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-γ) 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. vels of IL-2, IL-4 and IFN-γ 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-γ 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-γ mRNA expression increased with an early peak at 8 h post-stimulation. alysis of the culture supernatants for secreted cytokines revealed a correlation between the levels of mRNA transcription and the respective secreted cytokines during the first 24 h of stimulation. After 24 h of stimulation, however, a decrease in IFN-γ 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. clusion, similar cytokine production profiles as defined by mRNA and protein, respectively are obtained only during the first 24 h after stimulation of the cell cultures.