Molecular characterization and immunological roles of avian IL-22 and its soluble receptor IL-22 binding protein.

As a member of the interleukin (IL)-10 family, IL-22 is an important mediator in modulating tissue responses during inflammation. Through activation of STAT3-signaling cascades, IL-22 induces proliferative and anti-apoptotic pathways, as well as antimicrobial peptides (AMPs), that help prevent tissue damage and aid in its repair. This study reports the cloning and expression of recombinant chicken IL-22 (rChIL-22) and its soluble receptor, rChIL22BP, and characterization of biological effects of rChIL-22 during inflammatory responses. Similar to observations with mammalian IL-22, purified rChIL-22 had no effect on either peripheral blood mononuclear cells (PBMCs) or lymphocytes. This was due to the low expression of the receptor ChIL22RA1 chain compared to ChIL10RB chain. rChIL-22 alone did not affect chicken embryo kidney cells (CEKCs); however, co-stimulation of CEKCs with LPS and rChIL-22 enhanced the production of pro-inflammatory cytokines, chemokines and AMPs. Furthermore, rChIL-22 alone stimulated and induced acute phase reactants in chicken embryo liver cells (CELCs). These effects of rChIL-22 were abolished by pre-incubation of rChIL-22 with rChIL22BP. Together, this study indicates an important role of ChIL-22 on epithelial cells and hepatocytes during inflammation.

Molecular cloning and functional characterization of the avian macrophage migration inhibitory factor (MIF).

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 role in innate and adaptive immunity, and potential use 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 0.01microg/ml rChMIF. qRT-PCR analysis revealed that the presence of rChMIF enhanced levels of IL-1beta and iNOS during PBMCs stimulation with LPS. Additionally, the Con A-stimulated lymphocytes showed enhanced interferon (IFN)-gamma 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 of macrophage migration, similar to mammalian MIF, and the mediation of inflammatory responses during antigenic stimulation.

Molecular cloning and functional characterization of avian interleukin-19.

The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chicken monocytes, and cloned into both prokaryotic (pET28a) and eukaryotic (pcDNA3.1) expression vectors. The confirmed avian IL-19 amino acid sequence has 66.5% homology with human and murine IL-19, with a predicted protein sequence of 176 amino acids. Analysis of avian IL-19 amino acid sequence showed six conserved, structurally relevant, cysteine residues as found in mammals, but only one N-glycosylation residue. The recombinant IL-19 (rChIL-19) expressed in the prokaryotic system was purified by Ni(+)-resin column followed by endotoxin removal. Using purified avian rChIL-19, expression of Th2 cytokines was measured in splenocytes using quantitative real-time PCR (qRT-PCR). In the presence of rChIL-19, expression levels of IL-4 and IL-13, as well as IL-10, were significantly increased after 6- and 12 h treatments. This was confirmed by treating splenocytes with supernatants from IL-19 transfected cells. Also, avian monocytes incubated with rChIL-19 displayed increased expression of IL-1beta, IL-6, and IL-19. This study represents the first report for the cloning, expression, and functional characterization of avian IL-19. Taken together, avian IL-19 function seems to be conserved and similar to that of mammals and may play an important role in responses to intracellular poultry pathogens like bacteria and protozoa.

Molecular cloning and characterization of chicken NK-lysin.

NK-lysin is an anti-microbial and anti-tumor protein expressed by NK cells and T lymphocytes. In a previous report, we identified a set of overlapping expressed sequence tags constituting a contiguous sequence (contig 171) homologous to mammalian NK-lysins. In the current report, a cDNA encoding NK-lysin was isolated from a library prepared from chicken intestinal intraepithelial lymphocytes (IELs). It consisted of an 850 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 and peripheral blood lymphocytes, and lowest levels in thymic and bursa lymphocytes. Following intestinal infection of chickens with Eimeria maxima, one of seven Eimeria species causing avian coccidiosis, NK-lysin transcript levels increased 3-4-fold in CD4+ and CD8+ intestinal IELs. However, cell depletion experiments suggested other T lymphocyte subpopulations also expressed NK-lysin. 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, a retrovirus-transformed B-cell line. We conclude that chicken NK-lysin plays important roles during anti-microbial and anti-tumor defenses.