High-Dose Methylprednisolone Therapy in Multiple Sclerosis Induces Apoptosis in Peripheral Blood Leukocytes

Abstract

Apoptosis is supposed to contribute to the elimination of T cells from the inflamed central nervous system in the natural disease course of multiple sclerosis (MS). In the animal model experimental autoimmune encephalomyelitis, T-cell apoptosis can be induced by high-dose glucocorticoid (GC) administration. To study the effects of intravenous high-dose GC therapy in MS on T-cell apoptosis ex vivo. Sixty-six patients with MS (28 with relapsing-remitting MS, 22 with secondary chronic progressive MS, and 16 with primary chronic progressive MS) and 16 control patients receiving corticosteroids for other disorders were included in the study. Blood samples were collected before and immediately after the first infusion of 500 to 1000 mg of methylprednisolone given during 2 hours in the early morning. Gradient-isolated peripheral blood leukocytes (PBLs) were cultured, unstimulated, with corticosteroids (positive control), the mitogen phytohemagglutinin, or anti-T-cell receptor monoclonal antibody. For investigation of apoptosis, PBLs were cultured overnight and analyzed by immunoflow cytometry using TUNEL (terminal transferase-mediated dUTP biotin nick end labeling) or annexin labeling in combination with CD4, CD8, CD22, CD56, or bcl-2 staining. Proliferation was measured by (3)H-thymidine incorporation. For cytokine determination, supernatants were collected after 48 hours of culture. After in vivo corticosteroid treatment, apoptosis of unstimulated PBLs was markedly and significantly augmented in all 3 MS subgroups. Fluorescence-activated cell sorter analysis showed that apoptosis affected predominantly CD4 T cells. Natural killer cells showed a relative increase after GC therapy without a change in the rate of apoptotic cells. Expression of bcl-2 in T-cell subpopulations was not significantly modified by high-dose GC therapy. Culture supernatants of T-cell receptor-stimulated PBLs after GC therapy contained lower concentrations of interleukin 2, interferon gamma, and tumor necrosis factor alpha than those from PBLs taken before pulse therapy. Similar changes in the rate of apoptosis and cytokine production were seen in controls. Corticosteroid pulse therapy is a strong inducer of leukocyte apoptosis. Induction of apoptosis might contribute to the down-regulation of T-cell activity and thereby terminate inflammation in the central nervous system.