Single-cell analysis of cytokine production shows different immune profiles in multiple sclerosis patients with active or quiescent disease

Abstract

Peripheral blood mononuclear cells of multiple sclerosis (MS) patients were stimulated with myelin basic protein (MBP) together with anti-CD28 monoclonal antibody and staphylococcal enterotoxin B to optimize cytokine production by antigen-specific cells. Type 1 (IL-2, IL-12, IFNγ) and pro-inflammatory (TNFα, IL-1β, IL-6) cytokines were augmented in CD4+, CD8+, and CD14+ cells of acute MS patients and of patients undergoing disease reactivation. These cytokines were reduced in IFNβ-treated and in stable MS patients; type 2 cytokines (IL-4, IL-10) were increased in these patients. Similar immune profiles are seen in MS patients in whom remission is naturally or pharmacologically (IFNβ) achieved. Cytokine alterations are particularly evident in CD14+ cells, underlying their critical role in the modulation of the immune response.

Introduction

Multiple sclerosis (MS) is a chronic neurologic disease characterized by multifocal inflammation and damage involving the myelin sheath. MS patients present a variety of clinical patterns, the most common of which is defined relapsing–remitting (RR): acute disease episodes and remission phases are typically observed in MS RR patients Lublin and Reingold, 1996, Thompson et al., 1991. MS is suggested to be associated with a multistep and multifactorial pathogenesis Poser, 1993, Martin and McFarland, 1995, Giovannoni and Hartung, 1996. In particular, MS could be due to an (auto) immune process triggered by one or more environmental factors, among which viruses are suspected of playing an important role Kurtzke, 1987, Dalgleish, 1997. An immunopathologic mechanism, mainly mediated by the activation of cell-mediated immunity (CMI), has repeatedly been linked to the destruction of the myelin sheath Martin and McFarland, 1995, Steinman, 1996. Cytotoxic T lymphocytes (CTL) specific for antigenic epitopes of myelin basic protein (MBP) are present but innocuous in peripheral blood of normal individuals Martin et al., 1990, Pette et al., 1990. It is postulated that because of molecular mimicry with cross-reacting and yet undefined epitopes, these CTL would be activated, tolerance broken, and disease initiated or reactivated Wucherpfenning and Strominger, 1995, Wucherpfenning et al., 1994. In particular, an autoimmune T cell attack against myelin antigens within the central nervous system (CNS) is considered to be the cellular onset of an inflammatory sequence resulting in MS in humans, and in experimental autoimmune encephalomyelitis (EAE) in susceptible strains of rats and mice Martin and McFarland, 1995, Steinman, 1996.

Activated lymphocytes produce cytokines, and cytokines have repeatedly been suggested to be involved in the pathogenesis of MS. To summarize: (1) increased levels of TNFα and IFNγ are present in MS patients with acute disease Olsson et al., 1990, Link et al., 1994, Rieckmann et al., 1995, Navikas et al., 1996; (2) the number of TNFα and IFNγ mRNA-expressing lymphocyte and dendritic cells is different in MS patients in different phases of disease activity (Rieckmann et al., 1994; Arnson, 1996); (3) MBP-stimulated T lymphocytes of MS patients produce high quantities of IFNγ Olsson et al., 1992, Sun et al., 1991a, Sun et al., 1991b; and (4) production of IL-12 p70 by PBMC is increased in MS patients Ferrante et al., 1998, van Boxel-Dezaire et al., 1999. Additionally, proteolipid protein-specific and IFNγ-producing peripheral T cells are greatly augmented in MS patients (Pelfrey et al., 2000). Finally, transcriptional profiling of a panel of genes in brain specimens from MS individuals showed that, along with several Th1 and proinflammatory molecules, some Th2 genes are up-regulated in the brain of these patients (Baranzini et al., 2000).

Multiple cell types including T lymphocytes and antigen presenting cells produce cytokines (rev. in Arai et al., 1990). In particular, cytokine production by APC plays a critical role in the initiation and modulation of the immune response (Moser and Murphy, 2000). Hence, IL-12-producing APC are suggested to initiate CMI responses (Trinchieri, 1995). In contrast, IL-10-producing APC have a tolerogenic effect on allogeneic T cell responses, and IL-10-constitutively-expressing-transgenic mice fail to mount antigen-specific T and B cell responses (rev. in Moore et al., 1993).

The frequency of cytokine-secreting antigen-specific immune cells is extremely low. To bypass this problem a European Concerted Action (European Union Concerted Action, 1999) suggested an experimental protocol finalized at the amplification of the frequency of these cells in the peripheral blood. Peripheral blood cells are stimulated by antigens in the presence of anti-CD28 (which adds an essential co-stimulatory signal) and of staphylococcal enterotoxin B (maximizing the function of antigen presenting cells)(Bright et al., 1999). This protocol results in an amplification of the frequency of cytokine-producing antigen-specific cells, which is directly proportional to their original frequency. We applied this protocol to examine whether stimulation of PBMC of MS patients would result in the detection of diverse cytokine profiles. In particular we examined the frequency of type 1 (IL-2, IL-12, IFNγ), type 2 (IL-4, IL-10), and pro inflammatory (TNFα, IL-1β, IL-6) cytokines—producing immune cells in active (AMS) and quiescent (SMS) MS. Additionally, in longitudinal studies, we verified whether disease relapse would result in changes in cytokine profiles of MBP-specific cells and whether pharmacological-induced control of disease (IFNβ) would be associated with the development of an immune pattern similar to the one detected in SMS patients.