Kinetic study of porcine GM-CSF expression in porcine alveolar macrophages and spleen cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator in inducing differentiation and proliferation of immune cells. The functional roles of porcine GM-CSF (pGM-CSF) have not yet been revealed. Therefore, expression patterns of pGM-CSF were investigated in immune cells after cloning and sequencing of whole pGM-CSF cDNA. Whole cDNA of pGM-CSF was amplified from porcine alveolar macrophages stimulated by lipopolysaccharide (LPS), using 5'- and 3'-rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) methods. The products of 5'- and 3'-RACE-PCR were cloned, and the nucleotide sequence of whole pGM-CSF cDNA was determined (GenBank accession number AY116504). The kinetics of pGM-CSF mRNA expression were studied in porcine immune cells such as alveolar macrophages and spleen cells, using a real-time quantitative PCR. The expression of pGM-CSF in LPS-, phytohemagglutinin (PHA)-, or concanavalin A (ConA)-stimulated cells was always higher as compared to the control cells. The expression levels of pGM-CSF in alveolar macrophages were highest at 5 h after LPS stimulation and then continuously decreased in the late phase. In spleen cells, the LPS-stimulated group showed the highest levels after 5 h, but the PHA- and the ConA-stimulated groups showed slightly increased expression levels at the early phase and peaked at 24 h. To our knowledge, this is the first published report describing the nucleotide sequence of whole cDNA and the expression pattern of pGM-CSF using real-time quantitative PCR. These results indicate that pGM-CSF has its own characteristic expression profile in different immune cells.