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Involvement of cathepsin E in exogenous antigen processing in primary cultured murine microglia
Tsuyoshi Nishioku1, Koichi Hashimoto, Keizo Yamashita
1Department of Pharmacology, Graduate School of Dental Science, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
Abstract:
We have attempted to elucidate an involvement of cathepsin E (CE) in major histocompatibility complex class II-mediated antigen presentation by microglia. In primary cultured murine microglia, CE was localized mainly in early endosomes and its expression level was markedly increased upon stimulation with interferon-gamma. Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266-281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-gamma-treated microglia. However, pepstatin A failed to inhibit the presentation of OVA-(266-281) peptide. The possible involvement of CE in the processing of native OVA into antigenic peptide was further substantiated by that digested fragments of native OVA by CE could be recognized by OVA-specific Th cells. Cathepsin D also degraded native OVA into antigenic peptide, whereas microglia prepared from cathepsin D-deficient mice retained an ability for antigen presentation. On the other hand, the requirement for cysteine proteases such as cathepsins S and B in the processing of invariant chain (Ii) was confirmed by immunoblot analyses in the presence of their specific inhibitors. In conclusion, CE is required for the generation of an antigenic epitope from OVA but not for the processing of Ii in microglia.
Insights
Cathepsin E (CE) is crucial for microglia to process native ovalbumin (OVA) into antigenic epitopes for major histocompatibility complex class II presentation. However, CE does not process the invariant chain (Ii) in this immune cell type.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Microglia play a key role in antigen presentation via major histocompatibility complex class II (MHC-II).
- The processing of antigens into peptides for MHC-II presentation involves various proteases within endosomal compartments.
Purpose of the Study:
- To investigate the role of cathepsin E (CE) in MHC-II-mediated antigen presentation by microglia.
- To determine if CE is involved in the processing of native ovalbumin (OVA) or the invariant chain (Ii).
Main Methods:
- Primary murine microglia cultures were used.
- Interferon-gamma (IFN-γ) stimulation was applied to upregulate CE expression.
- Pepstatin A, a CE inhibitor, was used to assess its impact on antigen presentation.
- OVA-specific T helper cell hybridomas were employed to measure interleukin-2 production.
- Immunoblot analyses were performed to confirm the role of cysteine proteases.
Main Results:
- CE expression increased in microglia upon IFN-γ stimulation.
- Pepstatin A inhibited the presentation of native OVA but not OVA peptide, indicating CE's role in initial OVA processing.
- CE-digested OVA fragments were recognized by OVA-specific T cells.
- Cathepsin D also degraded OVA, but its deficiency did not impair microglial antigen presentation.
- Cysteine proteases (cathepsins S and B) were confirmed to be essential for invariant chain (Ii) processing.
Conclusions:
- Cathepsin E is essential for generating antigenic epitopes from native OVA for MHC-II presentation by microglia.
- Cathepsin E is not involved in the processing of the invariant chain (Ii) in microglia.
- These findings highlight CE's specific function in the antigen processing pathway within microglia.