimulatory molecules in blood and chemokine receptors in blood and CSF; (2) In situ hybridization to detect MCP-1 and RANTES mRNA expression by blood and CSF MNC; (3) ELISA to determine concentrations of RANTES and IP-10 in plasma and CSF; (4) In vitro generation of moDC in the presence of GM-CSF and IL-4; and (5) Chemotaxis assay to study chemotactic ability of moDC.

Results: MS patients had elevated numbers of CD40L+ blood T cells compared to OND and healthy subjects. Patients with MG had increased numbers of T cells expressing CD80 and CD86, and monocytes expressing CD80, in blood compared to healthy subjects. Elevated expression of co- stimulatory molecules in MS and MG may suggest continuous T cell activation in both diseases. MS and ON patients, representing early MS, had elevated numbers of CCR5+ (receptor for RANTES) and CXCR3+ (receptor for IP-10) T cells vs. healthy subjects in blood, and vs. OND patients in CSF, indicating that expression of CCR5 and CXCR3 is altered already at the early stage of the disease. MS patients also had elevated numbers of CCR5+moDC compared to healthy subjects, resulting in increased chemotaxis in response to RANTES. Elevated expression of chemokine receptors by T cells and moDC in MS may lead to their active recruitment to the MS lesions in response to inflammatory chemokines.

IFN-beta in vivo and in vitro modulated expression of CD40L and chemokine receptors. Augmented levels of CD40L+, CCR5+ and CXCR3+ T cells were confined to untreated MS patients, and not observed in patiens treated with IFN-beta. Follow-up study of patients with MS before and during treatment with IFN-beta showed decreasing numbers of CD40L+ T cells. IFN-beta in vitro reduced CCR1 and CXCR1 chemokine receptor expression by moDC and chemotactic responses.

Conclusion. (1) NIS is associated with augmented expression of co-stimulatory molecules and chemokine receptors, which may indicate their role in initiation and progression of immune responses in MS. IFN-beta treatment in vivo is associated with decreased co-stimulatory molecule expression. IFN-beta treatment both in vivo and in vitro is also associated with decrease of chemokine receptor expression. (2) Elevated expression of co-stimulatory molecules may have an implication for immune cell activation and tolerance breakdown in MG.