Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques.
BACKGROUND
We investigated the relation between intracellular cytokine production and extracellular cytokine levels by using two flow cytometric techniques.


METHODS
A two-color flow cytometric technique was used to measure interleukin (IL)-1beta, IL-6, tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-12 production blocked intracellularly with brefeldin A in lipopolysaccharide (LPS)-stimulated CD14(+) monocytes and IL-2, IL-4, and IFN-gamma production in phorbol-12-mirystate-13-acetate (PMA)-stimulated CD3(+) T lymphocytes in samples from patients with rheumatoid arthritis. A flow cytometric microsphere-based immunoassay was performed to detect cytokine secretion in plasma of PMA- and LPS-stimulated whole blood samples.


RESULTS
There was a strong linear correlation between extracellular quantitative (pg/ml) and intracellular semiquantitative detection of LPS-stimulated IL-1beta, IL-6, IL-10, and IL-12 production (r > 0.9). For lymphocytes, extracellularly detected IL-2 and IFN-gamma correlated well with percentages of cytokine-producing cells (r > 0.8). The percentages of IL-4-positive T cells were moderately correlated with the secreted amounts of IL-4 as detected with the microsphere-based immunoassay (r = 0.7).


CONCLUSIONS
Overall, there was a good correlation between semiquantitative intracellular detection of cytokines and the secreted amounts of cytokines detected with the microsphere based immunoassay.
Schuerwegh
A J
AJ
Department of Immunology, Allergology and Rheumatology, University of Antwerp, Belgium.
De Clerck
L S
LS
Bridts
C H
CH
Stevens
W J
WJ
D001172:Arthritis, Rheumatoid / Q000276:immunology; D017252:CD3 Complex / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis* / Q000187:drug effects; D045604:Extracellular Fluid / Q000737:chemistry*; D005434:Flow Cytometry; D006801:Humans; D007118:Immunoassay; D007424:Intracellular Fluid / Q000737:chemistry*; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008863:Microspheres; D009000:Monocytes / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D013601:T-Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D017252:CD3 Complex; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D013755:Tetradecanoylphorbol Acetate