Comparative analysis of porcine cytokine production by mRNA and protein detection.
To analyze the correlation between cytokine mRNA transcription and secretion, interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) were quantified by enzyme-linked immunosorbent assay and IL-2 by bioassay and compared with their mRNA levels, determined by reverse transcription polymerase chain reaction (RT-PCR). For this purpose, peripheral blood monomorphonuclear cells (PBMC) were stimulated in vitro with the lectins pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (ConA), respectively, and with F4 fimbriae in an antigen-specific assay. Analyses were performed 4, 8, 16, 24 and 48 h after stimulation on the stimulated PBMC for mRNA and on the respective culture supernatants for proteins. RT-PCR products were quantified by densitometric scanning of the electrophoresis bands and related to the band intensity of the housekeeping gene, cyclophilin. Low levels of IL-2, IL-4 and IFN-gamma mRNA were detected in unstimulated PBMC. Stimulation with all three mitogens (PWM, ConA, PHA) led to an increase in mRNA transcription. In contrast, substantial IL-10 mRNA levels were detected in both unstimulated and stimulated cells with practically no difference between the three mitogens used. IL-2 mRNA expression tended to peak after 8-16 h for all three mitogens. The cells stimulated with PWM and ConA showed higher levels of gene expression for IFN-gamma and lower for IL-4 then the cells stimulated with PHA, however, differences were not statistically significant. For cells stimulated with F4 fimbriae only the IFN-gamma mRNA expression increased with an early peak at 8h post-stimulation. The analysis of the culture supernatants for secreted cytokines revealed a correlation between the levels of mRNA transcription and the respective secreted cytokines during the first 24h of stimulation. After 24h of stimulation, however, a decrease in IFN-gamma and IL-2 mRNA levels was accompanied by an increase or a less pronounced decrease in cytokine concentration; only the ConA induced IL-2 mRNA and protein concentration slopes showed similar profiles. In conclusion, similar cytokine production profiles as defined by mRNA and protein, respectively are obtained only during the first 24h after stimulation of the cell cultures.
Verfaillie
T
T
Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Universiteit Gent, Salisburylaan 133, B-9820 Merelbeke, Belgium. tine.verfaillie@rug.ac.be
Cox
E
E
To
L T
LT
Vanrompay
D
D
Bouchaut
H
H
Buys
N
N
Goddeeris
B M
BM
D000818:Animals; D016207:Cytokines / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D016207:Cytokines; D012333:RNA, Messenger