Molecular Cloning and Functional Characterization of the Avian Macrophage Migration Inhibitory Factor (MIF)

Authors: KIM, SUNGWON - Virginia Polytechnic Institution & State University; COX, CHASITY - Virginia Polytechnic Institution & State University; STUARD, LINDSAY - Virginia Polytechnic Institution & State University; DALLOUL, RAMI - Virginia Polytechnic Institution & State University; Miska, Kate; Jenkins, Mark; Fetterer, Raymond

Submitted to: Developmental and Comparative Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2010
Publication Date: 5/20/2010
Citation: Kim, S., Cox, C.M., Stuard, L.H., Dalloul, R.A., Miska, K.B., Jenkins, M.C., Fetterer, R.H. 2010. Molecular Cloning and Functional Characterization of the Avian Macrophage Migration Inhibitory Factor (MIF). Developmental and Comparative Immunology. 34:1021-1032.

Interpretive Summary: Poultry coccidiosis is the result of several different species of a protozoan intestinal parasite which causes considerable annual losses to the poultry industry. The primary control for the disease is through application of medications in the feed as birds are raised in confinement housing. The controls by medications are becoming less effective because of increased resistance to the drugs and less desirable due to concerns about drugs possibly remaining in the meat and within the environment. Key to developing new controls is a more complete understanding of the interaction of the parasite with the chicken immune system. Unlike mammals many of the immune components of chickens are poorly characterized. The current study presents the first characterization of macrophage inhibitory factor (MIF) an important regulatory molecule. Using molecular techniques a recombinant MIF was produced and the structure characterized. The physiological and immunological properties were characterized by a variety of cellular assays. This study gives the first characterization of this important molecule from an avian species and provides an important tool to study the interaction of the chicken immunes system with avian coccidia.

Technical Abstract: Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor produced by sensitized T lymphocytes and inhibits the random migration of macrophages. Recent studies have revealed a more prominent role for MIF as a multi-functional cytokine mediating both innate and adaptive immune responses. This study describes the cloning and functional characterization of avian MIF in an effort to better understand its function and potential in poultry health applications. The full-length avian MIF gene was amplified from stimulated chicken lymphocytes and cloned into a prokaryotic expression vector. The confirmed 115 amino acid sequence of avian MIF has 71% identity with human and murine MIF. The bacterially expressed avian recombinant MIF (rChMIF) was purified, followed by endotoxin removal, and then tested by chemotactic assay and quantitative real-time PCR (qRT-PCR). Diff-Quick staining revealed a substantial decrease in migration of macrophages in the presence of 0.01 µg/ml rChMIF. qRT-PCR analysis revealed that the presence of rChMIF enhanced levels of IL-1' and iNOS during PBMCs stimulation with LPS. Additionally, the Con A-stimulated lymphocytes showed enhanced interferon (IFN)-' and IL-2 transcripts in the presence of rChMIF. Interestingly, addition of rChMIF to the stimulated PBMCs, in the presence of lymphocytes, showed anti-inflammatory function of rChMIF. To our knowledge, this study represents the first report for the functional characterization of avian MIF, demonstrating the inhibition macrophage migration, similar to mammalian MIF, and the mediation of inflammatory responses during antigenic stimulation.