11/28/2023 0 Comments Molecular cloning serial clonerThe mammalian cell line COS-7 (African green monkey kidney cells) and the chicken DT-40 B cell line (a kind gift from P. ![]() So far only a limited number of avian cytokines have been identified (for two recent reviews, see Refs. As a result of this poor homology, cloning and sequencing of avian cytokines lags dramatically behind similar work achieved in mammals. The identification by PCR amplification using primers based on mammalian sequences proved to be very difficult and unpredictable ( 28, 29). This explains the disaster of classical approaches to identify avian homologues of mammalian cytokines. One of the major reasons for the failure to identify avian IL-12 thus far is the low sequence homology, usually restricted to ∼30–50%, between avian and mammalian cytokine sequences. Yet, the existence of a Th1-like cytokine network in birds was, in part, evidenced some years ago by the identification of chicken IFN-γ ( 26) and the recent discovery of chicken IL-18 ( 27). It is also very remarkable that there is no evidence for a balanced Th1/Th2 system in nonmammalian species. In fact, no nonmammalian IL-12 molecule has been identified or isolated so far. ![]() Unfortunately, no avian IL-12 subunit has been cloned yet. Th1-biased immune responses are essential in the control of mostly intracellular infections of bacterial, parasitic, fungal, or viral nature (for a review, see Ref. Furthermore, IL-12 plays an important and crucial role in mammals in the regulation of IFN-γ characteristic Th1-type immune responses ( 12, 13), while it inhibits the Th2 pathway. These effects mainly include induction of IFN-γ and TNF production by resting and activated T and NK cells and induction of activated T and NK cell proliferation ( 5). IL-12, also known as NK cell stimulatory factor ( 5) or cytotoxic lymphocyte maturation factor ( 6), is a pleiotropic cytokine that is produced by monocytes ( 7), macrophages ( 8), and dendritic cells ( 9) and has been shown to mediate multiple effects on T cells and NK cells ( 10, 11). In addition, p40 may form the composite cytokine IL-23, after combining with a 19-kDa protein (p19) ( 4), a molecule structurally related to IL-6, p35, and G-CSF. The mammalian p40 subunit links covalently, by disulfide binding, with a 35-kDa protein (p35) to form the heterodimeric p40-p35 IL-12 (IL-12p70 or IL-12) molecule ( 1, 2, 3). In mammals, a group of composite heterodimeric cytokines has been identified based on complexes of a 40-kDa protein subunit (p40). ![]() The group of soluble secreted molecules, collectively termed cytokines, represents critical communication signals among the cells of the immune system and between immune and nonimmune system cells. ![]() In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-γ synthesis and proliferative activity of freshly exposed chicken splenocytes. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. These molecules are the first discovered nonmammalian IL-12 subunits. By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains.
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