Deletion of the ST2 proximal promoter disrupts fibroblast-specific expression but does not reduce the amount of soluble ST2 in circulation.

IL-33 signals through ST2, which is expressed either as a full-length signaling receptor or a truncated soluble receptor that can suppress IL-33 activity. Previous data suggest that soluble ST2 mRNA in fibroblasts is coupled to a serum-inducible proximal promoter, while full-length ST2 expression in immune cells is directed from a distal promoter. In order to better understand the function of the alternative promoters and how they ultimately affect the regulation of IL-33, we generated a mouse in which the ST2 proximal promoter is deleted. Promoter deletion had no impact on ST2 expression in mast cells or their ability to respond to IL-33. In contrast, it resulted in a complete loss of both soluble and full-length ST2 mRNA in fibroblasts, which corresponded with both an inability to secrete soluble ST2 and a defect in IL-33 responsiveness. Importantly, in spite of the fibroblast defect, soluble ST2 concentrations were not reduced in the serum of naïve or allergen-exposed knockout mice. In summary, we found that ST2 promoter usage is largely cell-type dependent but does not dictate splicing. Moreover, the proximal promoter is not a major driver of circulating soluble ST2 under the conditions tested.

IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells.

IL-33 is an IL-1 family member recently identified as the ligand for T1/ST2 (ST2), a member of the IL-1 receptor family. ST2 is stably expressed on mast cells and T(h)2 effector T cells and its function has been studied in the context of T(h)2-associated inflammation. Indeed, IL-33 induces T(h)2 cytokines from mast cells and polarized mouse T cells and leads to pulmonary and mucosal T(h)2 inflammation when administered in vivo. To better understand how this pathway modulates inflammatory responses, we examined the activity of IL-33 on a variety of human immune cells. Human blood-derived basophils expressed high levels of ST2 receptor and responded to IL-33 by producing several pro-inflammatory cytokines including IL-1 beta, IL-4, IL-5, IL-6, IL-8, IL-13 and granulocyte macrophage colony-stimulating factor. Next, utilizing a human T(h)2-polarized T cell culture system derived from allergic donor blood cells, we found that IL-33 was able to enhance antigen-dependent and -independent T cell responses, including IL-5, IL-13 and IFN-gamma production. IL-33 activity was also tested on V alpha 24-positive human invariant NKT (iNKT) cells. In the presence of alpha-galactosylceramide antigen presentation, IL-33 dose dependently enhanced iNKT production of several cytokines, including both IL-4 and IFN-gamma. IL-33 also directly induced IFN-gamma production from both iNKT and human NK cells via cooperation with IL-12. Taken together, these results indicate that in addition to its activity on human mast cells, IL-33 is capable of activating human basophils, polarized T cells, iNKT and NK cells. Moreover, the nature of the responses elicited by IL-33 suggests that this axis may amplify both T(h)1- and T(h)2-oriented immune responses.

The IL-1 receptor accessory protein (AcP) is required for IL-33 signaling and soluble AcP enhances the ability of soluble ST2 to inhibit IL-33.

Interleukin (IL)-33 (or IL-1F11) was recently identified as a ligand for the orphan IL-1 receptor family member T1/ST2 (ST2). IL-33 belongs to the IL-1 cytokine family and, upon binding to ST2, induces intracellular signals similar to those utilized by IL-1. The effects of other IL-1 family cytokines are mediated by their binding to a specific receptor and the recruitment of a co-receptor required for elicitation of signaling. The aim of this study was to characterize the co-receptor involved in IL-33 signaling. Immunoprecipitation confirmed that IL-33 specifically binds ST2 and revealed that cellular IL-1 receptor accessory protein (AcP) associates with ST2 in a ligand-dependent manner. Receptor binding measurements demonstrated that the affinity of mouse (m)IL-33 for ST2 is increased by 4-fold in presence of AcP. IL-33 dose-dependently stimulated IL-6 secretion from wild-type (WT) mast cells, while no effect of IL-33 was observed with mast cells derived from AcP-deficient mice. Finally, soluble (s)ST2-Fc and sAcP-Fc acted synergistically to inhibit IL-33 activity. These observations identify AcP as a shared co-receptor within the IL-1 family that is essential for IL-33 signaling and suggest a novel role for sAcP in modulating the activity of IL-33.

Use of ProteinChip array surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify thymosin beta-4, a differentially secreted protein from lymphoblastoid cell lines.

The identification of proteins differentially expressed between cancer and normal cells is vital for the development of cancer diagnostics, therapeutics and vaccines. Using a ProteinChip Biomarker System (Ciphergen Biosystems, Fremont, CA) which combines ProteinChip technology with time-of-flight mass spectrometry, we have developed a simple method to screen and identify differentially secreted proteins from tumor cell lines. Mass spectra of the range of proteins secreted from normal B-cells were generated along with those secreted from Epstein-Barr virus transformed B-cells. A mass peak at m/z = 4972.1 that was highly over-represented in the transformed B-cell line was chosen for identification and purified by reversed phase chromatography with concomitant monitoring of fractions by SELDI-TOF MS. The resulting purified protein was digested with trypsin and the peptide masses derived from the SELDI-TOF spectrum were used to search the public databases for protein identification. Fragment matching of the resulting peptides identified the protein as thymosin beta-4. Using LC-electrospray ionization MS/MS, the identity of this protein was confirmed. Thymosin beta-4 is a known marker in LCLs establishing the utility of this method to discover and identify proteins differentially expressed between cancers and their matched normal counterparts.