Interferon-γ-induced calcium influx in T lymphocytes of multiple sclerosis and rheumatoid arthritis patients: a complementary mechanism for T cell activation?

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Abstract

Autoreactive T lymphocytes are considered to play a crucial role in orchestrating a chronic inflammation in the central nervous system (CNS) of multiple sclerosis (MS) patients and in the joints of rheumatoid arthritis (RA) patients. However, it has been suggested that the majority of T cells in the immune infiltrate are nonspecifically recruited into the CNS and into the inflamed joint. In addition, several lines of evidence suggest an important role for interferon-γ (IFN-γ) in the pathogenesis of MS and RA. We have studied whether peripheral blood T cells from patients with autoimmune diseases are more susceptible to activation in the presence of IFN-γ. The results indicate that IFN-γ mediates a sustained elevated [Ca2+]i in T cells of (active) MS and RA patients as compared to healthy controls and patients with common viral infections. No [Ca2+]i increase was observed in Ca2+-free medium, excluding an effect of IFN-γ on Ca2+-release from intracellular stores. Although the IFN-γ-activated Ca2+-influx is insufficient to induce T cell proliferation in vitro, our data indicate a significantly augmented proliferation in response to suboptimal doses of PHA in the presence of IFN-γ. This study suggests that the IFN-γ-induced Ca2+-influx can act as a complementary mechanism in the activation of blood T lymphocytes from MS and RA patients.

Introduction

Although the pathogenesis of multiple sclerosis (MS) is still unresolved, several lines of evidence indicate that a T cell-mediated pathway directed against myelin antigens may be responsible for the initiation of the inflammation in the central nervous system (CNS). Myelin reactive T lymphocytes are considered to play a crucial role in orchestrating an immunopathological cascade that culminates in damage to the myelin sheath, oligodendrocytes and axons (Stinissen et al., 1997). Activated autoreactive T lymphocytes may also play a crucial role in the development of rheumatoid arthritis (RA), a chronic inflammatory disease of the joints. The crucial site of destruction in RA is the pannus, a hypertrophic, highly vascularized synovial membrane consisting of hyperplastic synoviocytes as well as activated macrophages, fibroblasts and lymphocytes Zvaifler and Firestein, 1994, Feldmann et al., 1996. As in MS, T cells specific for unidentified joint antigens may initiate and maintain chronic inflammation in the joints of patients with RA.

It has been noted that the autoimmune infiltrate in experimental autoimmune encephalomyelitis during exacerbations of disease contains a highly diverse collection of T and B cells and macrophages. Autoreactive T cells appear to be only a small component of this infiltrate (Steinman, 1996). Similar observations were made in RA patients. T cell receptor analysis of RA patients indicates a highly diverse T cell repertoire in the joints of affected patients (Dedeoglu et al., 1993). It has been suggested that the majority of T lymphocytes are nonspecifically recruited into the inflamed joint of RA patients, and into the CNS of MS patients. However, the mechanisms underlying the peripheral activation of T lymphocytes in MS and RA and their clinical relevance remain largely unknown. Recent reports suggest that some cytokines may promote activation of lymphocytes, via a mechanism distinct from the TCR-triggered pathway. The combination of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) can activate resting CD4+ T cells (Unutmaz et al., 1994) while interferons can activate CD8+ T cells (Tough and Sprent, 1996). In this respect, interferon-γ (IFN-γ) may play a role in the pathogenesis of MS and RA. Increased production of IFN-γ by cerebrospinal fluid (CSF) cells as well as by peripheral blood (PB) lymphocytes precedes clinical attacks in MS Rieckmann et al., 1994, Olsson et al., 1990, Hohnoki et al., 1998, and administration of recombinant IFN-γ to MS patients leads to a worsening disease course (Panitch et al., 1987a). These data strongly suggest a pivotal role of this Th1 cytokine in disease progression in MS Popko and Baerwald, 1999, Benveniste, 1997. IFN-γ may also be involved in the pathophysiological processes of RA, since IFN-γ-producing cells are found at higher levels in synovial fluid (SF) than in peripheral blood (PB) of RA patients Bucht et al., 1996, Dolhain et al., 1996a. Significantly higher percentages of IFN-γ-producing T cells were found in the PB CD4+ subset of RA patients as compared to healthy controls (van der Graaff et al., 1999). Finally, evidence for an increased IFN-γ/IL-4 ratio in SF compared to PB supports a predominantly Th1 environment in the RA synovium Dolhain et al., 1996b, Steiner et al., 1999, Canete et al., 2000.

