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Updated: Nov 19, 2025
Isolation, Transfection, and Culture of Primary Human Monocytes
Published on: December 16, 2019
In vitro Phenotype Induction of Circulating Monocytes: CD16 and CD163 Analysis
Claudio Karsulovic1,2, Fabian Tempio3,4, Mercedes Lopez3,4
1Laboratorio de Inmunomodulación Neuroendocrina, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Introduction:
CD14 (monocyte differentiation antigen, LPS binding protein - endotoxin receptor) and CD16 (FcγRIII, Low-affinity receptor for IgG) define three subpopulations of circulating monocytes with different inflammatory and phagocytic capabilities. Contradictory reports exist regarding both in vivo monocyte phenotype-disease association and response of these circulating monocytes to in vitro stimulation. We analyzed phenotypic changes in circulating monocytes when stimulated with LPS (pro-inflammatory stimulus) and IL-4 (alternative inflammatory stimulus).
Methods:
Mononuclear cells from nine healthy donors were extracted and studied for surface and intracellular markers using flow cytometry. PBMC were extracted using Ficoll technic and immediately analyzed using flow cytometry. Pro-inflammatory interleukin IL-1β and IL-6 were measured by intracellular cytometry. Mononuclear cells were stimulated using LPS and IL-4 as previously described. Changes against non-stimulated populations were statistically analyzed.
Results:
Compared to non-stimulated and IL-4 stimulated monocytes, LPS-stimulated cells display a singular pattern of markers, with higher levels of intracellular IL-1β and IL-6 directly correlating with CD14+CD163- cell frequency and diminishing membrane CD163 fluorescence. CD14+CD16- classical monocytes show greater percentage of CD163- cells upon LPS stimulation. CD86 levels on monocytes' surface did not change with LPS or IL-4 stimulation.
Conclusions And Discussion:
We showed that CD14+CD16- classical monocytes display higher sensitivity to LPS stimulation, with more IL-1β and IL-6 levels than intermediate and non-classical monocytes. This subset also diminishes its CD163 levels on the membrane after LPS stimulation with a contemporary raise in CD163- cells, suggesting that classical monocytes preferentially acquire CD163- defined M1 characteristics upon in vitro LPS stimulation. Intermediate and non-classical monocytes respond with lower levels of interleukins and display surface proteins in an M2-type profile (CD163+).
Insights
Classical monocytes (CD14+CD16-) show heightened inflammatory responses to LPS, increasing IL-1β and IL-6. These monocytes also shift towards an M1-like phenotype, unlike intermediate and non-classical monocytes.
Area of Science:
- Immunology
- Cell Biology
Background:
- Circulating monocytes, defined by CD14 and CD16 expression, comprise distinct subpopulations with varied functions.
- Existing research presents conflicting data on monocyte phenotype-disease associations and in vitro responses.
- This study investigates phenotypic shifts in monocyte subsets upon stimulation with lipopolysaccharide (LPS) and interleukin-4 (IL-4).
Purpose of the Study:
- To analyze the phenotypic alterations of circulating monocyte subpopulations in response to pro-inflammatory (LPS) and alternative (IL-4) stimuli.
- To elucidate the differential responses of classical, intermediate, and non-classical monocytes to in vitro stimulation.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll density gradient centrifugation.
- Flow cytometry was employed to assess surface and intracellular markers, including IL-1β and IL-6.
- Monocytes were stimulated with LPS and IL-4, and changes in marker expression were statistically analyzed against non-stimulated controls.
Main Results:
- LPS stimulation induced a distinct monocyte phenotype, characterized by elevated intracellular IL-1β and IL-6.
- Higher frequencies of CD14+CD163- cells and decreased membrane CD163 fluorescence were observed upon LPS stimulation.
- Classical monocytes (CD14+CD16-) exhibited a greater percentage of CD163- cells and increased pro-inflammatory cytokines post-LPS exposure, while CD86 expression remained unchanged.
Conclusions:
- Classical monocytes (CD14+CD16-) demonstrate heightened sensitivity to LPS, producing more IL-1β and IL-6 compared to other subsets.
- LPS stimulation promotes a shift in classical monocytes towards an M1-like phenotype (CD163-), indicated by reduced membrane CD163.
- Intermediate and non-classical monocytes responded with lower cytokine levels and displayed an M2-type profile (CD163+).
