Mouse interleukin‐12 (IL‐12) p40 homodimer: a potent IL‐12 antagonist

Abstract

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide-linked p40 dimer [(p40)2]. The (p40)2 was 25- to 50-fold more active than the p40 monomer in causing specific, dose-dependent inhibition of IL-12-induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL-12-induced interferon-gamma (IFN-gamma) secretion by mouse splenocytes and IL-12-dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL-12 in competing with 125I-labeled moIL-12 ([125I]moIL-12) for binding to mouse Con A blasts. However, in contrast to moIL-12, mouse (p40)2 displayed little ability to compete with 125I-labeled human IL-12 (huIL-12) for binding to high-affinity IL-12 receptors (IL-12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL-12-induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL-12 in competing with [125I] huIL-12 for binding to COS cells transfected with the human IL-12R beta subunit and expressing low-affinity IL-12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL-12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL-12 interacts with the beta subunit of IL-12R, and (iii) (p40)2 may be a suitable IL-12 antagonist for studying the role of IL-12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL-12 activity.

Transduction of the IL-21 and IL-23 genes in human pancreatic carcinoma cells produces natural killer cell-dependent and -independent antitumor effects

“…14 However, retarded growth of AsPC-1/IL-23#10 tumors was not due to the P40 complexes because growth of AsPC-1/p40 tumors was rather increased compared with that of parent tumors. Since the P40 complexes could be antagonistic to IL-12-mediated immune responses by binding to IL-12 receptors, 14,16,17 antitumor responses against AsPC-1 cells in nude mice is currently unknown and a possible antagonistic activity of the P40 complexes to IL-23 needs further investigation. IL-23-mediated antitumor effects observed in the present study was independent of NK cells activity, since treatment of anti-asialo GM 1 antibody did not affect the growth of AsPC-1/IL-23 tumors in nude mice.…”

Polymorphisms in the Il12b gene affect structure and expression of IL-12 in NOD and other autoimmune-prone mouse strains

“…Although human IL12p40 also exists in homodimeric form, it is much less efficient than mouse IL-12(p40) 2 as a competitive antagonist of IL-12 functions. 8 Therefore, the lower production of IL-12p40, both basal and after induction, may point to a decreased capacity of the NOD mouse, as well as other autoimmune-prone strains, to regulate IL-12 activity. In spite of a lower level of IL-12p40 induced after LPS administration, NOD mice produced levels of IL-12p75 similar to those attained by B6 mice, suggesting the induction of a lower amount of the potentially inhibitory p40 chain coupled with a comparable level of the biologically active IL-12 heterodimer.…”

“…5,6 The IL-12p40 homodimer binds to the ␤1 subunit of the IL-12 receptor but is unable to mediate the biological actions of IL-12p75. [7][8][9] It is therefore a potent antagonist of bioactive IL-12 and could play a role in immune regulation.…”

Deficient IL-12p70 secretion by dendritic cells based on IL12B promoter genotype

“…Whereas IL-12p70 appears to be the prototypical Th1-biasing cytokine capable of stimulating naïve T cells and inducing IFN-g secretion, IL-23 has a profound effect on memory T cells and the effective generation of an immune response, at least in the murine system. 9,10 Homodimers of p40 may antagonize the effect of IL12p70 in vitro, both in mice and humans, 11,12 but an agonist property for murine p40 homodimer has also been shown. 13 Recently, an association has been described between a complex bi-allelic polymorphism in the promoter region of IL12B (IL12Bpro) and mortality of children suffering from cerebral malaria.…”