Kinetics of intracytoplasmic Th1 and Th2 cytokine production assessed by flow cytometry following in vitro activation of peripheral blood mononuclear cells.
BACKGROUND
Standard cytokine detection methods are unable to determine which cells are the producing cells. We report on the extent and under which conditions the multilabeling capability of flow cytometry (FCM) can bring new advances into the field.


METHODS
Five different cytokines, interleukin-2 (IL-2), -4, -5, -10 and interferon-gamma (IFN-gamma), were assessed simultaneously under five ex vivo stimulation conditions in peripheral blood mononuclear cells from five healthy volunteers in a 5-day kinetic study. A second group of 35 volunteers was assessed for IFN-gamma and IL-2 production.


RESULTS
This study showed that (a) intracytoplasmic cytokines were almost undetectable within unstimulated cells, (b) intracytoplasmic cytokines were detected only in CD69(+) T lymphocytes, and (c) intracytoplasmic IL-2 and IFN-gamma were dramatically upregulated after stimulation with phorbol myristate acetate (PMA)-ionomycin in a biphasic response or with PMA-phytohemagglutinin (one major peak only at 18 h) but to a lesser extent with other stimuli such as monoclonal antibodies. Th2 cytokines were detected at a later time point and at lower levels. PMA/ionomycin stimulation after 4 h and 18 h of culture in 35 other volunteers individualized several subgroups according to the frequency of IFN-gamma- or IL-2-producing cells--IFN-gamma delayed producers (n = 10/35), IFN-gamma low producers (n = 8/35), and IL-2 delayed producers (n = 16/35)--as opposed to IFN-gamma or IL-2 normal producers.


CONCLUSIONS
FCM appears to be a good tool to examine cell cytokine status in pathology (allergy, autoimmune disease, etc.) provided that optimal stimulation conditions and multiple time-point cultures are used. It also seems to be a relevant method to define new Th subsets further.
Rostaing
L
L
Department of Multiorgan Transplantation, CHU Rangueil, Toulouse, France.
Tkaczuk
J
J
Durand
M
M
Peres
C
C
Durand
D
D
de Préval
C
C
Ohayon
E
E
Abbal
M
M
D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D018801:CD2 Antigens / Q000378:metabolism; D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis*; D003593:Cytoplasm; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D016753:Interleukin-10 / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D015848:Interleukin-5 / Q000096:biosynthesis; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D037181:Lectins, C-Type; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008213:Lymphocyte Activation; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018417:Th1 Cells / Q000187:drug effects / Q000378:metabolism*; D018418:Th2 Cells / Q000187:drug effects / Q000378:metabolism*; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D018106:CD28 Antigens; D017252:CD3 Complex; D016207:Cytokines; D007371:Interferon-gamma; D016753:Interleukin-10; D007376:Interleukin-2; D015847:Interleukin-4; D015848:Interleukin-5; D015759:Ionomycin; D037181:Lectins, C-Type; D013755:Tetradecanoylphorbol Acetate; D000099192:CD69 Antigens