CLONING OF PORCINE CYTOKINE-SPECIFIC CDNAS AND DETECTION OF PORCINE TUMOR-NECROSIS-FACTOR-ALPHA, INTERLEUKIN-6 (IL-6), AND IL-1-BETA GENE-EXPRESSION BY REVERSE TRANSCRIPTION PCR AND CHEMILUMINESCENCE HYBRIDIZATION

A reverse transcription PCR assay with porcine cytokine-specific prime rs was developed to clone cDNA fragments and generate cDNA probes that were specific for porcine tumor necrosis factor alpha (TNF-alpha), in terleukin 6 (IL-6), and IL-1 beta. The specificities of the cDNA PCR p roducts were confirmed by sequence analysis on the basis of known porc ine cytokine gene sequences. The reverse transcription PCR assay was a lso used to study cytokine mRNA expression in lipopolysaccharide (LPS) -stimulated and control unstimulated porcine alveolar macrophages. The cDNA products were analyzed in ethidium bromide-stained agarose gels, and the transcription level of each cytokine was determined relative to the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA level of each cytokine by measuring the intensity of the chemilumines cence hybridization signals by densitometric scanning. Various levels of cytokine mRNAs were detected in both LPS-stimulated and control uns timulated cells. Thus, TNF-alpha mRNA levels were enhanced in the cell cultures stimulated for 6 h with LPS compared with those in control c ell cultures. No differences in TNF-alpha transcription levels between LPS-stimulated and control cells were observed after incubation for 2 4 or 55 h. Enhancements of IL-6 and IL-1 beta mRNA levels were also ob served in the cultures stimulated with LPS for 6 and 24 h compared wit h the cytokine mRNA levels in control cell cultures. The presence of c ytokine mRNA transcripts in the LPS-stimulated macrophage cultures cor related with the detection of these soluble cytokines by the bioassays . In contrast, no soluble cytokine was detected in control macrophage culture supernatants in the presence of cytokine mRNA transcripts.