1: Distinct tissue factors bind free IL-12p40 monomer extracellularly to generate novel cytokines

Abstract

Background IL-12 and IL-23 are heterodimeric cytokines formed by the association of a common p40 subunit with either p35 or p19. It is currently thought that these bioactive heterodimers must be assembled within the cell by covalent linkage before being secreted. However, we and others have shown that both human and mouse antigen presenting cells exposed to inflammatory signals secrete high levels of p40 in a free monomeric form. The biological significance of this is unknown. We have recently proposed that free IL-12p40 functions as a “cytokine adaptor” by binding to other protein(s) from its environment extracellulary and forming novel cytokines. Aim This study poses two questions: first, is free p40 capable of generating IL-12-like activities in concert with p35 extracellularly? Second, does circulating serum p40 specifically associate with other proteins to potentially form new cytokines? Methods We used an in vitro association assay using the p40 monomer in combination with p35 released from necrotic cells, together with a novel unbiased ex vivo proteomics approach to address these questions. Results Here we show for the first time that secreted free p40 monomers can combine with p35 extracellularly to generate biologically active IL-12. Both recombinant p40 and that from the serum of p35 −/− mice could combine with p35 released from necrotic cells to generate IL-12. Furthermore, we used an unbiased ex vivo proteomic approach to extract multiple novel proteins that were coeluted with p40 from the serum of mice undergoing sepsis. Conclusions Our data suggest that the rapid secretion of p40 monomer from APCs and its association with novel polypeptides from the local inflammatory “soup” generates new cytokines. We propose that such cytokines could regulate subsequent development of effector class during the innate phase of the immune responses before the recruitment of adaptive immunity.