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Detection of intracellular interleukin-8 in human mast cells: flow cytometry as a guide for immunoelectron microscopy
A Grützkau1, S Krüger-Krasagakes, H Kögel
1Department of Dermatology, Virchow Clinics, Humboldt University of Berlin, Germany.
Insights
Interleukin-8 (IL-8) is stored in cytoplasmic granules of human mast cells (HMC-1). This study confirms IL-8 production and intracellular localization in HMC-1 cells using flow cytometry and electron microscopy.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Interleukin-8 (IL-8) is a chemokine crucial for neutrophil recruitment in inflammation.
- Previous studies demonstrated IL-8 production and release by human mast cells (HMC-1).
- The intracellular storage and localization of IL-8 within HMC-1 cells remained to be fully elucidated.
Purpose of the Study:
- To investigate the intracellular storage and localization of IL-8 in HMC-1 cells at the single-cell level.
- To establish optimal conditions for fixation and permeabilization of HMC-1 cells for intracellular antigen detection.
- To confirm IL-8 expression in HMC-1 cells using advanced microscopy techniques.
Main Methods:
- Combined use of flow cytometry (FACS) and immunoelectron microscopy for single-cell analysis.
- Optimization of microwave fixation (paraformaldehyde/glutaraldehyde) and saponin permeabilization for HMC-1 cells.
- Detection of IL-8 using a monoclonal antibody in stimulated HMC-1 cells.
Main Results:
- FACS analysis showed time-dependent IL-8 synthesis, with up to 50% positive cells 8-12 hours post-stimulation.
- Immunoelectron microscopy revealed IL-8 localized within specific, electron-dense cytoplasmic granules in stimulated HMC-1 cells.
- Established protocols for fixation and permeabilization were validated for intracellular antigen detection.
Conclusions:
- Human mast cells (HMC-1) store interleukin-8 (IL-8) in distinct cytoplasmic granules.
- Quantitative FACS is a reliable and time-saving method for optimizing conditions for pre-embedding immunoelectron microscopy of intracellular antigens.
- These findings confirm and extend previous knowledge on IL-8 expression and localization in HMC-1 cells at the single-cell level.
Abstract:
The chemokine interleukin-8 (IL-8) mediates infiltration and adhesion of neutrophils during inflammatory processes. We have previously shown that this cytokine can be produced and released by normal and leukemic human mast cells (HMC-1 cells). To assess whether and to what extent this cytokine is stored intracellularly, we investigated production and localization of IL-8 at the single-cell level by combined use of flow cytometry (FACS) and immunoelectron microscopy. Conditions necessary for optimal fixation and permeabilization of HMC-1 cells were determined by measuring changes in cell-specific light scatter parameters and by estimating cellular uptake of propidiumiodide (PI). In this way, we were able to detect IL-8 with a monoclonal antibody in stimulated cells that were microwave-fixed with a combination of paraformaldehyde (4%) and glutaraldehyde (0.1%), followed by permeabilization with saponin (0.025%). FACS analysis revealed time-dependent synthesis of IL-8 with at most 50% positively stained cells at 8-12 hr after stimulation. For pre-embedding immunogold electron microscopy, cells were treated according to the protocol established by flow cytometry. IL-8 was found to be located in specific cytoplasmic, electron-dense granules of stimulated HMC-1 cells. These results confirm and extend our previous findings by demonstrating IL-8 expression in HMC-1 cells at the single-cell level. In addition, we propose that quantitative FACS can be reliably used in a timesaving manner to establish appropriate conditions for pre-embedding immunoelectron microscopy of intracellular antigens.
