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|Title: ||Study of Ca2+-fluxes in IFN-gamma-exposed T cells of patients with MS and control subjects|
|Authors: ||Buntinx, M.|
|Issue Date: ||2000|
|Citation: ||Multiple sclerosis, 6 (Supplement 1), p. S40-S40|
|Abstract: ||Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. T-lymphocytes are important mediators in the pathogenesis of MS. Their activity is modulated by a complex network of cytokines, in which interferon-gamma (IFN-gamma) is considered essential. We investigated the presence of an IFN-gamma-activated Ca2+-influx in T-cells of MS patients and control subjects. The study group (n=63) consisted of patients with stable and active RR-MS and CP-MS. Stable and active rheumatoid arthritis (RA) patients, flu patients and normal subjects (NS) were studied as controls. T-cells were isolated from PBMC by removing B cells and monocytes using anti-CD19 and anti-CD14 monoclonal antibodies and dynabeads. The depletion was confirmed by flow cytometry. T-cells loaded with the Ca2+-indicator FURA-2 AM were consecutively incubated in Hank's Balanced Salt Solution (2 mM Ca2+), HBSS supplemented with 3 mM EGTA, HBSS + 1 pg/ml IFN-gamma +3 mM EGTA and finally in HBSS + 1 pg/ml IFN-gamma. We used fluorescence imaging microscopy to measure the intracellular [Ca2+]i. No [Ca2+] increase was observed in Ca2+-free medium. As T-cells were incubated in Ca2+-containing medium, we observed a Ca2+-influx peak within 3 min, which dropped to a specific [Ca2+]i-level depending on the patient's diagnosis. Peak values observed within three minutes after Ca2+-reintroduction were higher in patients than in NS. The peak size is probably controlled by CRAC-channels. Experiments with and without IFN-gamma stimulation show that IFN-gamma induces a sustained higher [Ca2+]i in RA and MS patients but not in NS and flu patients. We defined D-influx for each individual cell as the difference between the [Ca2+]i 6-10min after Ca2+-reintroduction and the initial [Ca2+]i. ANOVA for nested unbalanced three-factor design was applied on the D-influx dataset. The Ca2+-influx in T-cells of MS and RA patients was significantly higher than in NS. The majority of stable MS patients showed lower Ca2+-influxes compared to active MS patients. We showed that T-lymphocytes from (active) MS and RA patients are more susceptible to IFN-gamma-induced activation. Our data argue for an increased activation status of T-cells in autoimmune disorders like MS and RA.|
|Type: ||Journal Contribution|
|Appears in Collections: ||Research publications|
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