136. New role for proteinase 3 in IL-16 bioactivity control in granulomatosis wit polyangiitis

Background: The immunomodulatory cytokine IL-16 is increased in several inflammatory and autoimmune diseases, such as granulomatosis with polyangiitis (GPA). IL-16 recruits and activates different immune cells such as T cells, dendritic cells, or monocytes via CD4 as the main receptor. IL-16 is produced by a variety of immune and non-immune cells, but synthesis and storage of IL-16 is regulated differently depending on the cell type and stimulation3. For the biological activity of IL-16, proteolytic cleavage by caspase-3 in apoptosis dependent- and independent manner is required. Necrotizing granulomatous inflammation is a hallmark of granulomatosis with polyangiitis (GPA) with neutrophil dysregulation as a putative driver of chronic inflammation and autoimmunity. Earlier studies showed correlation of increased serum IL-16 with clinical parameters reflecting chronic inflammation in GPA, but functional evidence for a direct link between IL-16 and neutrophils in granulomatous inflammation is missing so far. In this study we aim to identify a functional link between increased release of IL-16, neutrophils, and the autoantigen proteinase 3 (PR3) regarding chronic inflammation and autoimmunity in GPA. Methods: IL-16 was measured in sera of GPA patients (n = 40) and healthy controls (HC, n = 50) by ELISA and correlated with clinical features, such as disease activity (BVAS), creatinine, GFR, VDI and PR3-ANCA status. IL-16 protein expression was analyzed in peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) from GPA patients and HC (n = 5, each) by SDS-PAGE and western blot. Binding affinity of recombinant pro-IL-16 to fluorescently labeled native human PR3 was assessed by microscale thermophoresis. Cleavage of pro-IL-16 by active human PR3 was performed at various time points at 37°C. Cleavage products were analyzed using SDS-PAGE and western blot. Results: Circulating IL-16 was significantly increased in GPA patients compared to HC. Elevated IL-16 levels positively correlated with BVAS, creatinine, VDI and PR3-ANCA status whereas there was a negative correlation with GFR. In isolated PMBC and PMN from GPA patients and HC we identified different expression patters of precursor and active forof IL-16. In healthy PBMC we found high amounts of precursor (80kD), pro-IL-16 (55kD) and active IL-16 (17kD). In contrast, PBMC from GPA patients had lower amounts of pro-IL-16 and no active IL-16, indicating activation and secretion of IL-16 due to inflammatory stimulation, as described earlier5. In GPA PMN we detected no precursor IL-16, but pro-IL-16 and its active form, in contrast to very low amounts of all IL-16 forin healthy PMN. Processing of IL-16 in neutrophils has been linked to apoptosis and the release of active IL-16 was dependent on secondary necrosis6. Interaction studies showed direct binding of recombinant pro-IL-16 to PR3 with a Kd of 10 nM. In a subsequent cleavage assay we could confirm that PR3 processed pro-IL16 in a time-dependent manner. Conclusions: Correlation of serum IL-16 with clinical features of GPA suggests that IL-16 could represent a marker of disease activity, tissue damage and autoreactivity. We showed that PBMC and neutrophils represent a source of IL-16 in GPA. By the identification of PR3 as an additional IL-16-processing enzyme we demonstrate a potential link between excessive PR3 expression and cell death dysregulation to IL-16 dependent mechanisas a driver of the chronic granulomatous inflammation and autoimmunity in GPA. Disclosures: none