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Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags
Published on: September 9, 2014
Differential stimulation of monocytic cells results in distinct populations of microparticles
M Bernimoulin1, E K Waters, M Foy
1Immune Disease Institute, Boston, MA 02115, USA.
Background:
Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known.
Methods:
We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy.
Results:
Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera.
Conclusion:
We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.
Insights
Stimulus type affects microparticle (MP) properties. Monocytic MPs differ in phosphatidylserine and antigen expression based on stimulation, impacting immune responses.
Area of Science:
- Cell biology
- Immunology
- Biochemistry
Background:
- Microparticles (MPs) are vesicles shed from stimulated cells, facilitating intercellular communication.
- MP composition reflects their cell of origin and production method (stimulation vs. apoptosis).
- The influence of specific stimuli on MP characteristics remains largely unknown.
Purpose of the Study:
- To investigate how different stimuli (lipopolysaccharide vs. soluble P-selectin chimera) affect the properties of microparticles derived from monocytic THP-1 cells.
- To determine if MP characteristics vary based on the specific activating stimulus.
Main Methods:
- Monocytic THP-1 cells were stimulated with lipopolysaccharide or a soluble P-selectin chimera.
- MP characterization involved proteomics, flow cytometry, western blotting, and electron microscopy.
- Calcein-AM staining was used as a novel criterion for defining MP populations.
Main Results:
- MP populations exhibited similarities in size, cytoskeleton, and certain antigen expressions, along with comparable procoagulant activity.
- Distinct differences were observed in phosphatidylserine expression and subcellular protein localization (e.g., mitochondrial proteins) based on the stimulus.
- Leukocyte-associated immunoglobin-like-receptor (LAIR)-1 was uniquely expressed upon stimulation with the soluble P-selectin chimera.
Conclusions:
- The properties of microparticles are stimulus-dependent.
- Monocytic MPs differentially modulate thrombosis, inflammation, and immune regulation based on the specific stimulus, highlighting their functional heterogeneity.
