Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits

Abstract

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-bonded sub-units, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40) 2 , has been shown to be a potent IL-12 antagonist. To study the interaction between (p40) 2 and the known IL-12 receptor (IL-12R) subunits, IL-12R g 1 and IL-12R g 2, we directly measured the binding activity of mouse (p40) 2 to ConA-activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (K d about 5 pM) and low affinity (K d about 15 nM) binding sites for mouse 125 I-labeled (p40) 2. To elucidate which of the IL-12R subunits binds mouse (p40) 2 , binding studies of mouse 125 I-labeled (p40) 2 to Ba/F3 cells expressing recombinant mouse IL-12R g 1 and/or mouse IL-12R g 2 were carried out. Mouse IL-12R g 1 bound mouse 125 I-labeled (p40) 2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL-12R g 2 bound mouse 125 I-labeled (p40) 2 very poorly. These data demonstrate that similar to IL-12, mouse (p40) 2 binds with both high and low affinity to Con A blasts and B cells, and that IL-12R g 1 is responsible for mediating the specific binding of mouse (p40) 2 .

IL-12 p40 Homodimer-Dependent Macrophage Chemotaxis and Respiratory Viral Inflammation Are Mediated through IL-12 Receptor β1

“…In contrast to studies suggesting that p80 functioned as a competitive antagonist of IL-12 (11,17,55,56), our current report demonstrates p80 provides its own immunomodulatory signal that is independent of IL-12. Mouse Con A-activated splenocytes display identical binding affinities for p80 and IL-12, and in these cells p80 competitively inhibited IL-12 binding and IL-12-dependent proliferation.…”

“…Previous data indicated expression of R␤1 in the mouse pro-B cell line BA/F3 is necessary and sufficient to generate a p80 binding affinity that is equivalent to Con A splenocytes, LPS-activated B cells, and Staphylococcus aureus cell-activated B cells (17). However, this study failed to identify an agonist function for p80 that was mediated through R␤1.…”

“…2A). Since previous studies demonstrated p80 binds to R␤1 (10,17) and peritoneal elicited macrophages express R␤1 as well as IL-12R␤2 (R␤2) (23), we examined the role of these proteins in mediating p80-dependent chemotaxis. Macrophages pretreated with anti-R␤1 Ab or obtained from a R␤1 Ϫ/Ϫ mouse displayed blunted p80-dependent chemotaxis (Fig.…”

Non-Canonical Interleukin 23 Receptor Complex Assembly

“…As described previously (21) and as depicted in Fig. 1B, p40 binds to IL-12R␤1, but not to IL-23R, in the absence of p19.…”

Pathological Role of Large Intestinal IL-12p40 for the Induction of Th2-Type Allergic Diarrhea

“…15 Thus, murine IL-12p40 inhibits IL-12-mediated responses by means of the competitive binding to IL-12 receptor with an affinity similar to that of IL-12p70. 16,17 Further, IL-12p40 can behave as an IL12p70 antagonist in vivo, delaying the allograft rejection of cardiac myoblast. 18 IL-12p40 transgenic mice also showed increased susceptibility to the malaria infection.…”