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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Published on: November 28, 2014
The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system
Pieter J A de Koning1, Kiki Tesselaar, Niels Bovenschen
1Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
Abstract:
The cytotoxic serine protease granzyme M (GrM) is one of the five human granzymes, which are mainly expressed by cytotoxic T lymphocytes and/or NK cells. Upon perforin-dependent entry into a target cell, GrM cleaves specific substrates resulting in the onset of a unique cell death mechanism. However, the role of GrM in pathophysiological conditions is not clear yet. Knowledge of the expression and regulation of GrM by lymphocyte populations is instrumental for a better understanding of the contribution of this unique granzyme in health and disease. Two previous studies demonstrated GrM protein expression by lymphocytes of the innate immune system, i.e., NK cells, NKT cells, and gammadelta T cells, whereas its expression by CD8(+) T cells remained controversial. In the present study, we have investigated the expression and regulation of GrM in lymphocyte subsets in more detail. Flow cytometry analysis with a novel specific antibody against human GrM confirmed high expression of this protease by NK cells, NKT cells, and gammadelta T cells. CD8(+) T cells also expressed GrM and comparing the naive to early effector-memory, to late effector-memory, to effector subset, this expression gradually increased during differentiation. In contrast, CD4(+) T cells hardly expressed GrM. Quantitative PCR analysis for GrM mRNA levels in the diverse lymphocyte sub-populations confirmed the FACS results. GrM protein expression by lymphocyte populations was not significantly affected by a panel of GrB-inducing cytokines, indicating that GrM expression is differentially regulated as compared to GrB. In conclusion, the human cytotoxic protease GrM is, besides by innate immune cells, also expressed by CD8(+) effector T cells, in particular by the differentiated effector CD27(-) CD45RO(-) subset. Our current findings support not only a role for GrM in the innate but also in the adaptive immune response.
Insights
Granzyme M (GrM) is expressed by innate immune cells and importantly, also by CD8(+) effector T cells during differentiation. This protease plays a role in both innate and adaptive immunity.
Area of Science:
- Immunology
- Cellular Biology
- Protease Function
Background:
- Granzyme M (GrM) is a cytotoxic serine protease primarily found in cytotoxic T lymphocytes and NK cells.
- Its precise role in disease remains unclear, necessitating a deeper understanding of its expression and regulation within immune cell populations.
- Previous studies showed conflicting results regarding GrM expression in CD8(+) T cells.
Purpose of the Study:
- To investigate the expression and regulation of Granzyme M (GrM) in various human lymphocyte subsets.
- To clarify the controversial findings on GrM expression in CD8(+) T cells.
- To determine if GrM expression is influenced by cytokines known to induce Granzyme B (GrB).
Main Methods:
- Flow cytometry using a novel GrM-specific antibody.
- Quantitative PCR to analyze GrM mRNA levels.
- Analysis of lymphocyte subsets including NK cells, NKT cells, gammadelta T cells, CD8(+) T cells (differentiated subsets), and CD4(+) T cells.
Main Results:
- High GrM expression was confirmed in NK cells, NKT cells, and gammadelta T cells.
- CD8(+) T cells demonstrated increasing GrM expression with differentiation, particularly in effector subsets.
- CD4(+) T cells showed minimal GrM expression, and GrM levels were unaffected by GrB-inducing cytokines.
Conclusions:
- Human Granzyme M is expressed by innate immune cells and CD8(+) effector T cells, especially differentiated subsets.
- Granzyme M is differentially regulated compared to Granzyme B.
- These findings suggest Granzyme M contributes to both innate and adaptive immune responses.
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