Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR.
The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.
Abdalla
A O
AO
Immune and Gene Therapy Lab, CCK, Karolinska Hospital, Stockholm, Sweden.
Kiaii
S
S
Hansson
L
L
Rossmann
E D
ED
Jeddi-Tehrani
M
M
Shokri
F
F
Osterborg
A
A
Mellstedt
H
H
Rabbani
H
H
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D016207:Cytokines / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis
D016207:Cytokines; D012333:RNA, Messenger