Interferon-γ induces T lymphocyte proliferation in multiple sclerosis via a Ca2+-dependent mechanism

The intracellular mechanisms underlying T lymphocyte activation leading to demyelination in multiple sclerosis (MS) have not yet been clarified. We have recently reported that interferon (IFN)-γ activates a novel trans-plasmalemma Ca²⁺ influx on T lymphocytes (mainly CD4⁺) from patients with MS which induces intracellular Ca²⁺ ([Ca²⁺]_i) elevation. Since Ca²⁺ is an essential second messenger in regulating transcription of T lymphocyte activation genes, we have evaluated how [Ca²⁺]_i elevation due to the activity of this particular influx affects T lymphocyte proliferative behaviour in 12 influx-positive relapsing-remitting MS (RR-MS) patients. Fourteen influx-negative RR-MS patients and 14 healthy donors were used as controls. In lymphocytes from healthy controls, a significant correlation (r = 0.62; P <0.001) was found between [Ca²⁺]_i levels and proliferation rate after phytohemagglutinin (PHA) stimulation. Sustained proliferation was induced in T lymphocytes by ≥ 10 μg/ml of PHA, a dose leading to a [Ca²⁺]_i increase of at least 45% over basal level. Similar [Ca²⁺]_i elevations were obtained when ≥ 10 μg/ml of PHA were used on cells from RR-MS patients. However, T lymphocytes from RR-MS patients, but not from healthy donors, proliferated also in response to 1 μg/ml of PHA, indicating a state of preactivation. Moreover, l μg/ml of PHA used in combination with suboptimal doses of IFN-γ (5 UI/ml) doubled the proliferation rate of influx-positive MS cells, but not influx-negative MS cells or cells from healthy donors compared to the values obtained using PHA alone (P <0.01). In two RR-MS patients followed serially for 5 or 7 months, respectively, [Ca²⁺]_i elevations were significantly associated with increased proliferation due to the IFN-γ-activated Ca²⁺ influx observed when 5 Ul/ml of IFN-γ were added to PHA (1 μg/ml)-stimulated cultures (P <0.001). Our data suggest that the IFN-γ-activated Ca²⁺ influx operates as a complementary mechanism for T lymphocyte [Ca²⁺]_i increase in MS patients. Its activity lowers the amount of [Ca²⁺ provided by the PHA-stimulated, 1, 4, 5-trisphosphate-mediated pathway and needed to trigger T lymphocyte activation. The presence of IFN-γ-activated influx-positive cells in patients with MS and their exaggerated responsiveness to cytokines on a background of immune pre-activation provides a potential explanation for the observation that common viral infections, able to stimulate IFN-γ production, often precede clinical relapses in patients with MS.