The aim of the present study was to analyze whether T lymphocytes from patients with autoimmune diseases are more susceptible to T cell activation in the presence of the pro-inflammatory cytokine IFN-γ. A key component in the activation of T lymphocytes is a prolonged increase of the intracellular free calcium concentration ([Ca2+]i). Activation of T cells in vivo generally results from the occupancy of the T cell receptor (TCR) by immunogenic peptides bound to MHC molecules and interaction with co-stimulating molecules. Subsequent molecular events lead to an elevation of [Ca2+]i. This elevation must be sustained for several hours to maintain NF-AT (nuclear factor-activated T cells) transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation Lewis and Cahalan, 1995, Timmerman et al., 1996, Crabtree and Clipstone, 1994. We studied the effects of IFN-γ on Ca2+-release from intracellular stores and Ca2+-influx from the extracellular environment in peripheral blood T lymphocytes from patients with autoimmune diseases (MS and RA). In the control group, we studied patients with common viral infections (acute inflammation) and healthy donors. Fluorescence imaging microscopy was used to monitor the intracellular [Ca2+]i signal in individual T cells. The results indicate that IFN-γ exerts a different effect on Ca2+-homeostasis with respect to the magnitude and the pattern of the [Ca2+]i signal in T cells of MS and RA patients as compared to control subjects. Although the IFN-γ-activated Ca2+-influx as observed in MS and RA T cells seems to be insufficient to induce T cell proliferation, our data indicate that proliferative responses caused by suboptimal doses of PHA can significantly be augmented in the presence of IFN-γ. This study suggests that the Ca2+-influx stimulated by IFN-γ can act as a complementary mechanism in the activation of T cells in the blood of patients affected by autoimmune disorders such as MS and RA.

Section snippets

Patients and controls

Peripheral blood mononuclear cells were obtained from 36 patients with definite MS and 15 patients with definite rheumatoid arthritis (RA), 8 patients with flu-like symptoms and 12 healthy controls. Table 1 lists the clinical characteristics of the MS and RA patients. Twenty-eight MS patients had relapse-remitting (RR) MS (mean EDSS=3.3), while eight patients had secondary progressive (SP) MS (mean EDSS=6.8). Twelve RR–MS patients were in a clinically active phase (relapse) while the remaining

Experimental design and definitions

When T cells were stimulated with 1 pg/ml IFN-γ in Ca2+-containing medium, we observed a gradual increase in [Ca2+]i signal in MS T cells, which did not appear in T cells of normal subjects (NS) (Fig. 1). Since these results were similar to the fluorimetric [Ca2+]i measurements in Ca2+-containing medium of Martino et al. (1994), we performed more experiments according to the Ca2+-free/Ca2+-reintroduction protocol of Martino et al. (1996) with minor modifications concerning incubation times.

Discussion

Fluorescence imaging microscopy was used to study the [Ca2+]i signal patterns in individual T cells of patients with autoimmune diseases and controls. The data demonstrate significantly higher Ca2+-influxes in T lymphocytes of patients with (active) MS and RA as compared to NS and flu patients after stimulation with IFN-γ in Ca2+-containing medium as well as in Ca2+-free/Ca2+-reintroduction conditions. Furthermore, the data show that exposure to IFN-γ does not signal via Ca2+-discharge from

Acknowledgements

We thank Dr. S. Despa, Dr. M. Vandeven, Dr. A. VanderBorght, Dr. N. Hellings and A. Van der Aa for helpful discussions. We also thank Dr. Bullen for providing the blood samples of the flu patients. This work was supported by grants from the Belgian ‘Wetenschappelijk Onderzoek Multiple Sclerose’ (WOMS) foundation and ‘Limburgs Universitair Centrum’ (LUC), Belgium.

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