ABSTRACT
Type I Interferon (IFN-alpha/beta) therapy has altered the natural course of multiple sclerosis. In this paper we evaluate the possible molecular mechanisms involved in the in vitro effects of IFN-alpha/beta on peripheral blood mononuclear cells from patients with clinically definite Relapsing-Remitting Multiple Sclerosis. The total RNA from IFN-alpha, IFN-beta treated cells and untreated cells was extracted and amplified for CD86, CD28, CTLA-4, TNF-alpha, IFN-gamma, CCL2, CCR5, IL-13, MMP-9, TIMP-1, CD25, TGF-beta, IL-10 and the transcriptional factor Foxp3 by Reverse Transcription-Polymerase Chain Reaction and the CD4+CD25high subset was evaluated using flow cytometry. In general, there were no significant differences concerning the modulation of the genes studied in the response to IFN-alpha and IFN-beta treatments, which suggest a similar mechanism of action for both interferons. However, we found a significant increment in IFN-gamma expression after IFN-alpha but not after IFN-beta treatments. The in vitro treatment of mononuclear cells from multiple sclerosis patients with both interferons significantly increased the CD25 mRNA. Furthermore, we observed a CD25/Foxp3 correlation and an increment of the CD4+CD25high subset, indicating that the induction of regulatory T cells could be a crucial mechanism involved in the type I interferon effects.
Subject(s)
Cytokines/metabolism , Interferon Type I/immunology , Leukocytes, Mononuclear/immunology , Multiple Sclerosis/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Antigen Presentation , Blood-Brain Barrier , Cells, Cultured , Cytokines/immunology , Gene Expression , Humans , Interferon Type I/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Multiple Sclerosis/metabolism , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/metabolismABSTRACT
In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3). The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity. Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors.
