OP0128 The Regulation of MRP8/14 in Pstpip1-Associated Myeloid-Related-Proteinaemia Inflammatory Syndrome (PAMI)
Authors: S.K. Fassl, D. Holzinger, J. Austermann, T. Vogl, M. Gattorno, A. Omenetti, J.J. Chae, I. Aksentijevich, J. Roth
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> MRP8 and MRP14 are phagocyte-derived Damage Associated Molecular Pattern (DAMP) proteins and can be used as biomarkers in inflammatory diseases e.g. juvenile idiopathic arthritis. We found that the MRP8/14 complex (calprotectin) is highly elevated in the serum of patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome and is even significantly higher in hypercalprotectinaemia and hyperzincaemia (Hz/Hc) [1]. For PAPA, mutations in the proline serine threonine phosphatase-interacting protein 1 (PSTPIP1) gene are described [2]. We have recently identified novel autosomal dominant mutations in <i>PSTPIP1</i> Hz/Hc patients. All are heterozygous carrier of an E250K or E257K substitution encoded by exon 11 of the PSTPIP1 gene. These patients show an excessively high serum concentration (0.9-12.0 g/l, normal range &lt;0.001 g/l) of MRP8/14 not present in any other inflammatory condition. The mechanism of secretion of MRP8/14 is not clear but depends on a tubulin-mediated alternative secretory pathway. MRP8/14 induces pro-inflammatory effects through interaction with Toll-like receptor 4 dependent signalling pathways [3]. <h3>Objectives</h3> The molecular link between <i>PSTPIP1</i> mutations and elevated MRP8/14 concentrations is currently unknown. Therefore we investigated the role of PSTPIP1 during release of MRP8/14. <h3>Methods</h3> MRP8/14 serum concentrations of patients were determined by ELISA. Monocytes from patients were isolated and MRP8/14 levels were measured in culture supernatants prior and after activation. Intracellular distribution of E250K and wildtype PSTPIP1 was analysed in transfected cells by immunofluorescence and interactions between PSTPIP1, MRP8/14 and microtubules were characterized via immunoprecipitations or microtubule binding assays. Protein interactions were further quantified <i>in vitro</i> by using a modified MRP8/14-ELISA with different constructs of PSTPIP1. <h3>Results</h3> Monocytes from patients with <i>PSTPIP1</i> mutations release significantly higher amounts of MRP8/14 than control cells. A co-localization of PSTPIP1 and MRP8/14 could be shown in monocytes and in vitro studies confirm this interaction as a calcium dependent and direct binding. By using deletion constructs of PSTPIP1 we could demonstrate that the MRP8/14 binding motif is mutated in PAPA and Hz/Hc. A mutual interference of PSTPIP1 and MRP8/14 on their interaction with microtubules could be shown which is altered by using the E250K mutated PSTPIP1. <h3>Conclusions</h3> Phagocytes seem to be the responsible cell type for the high serum concentration of MRP8/14 in Hz/Hc and PAPA. The alarmin MRP8/14 interacts directly with PSTPIP1 in a calcium-dependent manner and mutations found in all patients are apparently located inside the PSTPIP1-MRP8/14 binding region. Interaction of these proteins seem to have a regulatory function on their tubulin binding capability and could be of important relevance for the tubulin-dependent secretion of MRP8/14 which may be a pathogenetic mechanism of MRP-driven inflammation in PAMI. <h3>References</h3> Sampson et al. (2002) Lancet 360(9347), 1742-1745. Wise et al. (2002) Hum. Mol. Gen. 11(8), 961-969. Rammes et al. (1997) J. Biol. Chem. 272, 9496-9502. <h3>Disclosure of Interest</h3> None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.2822