| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
Cathepsin E (CE, EC 3.4.23.34) is an intracellular aspartic protease of pepsin family, which is highly homologous to the lysosomal aspartic protease cathepsin D (CD, EC 3.4.23.5). In contrast to CD, CE has a limited distribution in tissues and cell types such as lymphoid tissues, gastrointestinal tracts, urinary organs and microglia. CE was mainly localized in the endosomal structures of microglia, whereas CE was localized in various cellular compartments such as the plasma membrane, the endoplasmic reticulum and the Golgi apparatus of the other cell types and tissues. Thus it is conceivable that the mature form of CE is closely associated with endosomal locarization of this enzyme because proform of CE appears to be converted into mature form only after entering in acidic compartments. Microglia are known to interact with ivaded CD4+ T helper cells in the central nervous system. Through these interactions, microglia may contribute to tissue damage and repair during autoimmune disease,
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viral infections and chronic inflammatory disease. Major histocompatibility complex (MHC) class II antigen presentation requires the participation of endosomal/lysosomal proteases in endocytic route to degrade exogenous antigens and invariant chain (li) associated with MHC class II.Despite the strategic localization of CE, no information is available about the involvement of CE in MHC class II antigen presentation of microglia. In the present study, we have investigated the effect of highly selective inhibitors of cathepsins including the aspartic protease inhibitor pepstain A on the presentation of ovalbumin (OVA) or OVA peptide (OVA 267-282) by primary cultured murine microglia to OVA-specific I-Ab restricted helper T (Th) cell hybridomas. Upon stimulation with IFN-g, expression level of CE mRNA was significantly increased without affecting the expression levels of other house keeing cathepsins including cathepisn D (CD) and cathepsin B (CB). When microglia were treated with pepstatin A, IL-2 production from an OVA-specific Th cell hybridomas upon stimulation with naive OVA presented by IFN-g-treated microglia was markedly suppressed. However, pepstatin A failed to inhibit the presentation of OVA peptide. CLIK-060, a specific inhibitor of cathepsin S (CS), and CA-074Me, a specific inhibitor of CB, showed an inhibitory effect on the presentation of both naive OVA and OVA peptide. The involvement of aspartic proteases in the processing of antigenic peptide of naive OVA was further supported by the fact that digested fragments of naive OVA by CE or CD could be recognized by OVA-specific Th cell hybridomas to produce IL-2. When microgla prepared from CD deficinet mice were used, the efficacy for presenting OVA antigenic peptide to T cells was only slightly reduced. The requirements for CS and CB in the final stage of li-degradation were further substantiated by immunoblot analyses by using anti-li antibody. These results indicate that CE rather than CD are necessary for the generation of an antigenic epitope from OVA but not for the processing of li in microglia. Less
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