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Title: CLONING AND CHARACTERIZATION OF CHICKEN NK-LYSIN AND LIPOPOLYSACCHARIDE-INDUCED TNF-ALPHA FACTOR (LITAF)

Author
item Lillehoj, Hyun
item DALLOUL, RAMI - VISITING SY
item LEE, SUNG - VISITING SY

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/30/2006
Publication Date: 10/21/2006
Citation: Hong, Y.H., Lillehoj, H.S., Dalloul, R.A., Lee, S.H. 2006. Cloning and characterization of chicken nk-lysin and lipopolysaccharide-induced tnf-alpha factor (litaf). Proceedigs of International Avian Immunology Research Group Meeting. Oct. 21-24. Paris, France. P 28.

Interpretive Summary:

Technical Abstract: The inflammatory response to parasites is mediated by multiple host factors. In this report, we present molecular and functional characterizations of two novel immune mediators whose gene expression increased following infection with Eimeria. NK-lysin is an anti-microbial and anti-tumor protein expressed by NK cells and T lymphocytes. Full-length clone encoding chicken NK-lysin was isolated from intestinal intraepithelial lymphocytes (IELs) cDNA library. NK-lysin is consisted of an 868 bp DNA sequence with an open reading frame of 140 amino acids and a predicted molecular mass of 15.2 kDa. Comparison of its deduced amino acid sequence showed less than 20% identity to mammalian NK-lysins. The tissue distribution of NK-lysin mRNA revealed highest levels in intestinal IELs, intermediate levels in splenic, peripheral blood lymphocytes and lowest levels in thymic, bursa lymphocytes. The kinetics of NK-lysin mRNA expression indicated that, whereas infection with E. acervulina induced maximum expression only at 7-8 days post-infection, E. maxima and E. tenella elicited biphasic responses at 3-4 and 7-8 days post-infection. Finally, recombinant chicken NK-lysin expressed in COS7 cells exhibited anti-tumor cell activity against LSCC-RP9. Another factor whose gene expression increased following Eimeria infection is LPS-induced TNF-alpha (LITAF). A full-length cDNA encoding the chicken homologue of LITAF, transcription factor, with an open reading frame of 148 amino acids with a predicted molecular mass of 16.0 kDa was cloned. Quantitative RT-PCR analysis showed that chicken LITAF mRNA was predominantly expressed in spleen and IELs. LITAF mRNA levels were up-regulated following in vitro stimulation of macrophages for 4 hr with E. coli or S. typhimurium endotoxin, and 18-48 hr after treatment with E. acervulina, E. maxima, or E. tenella. LITAF mRNA was up-regulated more than 700-fold in IELs isolated from E. maxima-infected birds. Purified recombinant LITAF protein expressed in E. coli or COS7 cells exhibited cytotoxic activity against chicken tumor cell lines in vitro, presumably through autocrine activation of TNF-alpha or its chicken homologue. This supposition was strengthened by the fact that treatment of chicken macrophages with recombinant LITAF induced the expression of TL1A (TNFSF 15), a member of the TNF ligand super family. We conclude that chicken LITAF may play an important role in the regulation of TNF-alpha gene expression during the course of coccidiosis or tumorigenesis. We conclude that chicken NK-lysin plays important roles during anti-microbial and anti-tumor defenses.