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Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays
Published on: November 29, 2014
Cell-type specificity of l-leucyl l-leucine methyl ester
Y Kobayashi1, A Takasaki, K Kurosaka
1Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, Japan. yoshiro@biomol.sci.toho-u.ac.jp
Abstract:
l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent which is converted to a membranolytic compound by dipeptidyl peptidase I and kills human leukocytes such as CD8+ T cells and monocytes but not B cells. The reagent has also been used in mice on the assumption that the cell-type specificity to murine leukocytes is the same as that to human leukocytes. During study on the effect of LeuLeuOMe on antigen-driven IL-2 production using murine splenocytes as antigen-presenting cells, however, we noticed that murine B cells were sensitive to LeuLeuOMe. We therefore examined the cell-type specificity using murine splenocytes and peritoneal macrophages. Flow cytometric analysis revealed that the most sensitive cells to LeuLeuOMe were CD8+ cells and that CD19+ cells (B cells) were as sensitive as CD3+ cells (T cells). Murine splenic B cells, which were either positively or negatively sorted with a cell sorter, were also sensitive to LeuLeuOMe, whereas human peripheral blood B cells, which were positively sorted, were not. Peritoneal macrophages were the most insensitive to LeuLeuOMe. Thus, this study demonstrated that the cell-type specificity to murine leukocytes is different from that to human leukocytes.
Insights
l-Leucyl l-leucine methyl ester (LeuLeuOMe) kills human leukocytes but not B cells. However, this study found murine B cells are sensitive to LeuLeuOMe, revealing species-specific differences in leukocyte sensitivity.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent known to lyse human CD8+ T cells and monocytes.
- It was previously assumed that LeuLeuOMe exhibits similar cell-type specificity in murine leukocytes as in human leukocytes.
- This assumption guided its use in mouse models for immunological studies.
Purpose of the Study:
- To investigate the cell-type specificity of l-Leucyl l-leucine methyl ester (LeuLeuOMe) in murine leukocytes.
- To determine if murine leukocyte sensitivity to LeuLeuOMe differs from that observed in human leukocytes.
- To clarify the potential impact of these differences on immunological research using mouse models.
Main Methods:
- Utilized murine splenocytes and peritoneal macrophages for cell-type specificity analysis.
- Employed flow cytometry to assess cell sensitivity to LeuLeuOMe.
- Used cell sorting to isolate and test specific murine leukocyte populations, including B cells (CD19+) and T cells (CD3+).
Main Results:
- Murine CD8+ cells were identified as the most sensitive to LeuLeuOMe.
- Murine B cells (CD19+) demonstrated sensitivity to LeuLeuOMe, comparable to T cells (CD3+).
- In contrast, human peripheral blood B cells showed no sensitivity to LeuLeuOMe, and murine peritoneal macrophages were the least sensitive.
Conclusions:
- The cell-type specificity of l-Leucyl l-leucine methyl ester (LeuLeuOMe) in murine leukocytes significantly differs from that in human leukocytes.
- Murine B cells are sensitive to LeuLeuOMe, contrary to observations in human B cells.
- These findings highlight crucial differences for interpreting immunological studies involving LeuLeuOMe in mouse models.
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