Differential processing of proenkephalin-A by human peripheral blood monocytes and T lymphocytes
W Kuis1, P M Villiger, H G Leser
1Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Abstract:
Human peripheral blood mononuclear cells are analyzed for preproenkephalin gene expression and peptide processing. Met-enkephalin immunoreactivity as detected with a specific antiserum is found in the cytoplasm of monocytes but not in T lymphocytes. Secretion of met-enkephalin was analyzed with an RIA that is specific for the met-enkephalin pentapeptide. Unfractionated PBMC spontaneously released 40 pg/ml met-enkephalin and this increased two- to fourfold after stimulation with PHA. Lower levels (less than 100 pg/ml) of met-enkephalin were detected in supernatants from purified T cells that were activated with PHA and IL-2. In contrast, stimulation of purified monocytes with LPS or PMA resulted in the release of up to 600 pg/ml of the processed peptide. To examine whether T cells can produce met-enkephalin precursor peptides, T cell conditioned media were treated with trypsin and carboxypeptidase-B, which is known to release met-enkephalin from the propeptide. This increased levels of met-enkephalin to 400 pg/ml, indicating that lymphocytes secrete the propeptide but do not process it to met-enkephalin. The 1.4-kb preproenkephalin mRNA is detected in activated blood mononuclear cells and in purified monocytes and T cells. To determine whether monocytes or lymphocytes express met-enkephalin in vivo, lymphoid tissues were analyzed by immunohistochemistry. In human spleen tissue, positive cells were found in the red pulp but not in the follicles, which is also consistent with met-enkephalin expression in monocytes. In summary, these results show that human peripheral blood mononuclear cells express preproenkephalin mRNA and that monocytes, but not T cells, process the propeptide to metenkephalin.
Insights
Human monocytes process and release met-enkephalin, while T cells secrete precursor peptides. This study clarifies the distinct roles of these immune cells in enkephalin production and processing.
Area of Science:
- Immunology
- Neuroendocrinology
- Molecular Biology
Background:
- Preproenkephalin (PPE) is a precursor to opioid peptides like met-enkephalin.
- The cellular source and processing of met-enkephalin within human peripheral blood mononuclear cells (PBMCs) remain incompletely understood.
- Understanding PPE gene expression and peptide processing in immune cells is crucial for neuroimmunology.
Purpose of the Study:
- To investigate preproenkephalin gene expression and met-enkephalin peptide processing in human peripheral blood mononuclear cells.
- To differentiate the roles of monocytes and T lymphocytes in the production and secretion of met-enkephalin.
- To determine the in vivo expression patterns of met-enkephalin in human lymphoid tissues.
Main Methods:
- Analysis of preproenkephalin mRNA expression in PBMCs, purified monocytes, and T cells.
- Quantification of secreted met-enkephalin using radioimmunoassay (RIA).
- Immunohistochemical analysis of human spleen tissue to localize met-enkephalin expression.
Main Results:
- Met-enkephalin immunoreactivity was localized to the cytoplasm of monocytes, not T lymphocytes.
- Stimulated monocytes released significantly higher levels of met-enkephalin compared to activated T cells.
- T cells secreted PPE precursor peptides, which could be processed to met-enkephalin by enzymatic treatment, while monocytes processed the peptide.
- Preproenkephalin mRNA was detected in activated PBMCs, monocytes, and T cells.
- Immunohistochemistry revealed met-enkephalin expression in the red pulp of human spleen, consistent with monocyte localization.
Conclusions:
- Human peripheral blood mononuclear cells express preproenkephalin mRNA.
- Monocytes are the primary cell type responsible for processing the propeptide to mature met-enkephalin within PBMCs.
- T lymphocytes primarily secrete the precursor peptide rather than the mature met-enkephalin.
