Cytokines of Birds: Conserved Functions

Targeted disruptions of the mouse genes for cytokines, cytokine receptors, or components of cytokine signaling cascades convincingly revealed the important roles of these molecules in immunologic processes. Cytokines are used at present as drugs to fight chronic microbial infections and cancer in humans, and they are being evaluated as immune response modifiers to improve vaccines. Until recently, only a few avian cytokines have been characterized, and potential applications thus have remained limited to mammals. Classic approaches to identify cytokine genes in birds proved difficult because sequence conservation is generally low. As new technology and high throughput sequencing became available, this situation changed quickly. We review here recent work that led to the identification of genes for the avian homologs of interferon-a/b (IFNa/b) and IFN-g, various interleukins (IL), and several chemokines. From the initial data on the biochemical properties of these molecules, a picture is emerging that shows that avian and mammalian cytokines may perform similar tasks, although their primary structures in most cases are remarkably different

Modulation of Functional Activities of Chicken Heterophils by Recombinant Chicken IFN-γ

The objective of the present studies was to examine the in vitro effects of recombinant chicken interferon-γ (rChIFN-γ) on shape change, phagocytosis, and the oxidative/nonoxidative killing activities of day-old chicken heterophils. Heterophils (4 × 106/ml) were incubated with various concentrations of recombinant ChIFN-γ from both Escherichia coli and transfected Cos cells for 2 h at 39°C. The incubation of the neonatal heterophils with rChIFN-γ resulted in significantly greater numbers of cells with membrane shape change when compared with the mock-treated heterophils. Both Cos cell-derived and E. coli-derived ChIFN-γ significantly increased (p < 0.01) the phagocytosis of opsonized or nonopsonized Salmonella enteritidis by the neonatal heterophils in a concentration-dependent manner. Incubation with ChIFN-γ induced no direct stimulation of the respiratory burst by the chicken heterophils but did prime the heterophils for a significantly strengthened respiratory burst to subsequent stimulation with opsonized zymosan (OZ). Lastly, incubation of the heterophils with ChIFN-γ primed the cells for a significant increase in the release of β-D-glucuronidase following stimulation with OZ. These results show that neonatal avian heterophils can respond to cytokine modulation with enhanced functional competence, suggesting that ChIFN-γ can enhance the immune competence of the innate defenses of chickens during the first week of life

cDNA Cloning of Biologically Active Chicken Interleukin-18

By searching a chicken EST database, we identified a cDNA clone that appeared to contain the entire open reading frame (ORF) of chicken interleukin-18 (ChIL-18). The encoded protein consists of 198 amino acids and exhibits approximately 30% sequence identity to IL-18 of humans and various others mammals. Sequence comparisons reveals a putative caspase-1 cleavage site at aspartic acid 29 of the primary translation product, indicating that mature ChIL-18 might consist of 169 amino acids. Bacterially expressed ChIL-18 in which the N-terminal 29 amino acids of the putative precursor molecule were replaced by a histidine tag induced the synthesis of interferon-γ (IFN-γ) in cultured primary chicken spleen cells, indicating that the recombinant protein is biologically active

Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable X P^ promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product

Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase. 14% of the tested sera (n=43) conttained insulin autoantibodies which strongly recognized the recombinant human preproinsulin. Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor a-chain (IL-7Ra) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Ra extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency

Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumine in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source

Chicken Toll-like Receptor 3 Recognizes Its Cognate Ligand When Ectopically Expressed in Human Cells

Recognition of pathogens by toll-like receptors (TLRs) causes activation of signaling cascades that trigger cytokine secretion and, ultimately, innate immunity. Genes encoding proteins with substantial homology to mammalian TLR1, TLR2, TLR3, TLR4, TLR5, and TLR7 are present in the chicken genome, whereas orthologs of TLR8, TLR9, and TLR10 seem to be defective or missing. Except for chicken TLR2 (ChTLR2), which was previously shown to recognize lipopeptides and lipopolysaccharides (LPS), the ligand specificity of ChTLRs had not been determined. We found that polyI:C, LPS, R848, S-28463, and ODN2006, which are specifically recognized by TLR3, TLR4, TLR7/8, and TLR9 in mammals, induced substantial amounts of type I interferon (IFN) and interleukin-6 (IL-6) in freshly prepared chicken splenocytes. To determine the ligand specificity of ChTLR3 and ChTLR7, we used a standard reporter assay frequently employed for analysis of mammalian TLRs. Neither S-28463 nor any other TLR ligand induced reporter activity in human 293 cells expressing ChTLR7. However, human 293 cells expressing ChTLR3 strongly and specifically responded to polyI:C, demonstrating that this chicken receptor represents a true ortholog of mammalian TLR3

Experimental Lymphatic Filariasis in Gerbils (Meriones Unguiculatus): Molecular Cloning and Expression of Gerbil Cytokines and Measurement of Cytokine Gene Expression During a Primary Infection of Brugia Pahangi.

Cytokines play an important role in regulation of T cell responses. Two T helper cell subsets have been identified in some species and are defined by their cytokine secretion profiles. Th1 cells produce IL-2 and IFN-$\gamma,$ whereas Th2 cells express IL-4, IL-5, and IL-10. Precursor T cells that express a mixture of these cytokines are considered Th0 cells. It is hypothesized that primary infections of Brugia pahangi in gerbils induce an initial Th0 or Th1 like response which with time becomes a Th2 like response. To test this dynamic parasitism induced shift in cytokine profile, it is necessary to measure the expression level of cytokines that differentiate the Th1 and Th2 responses. For this purpose, the full length cDNAs of gerbil IL-2, IL-4, IL-5, IL-10, IFN-$\gamma$ and HPRT were first isolated using cross-species PCR, inverse PCR or RACE and conventional PCR techniques. Subsequently, a competitive RT-PCR ELISA for quantitation of gerbil cytokine mRNAs was developed. Using this assay, the profile of gerbil cytokine response was measured during a primary infection of Brugia pahangi. Increased levels of IL-2, and IL-5 were seen during the initial stages of the infection at 14 to 28 days post-infection (DPI). An increase in IL-4 was first detected 28 DPI in renal lymph nodes and continued to increase during the infection. Increased levels of IL-10 were first seen in the spleen 56 DPI and in all tissues at 150 DPI. These initial results are in agreement with the hypothesis proposed. In addition to the development of this cytokine measuring method, gerbil IL-2 cDNA was expressed in both eucaryotic and procaryotic expression systems. Pure and functional recombinant gerbil IL-2 (gIL-2) was obtained using the pMAL procaryotic system. Further, neutralizing anti-gIL-2 antibodies were raised in rabbits. The immunological reagents produced plus the cytokine quantitation assay established make it possible to initially measure immunologic responses in the gerbil. These types of measurements will advance the understanding of immunologic and pathologic responses of filariasis, using the gerbil-Brugia model. These methodologies also will expand the usefulness of this unique laboratory animal, in studies of other diseases

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzmes A and B in the cytotoxic response in vivo