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Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation
Published on: December 15, 2017
Intracellular detection of macrophage migration inhibitory factor in peripheral blood leukocytes
Lutz E Lehmann1, Stefan U Weber, Dagmar Fuchs
1Klinik und Poliklinik für Anästhesie und Intensivmedizin der Universität Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. lutz.lehmann@ukb.uni-bonn.de
Abstract:
The oxidoreductase MIF is currently discussed as a new promising target of immunomodulatory therapy in patients with severe sepsis. An increasing body of evidence attributes an important role especially to intracellular MIF for regulation of endotoxin responsiveness as well as regulation of nuclear transcription factors. Up to now, measurement of MIF relied on ELISA techniques, lacking the ability to directly measure intracellular MIF and distinguish between different leukocyte subpopulations. Therefore, we developed a sensitive and robust flow cytometry-based method to reliably detect intracellular levels of MIF. This method can readily be applied in cultured cells as well as in subsets of human leukocytes in whole blood. Intracellular MIF content of whole-blood leukocyte subsets is detected simultaneously, and is individually determined for T-lymphocytes, B-lymphocytes, macrophages, and granulocytes, respectively. When tested in an ex vivo whole-blood stimulation system using PMA/Ionomycin the intracellular MIF content doubled in CD3+ T-lymphocytes and increased threefold in CD14+ macrophages. Baseline intracellular MIF levels of different leukocyte subpopulations were quantified in 22 healthy blood donors. Baseline intracellular MIF levels in T-lymphocytes are twice as high compared to those in B-lymphocytes and macrophages.
Insights
A new flow cytometry method accurately measures intracellular MIF (Macrophage Migration Inhibitory Factor) in various immune cells. This technique is crucial for understanding MIF
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Macrophage Migration Inhibitory Factor (MIF) is a key mediator in inflammatory responses, particularly in severe sepsis.
- Intracellular MIF plays a critical role in regulating endotoxin responsiveness and nuclear transcription.
- Existing ELISA methods for MIF lack the sensitivity to measure intracellular levels and differentiate leukocyte subpopulations.
Purpose of the Study:
- To develop and validate a sensitive flow cytometry-based method for detecting intracellular MIF.
- To enable simultaneous quantification of intracellular MIF in distinct human leukocyte subsets.
- To establish baseline intracellular MIF levels in various immune cell populations from healthy donors.
Main Methods:
- Development of a robust flow cytometry assay for intracellular MIF detection.
- Application of the method to cultured cells and human whole blood leukocyte subsets (T-lymphocytes, B-lymphocytes, macrophages, granulocytes).
- Ex vivo whole-blood stimulation assay using PMA/Ionomycin to assess MIF response.
Main Results:
- The developed flow cytometry method reliably detects intracellular MIF in various cell types.
- Intracellular MIF levels significantly increased in T-lymphocytes and macrophages upon stimulation.
- Baseline intracellular MIF levels were quantified, revealing higher concentrations in T-lymphocytes compared to B-lymphocytes and macrophages.
Conclusions:
- A novel flow cytometry method provides a sensitive and specific tool for intracellular MIF measurement in leukocyte subsets.
- This method facilitates a deeper understanding of MIF's role in immune cell function and inflammatory diseases like sepsis.
- The findings establish baseline intracellular MIF levels, crucial for future clinical and research applications.
