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Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes
Seyed K Tayebati1, Dahuk El-Assouad, Alberto Ricci
1Sezione di Anatomia Umana, Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Università di Camerino, Via Scalzino 3, 62032 Camerino, Italy.
Insights
Human lymphocytes express key components of the cholinergic system, including acetylcholine (ACh) and muscarinic receptors. This finding suggests a potential role for the lymphocyte cholinergic system in regulating immune function.
Area of Science:
- Neuroimmunology
- Molecular Biology
- Cell Biology
Background:
- The cholinergic system, primarily known for neurotransmission in the nervous system, has increasingly been recognized for its roles in peripheral functions.
- Understanding the presence and function of cholinergic markers in immune cells like lymphocytes is crucial for elucidating neuro-immune interactions.
Purpose of the Study:
- To investigate the expression and localization of key cholinergic markers and muscarinic receptor subtypes (M(2)-M(5)) in human peripheral blood lymphocytes.
- To determine if these markers are present in both T and B lymphocytes.
Main Methods:
- Western blot analysis was employed to detect proteins including acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular ACh transporter (VAChT), and M(2)-M(5) muscarinic receptors.
- Immunocytochemistry and confocal laser immunofluorescence microscopy were used to determine the cellular localization of these markers.
- T and B lymphocytes were analyzed separately.
Main Results:
- Human peripheral blood lymphocytes express immunoreactivity for ACh, ChAT, AChE, VAChT, and M(2)-M(5) muscarinic receptors.
- Western blot analysis confirmed the presence of specific protein bands for ChAT, AChE, VAChT, and muscarinic receptors in lymphocytes, with patterns comparable to brain tissue.
- Immunofluorescence revealed cytoplasmic and plasma membrane localization for ChAT, AChE, and muscarinic receptors, and vesicular localization for ACh and VAChT.
Conclusions:
- The presence of comprehensive cholinergic markers in human T and B lymphocytes indicates a functional cholinergic system within these immune cells.
- This discovery supports the hypothesis that the lymphocyte cholinergic system may play a role in regulating immune responses.
- Further characterization of these markers could potentially serve as indicators for the status of the central nervous system's cholinergic system.
Abstract:
Cholinergic markers and the expression of M(2)-M(5) muscarinic cholinergic receptor subtypes were investigated in human peripheral blood lymphocytes by Western blot analysis and immunocytochemistry. The totality of peripheral blood lymphocytes express acetylcholine (ACh) immunoreactivity, choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular ACh transporter (VAChT) and M(2)-M(5) muscarinic cholinergic receptor protein immunoreactivity. Western blot analysis performed independently on T and B lymphocytes using anti-ChAT and anti-AChE antibodies revealed labelling of single bands of approximately 68-70 and 70 kDa, respectively, whereas VAChT was bound to two bands of approximately 80 and 45 kDa. The pattern of immunoblotting was similar in membranes of lymphocytes and striatum, used as a reference brain tissue. Western blot analysis using anti M(2)-M(5) receptor antibodies revealed labelling of single bands of approximately 55, 85-90, 50 and 81 kDa, respectively. Confocal laser immunofluorescence showed the localization of ACh and VAChT immunoreactivity in punctiform areas likely corresponding to cytoplasmic vesicles. ChAT and AChE were diffused to the cytoplasm and plasma membrane. Muscarinic receptor immunoreactivity was located in lymphocyte plasma membrane. Although the role of lymphocyte cholinergic system is still unclear, the demonstration of cholinergic markers in T and B human blood lymphocytes supports the view that a cholinergic systems may contribute to the regulation of immune function. The characterization of these cholinergic markers may also contribute to define if their evaluation can be used for assessing the status of brain cholinergic system.
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