Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity Its activities are mediated through a high-affinity receptor composed of two subunits, designated beta 1 and beta 2 Of these two subunits, beta 2 is more restricted in its distribution, and regulation of its expression is likely a central mechanism by which IL-12 responsiveness is controlled Studies with neutralizing anti-IL-12 antibodies and IL-12-deficient mice have suggested that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens However, IL-12 appears also to play a central role in the genesis of some forms of immunopathology Inhibition of IL-12 synthesis or activity may be beneficial in diseases associated with pathologic Th1 responses, such as multiple sclerosis or Crohn's disease On the other hand, administration of recombinant IL-12 may have utility in the treatment of diseases associated with pathologic Th2 responses such as allergic disorders and asthma

THE INTERLEUKIN-12/INTERLEUKIN-12-RECEPTOR SYSTEM: Role in Normal and Pathologic Immune Responses

Publisher Summary Although interleukin-12 (IL-12) has been discovered only relatively recently, a remarkable level of knowledge has been obtained for this cytokine, and many possible therapeutic applications are being tested or have been proposed. It performs the dual function as a proinflammatory cytokine and as an immunomodulatory molecule. This chapter describes the structure, genetics, induction, and biological functions of IL-12. Being produced at the early times of inflammation in response to infection or other stimuli, IL-12 contributes via the production of IFN-γ and other cytokines to the inflammatory process itself and particularly to the activation of macrophages. IL-12 represents a bridge between innate resistance and adaptive immunity, with a central role in the regulation of the response to infection and tumor cells, as well as in autoimmunity and allergy. Because of these activities, its clinical use has been proposed in infections, both as an immunopotentiating agent or as an adjuvant in vaccination, against tumors, and for the prevention of severe allergic syndromes. The study of IL-12 has revealed much about the physiology of innate and adaptive immunity; however, harnessing its potent biological activities for efficient therapeutical applications is a challenge.

Interleukin-12: A Cytokine at the Interface of Inflammation and Immunity

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light alpha-chain, formerly defined as p35, disulfide-linked to a heavier beta-chain, formerly defined as p40. The beta-chain is also produced in large excess in a free form, and disulfide-linked beta-chain homodimers with anti-inflammatory effects are produced in the mouse. We analyzed the biosynthesis and glycosylation of IL-12 in human monocytes, and in a cell line stably transfected with IL-12 alpha and beta genes (P5-0.1). The IL-12 heterodimer and free beta-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the beta-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the alpha-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs extracellular IL-12 heterodimer revealed that the alpha-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free beta-chain secretion, while preventing the IL-12 heterodimer assembling and secretion. Pulse-chase experiments indicated that IL-12 persists intracellularly for a long period as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion. beta-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.

Biosynthesis and Posttranslational Regulation of Human IL-12

Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-gamma) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-gamma production from CD4+ T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-gamma production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.

Hydrolysis of interleukin-12 by Porphyromonas gingivalis major cysteine proteinases may affect local gamma interferon accumulation and the Th1 or Th2 T-cell phenotype in periodontitis.

DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were compared in vitro in human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration.

https://www.pnas.org/content/pnas/100/3/1163.full.pdf

Optimized expression and specific activity of IL-12 by directed molecular evolution

Molecular model of interleukin 12 that highlights amino acid sequence homologies with adhesion domains and gastrointestinal peptides

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Molecular model of interleukin 12 that highlights amino acid sequence homologies with adhesion domains and gastrointestinal peptides