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Title: CONSTRUCTION OF INTERNAL CDNA COMPETITORS FOR MEASURING IL-10 AND IL-12 CYTOKINE GENE EXPRESSION IN SWINE

Author
item MANSFIELD, L - MICHIGAN STATE UNIV
item Urban, Joseph
item Holley Shanks, Rhonda
item MURTAUGH, M - UNIV OF MINNESOTA
item Zarlenga, Dante
item Canals, Ana
item FOSS, D - UNIV OF MINNESOTA
item GAUSE, W - USUHS
item Lunney, Joan

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Detailed analyses of protective immune responses to infectious diseases require that the scientist be able to measure not only serological antibodies but also cellular products called cytokines that are now known to regulate immune cell interactions. Certain cytokines, such as interleukin-10 (IL-10) and IL-12, can completely shift the animal's immune response so that only certain cells are able to respond against an infection. This manuscript reports the development of molecular assays to quantitate IL-10 and IL-12 expression in pig tissues. Partial cDNAs for these genes and a reference, "housekeeping" gene termed HPRT were cloned and, using polymerase chain reaction techniques, 100 basepairs were specifically deleted. This resulted in a competitor deleted cDNA (DcDNA) to use for assaying, on a semiquantitative basis, mRNA expression in tissues from parasite infected pigs. Pigs exposed to the intestinal worm, Trichuris suis, clearly responded to the infection by producing IL-10, but no IL-12, mRNA; however, if pigs were simultaneously infected with T. suis and bacterial pathogens, by exposure to a contaminated dirt lot, they then expressed high levels of both IL-10 and IL-12. These changes in cytokine expression may account for the observed differences in the pig's ability to clear these infections and to prevent the associated intestinal pathology. Our quantitative PCR assay has thus permitted the determination of specific cell mediated immune responses to infection and will be useful to scientists as they design new vaccines for a wide range of infectious diseases.

Technical Abstract: A competitive PCR assay was used to quantify pig cytokine responses to parasite infection. Internal standards (cDNA competitor molecules) were produced and tested for swine Interleukin-12 (IL- 12), IL-10 and HPRT from PCR generated cytokine cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated by PCR and their authenticity verified by 1) amplification of the expected smaller PCR product, 2) appropriate fragment size, and 3) correct sequence. Resulting PCR amplification products were readily distinguished by agarose gel electrophoresis from PCR amplified wild type mRNA from lymphoid cells or tissues. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental infections of swine with the nematode Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (40 dpi). A competitive PCR (cPCR) reaction was performed in which cytokine cDNA content in these samples was quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN of infected pigs increased 10-fold at day 40 compared to control pigs; IL-12 gene expression was only detectable in MLN of pigs exposed to T. suis and other bacterial pathogens on a contaminated dirt lot. Our quantitative PCR assay has permitted the determination of specific cell mediated immune responses to parasite infection. The cPCR assay will enable us quantitate cytokine gene expression in swine and determine the details of swine immune responses to important infectious diseases.