Two pivotal aspects of the IL-33 system are characterized in this study: First the composition and function of the IL-33 receptor complex on IL-33 responder cells and second the generation of biologically active IL-33 by the IL-33 producer cell. IL-33 binds to the IL-33 receptor alpha-chain (ST2) and as is shown here, this results in the recruitment of interleukin-1 receptor accessory protein (IL-1RAcP) as the co-receptor. The IL-33-dependent interaction of membrane bound and soluble form of IL-1RAcP and IL-33Ralpha-chain was demonstrated in co-immunoprecipitation assays. Lack of the IL-1RAcP abrogated responses to IL-33 and IL-1 in the mouse thymoma clone EL-4 D6/76. Responsiveness to IL-33 and IL-1 was restored with the expression of full length IL-1RAcP, while a mutant protein lacking the Toll/IL-1 Receptor domain (TIR) was not sufficient to restore IL-33 or IL-1 responsiveness in EL-4 D6/76 cells. Moreover the monoclonal antibody 4C5, which neutralizes IL-1beta effects by blocking of murine IL-1RAcP, inhibited IL-33-stimulated signaling in mouse thymoma cells and bone marrow-derived mast cells. In search for putative further components of the IL-33 receptor complex, TIR8 / SIGIRR was studied, as it was proposed to be a candidate IL-33 receptor component. Using wild type TIR8 and chimeric fusion proteins it could be excluded that TIR8/SIGIRR can substitue for IL-1RAcP as a co-receptor. Furthermore, no experimental proof could be generated to show that this molecule is participating in the signaling IL-33 receptor complex. Like IL-18 and IL-1beta, IL-33 was found to have strong immunomodulatory functions. However, whereas IL-1beta and IL-18 promotes pro-inflammatory and Th1-associated responses, IL-33 induces Th2-associated cytokines. In order to find the differential gene regulation in response to these cytokines, IL-1-, IL-18- and IL-33 -induced gene expression profiles were compared by microarray from a triple responsive T cell line. Although a large number of genes were regulated by the IL-1 family members, no differentially regulated gene could be identified, suggesting the use of the same signaling molecules/pathways by all three cytokines, at least in this cellular system. Common notion in the field is that IL-33, like IL-1beta and IL-18, requires processing by caspase-1 to a mature form, in order to achieve biological activity as a cytokine. Contrary to the current dogma, here it is described that IL-33 is biologically active as unprocessesed full length molecule. Full length IL-33 binds to the IL-33 receptor-alpha chain and mediates the recruitment of IL-1RAcP to activate cells. IL-33 is processed in cells, however not by caspase 1 to a mature, biologically active, IL-33 but instead it is cleaved by caspase 3 at aa175 to yield two products which are both unable to bind to the IL-33 receptor. Thus caspase 3 processing inactivates IL-33 as a cytokine. Full length IL-33 and its N-terminal caspase 3 breakdown product, however, translocate to the nucleus of the producing cell. Interestingly, bioactive IL-33 is not released by cells constitutively or after activation of the inflammasome as is the case with other IL-1 family members, like IL-1beta and IL-18. Thus, it is tempting to speculate that IL-33 is not a classical cytokine but a molecule with dual function which normally exerts its function in the nucleus of intact cells and only activates others cells via the IL-33 receptor complex if cells are destroyed. Thus IL-33 may act as an endogenous danger signal, to alert cells of the innate immune system of the destruction of the IL-33 producing cells during hypoxia or after mechanical injury to initiate a sterile inflammation.
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