title: Intracellular IL-10 detection in T cells by flowcytometry: the use of protein transport inhibitors revisited.
abstract: In the past two decades, interleukin-10 (IL-10) has gained much attention as an important regulatory cytokine involved in self-tolerance. Functional assessment of IL-10 producing immune cells is traditionally done by stimulation and measurement of cytokine production by flowcytometry. Thereby a protein transport inhibitor like monensin is used to accumulate the cytokine of interest intracellularly. In this study we elaborated on the monensin effect on cytokine detection and focused on IL-10 detection in human T cells. Peripheral blood mononuclear cells (PBMC) of 32 study subjects were isolated and stimulated with PMA/ionomycin, in the absence and presence of monensin, and stained intracellularly for IFN-γ, IL-4, IL-10 and IL-17A. Our results re-established that detection of IFN-γ+ and IL-4+ T cells benefited from the presence of monensin during stimulation. However, stimulation in the presence of monensin yielded lower proportions of IL-10+ T cells (0.45% (0.28-0.80) versus 0.80% (0.50-1.50) of CD4+ T cells, p<0.01), although monensin addition did result in an increased MFI (2431 (1273-4959) versus 1928 (1147-3760), p<0.01). Detectable fractions of IL-17A+ CD4+ T cells were not affected by monensin. A shorter incubation time, but not lower monensin concentrations, was effective in improving the detection of IL-10+ T cells. We found a strong correlation between the fraction of IL-10+ CD4+ T cells in the presence and absence of monensin (R=0.80 p<0.01). Next to this, also the detection of IL-10+ NK-T cells and IL-10+ monocytes, but not IL-10+ B cells, is impaired in the presence of monensin. This study shows that the effect of monensin on cytokine accumulation is time and cytokine dependent. Due to the use of monensin, previous research may have underestimated the number of IL-10+ leukocytes or may even have not been able to detect them at all. It is important to consider this for future research or when interpreting historical IL-10 data.
authors.affiliation: School for Mental Health and Neuroscience, Maastricht University Medical Center, Maastricht, The Netherlands. a.muris@maastrichtuniversity.nl
authors.forename: Anne-Hilde
authors.initials: AH
authors.lastname: Muris
authors.forename: Jan
authors.initials: J
authors.lastname: Damoiseaux
authors.forename: Joost
authors.initials: J
authors.lastname: Smolders
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Cohen Tervaert
authors.forename: Raymond
authors.initials: R
authors.lastname: Hupperts
authors.forename: Mariëlle
authors.initials: M
authors.lastname: Thewissen
mesh_terms: D000328:Adult; D000368:Aged; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism*; D020381:Interleukin-17 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008985:Monensin / Q000494:pharmacology*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D055611:Natural Killer T-Cells / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D061209:Proton Ionophores / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D055815:Young Adult
chemical_list: D020381:Interleukin-17; D061209:Proton Ionophores; D016753:Interleukin-10; D015847:Interleukin-4; D015759:Ionomycin; D007371:Interferon-gamma; D008985:Monensin; D013755:Tetradecanoylphorbol Acetate