Aldosterone Stimulates a Degranulation Response in Human Neutrophils: Role of Protein Disulfide Isomerase

Abstract 1034

There is growing evidence for an important role of aldosterone (ALDO) in inflammatory responses in addition to its well-described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). We studied the effects of ALDO on activation of ex vivo human polymorphonuclear leukocytes (PMNC). We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses and when incubated with ALDO (10⁻⁹ −10⁻⁷ M) showed a dose-dependent rise in cytosolic Ca²⁺ that peaked within 2 min using FURA-2AM fluorescence. We then studied the effect of ALDO on PMNC degranulation following incubations with ALDO (10⁻⁹ −10⁻⁷ M) for 30 min and observed a significant increase in β–glucuronidase release (P<0.001, n=3) by established fluorescent detection methods, an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). PMA and N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used as positive controls for PMNC activation. We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcripts by quantitative RT-PCR using TaqMan detection probes in these cells and as reported in kidney and endothelial cells. Cells incubated with ALDO (10⁻⁸-10⁻⁷ M) showed a dose-dependent rise in cytosolic Ca²⁺ that peaked within 3 min using FURA-2AM fluorescence. To assess the degranulation response of these cells we quantified the in vitro release of myeloperoxidase (MPO) and observed that 10⁻⁸M ALDO was likewise associated with increased degranulation when compared to vehicle treated cells (AUC: 590±14 to 185±11, P<0.01, n=6). To characterize the mechanisms by which ALDO regulates the degranulation responses of these cells we studied the effects of Protein Disulfide Isomerase (PDI) on ALDO-stimulated cells. PDI catalyzes the oxidation or reduction of thiol/disulfide groups and modulates leukocyte function. Our results show that blockade of PDI, by bacitracin, led to a blunted ALDO-stimulated degranulation response in both cell types. Consistent with these observations, we show that in differentiated HL-60 cells, siRNA against PDI likewise led to reduced MPO responses (AUC: 590±14 to 290±13, P<0.01, n=6) that were associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls (0.55 ± 0.02, ΔΔCT of PDI siRNA relative to scrambled transfected cells, P<0.01, n=6). These results suggest that ALDO stimulates MPO release. MPO has been shown to be one of the predominant granule proteins associated with Neutrophil Extracelullar Traps (NETs), extracellular structures that contain chromatin (DNA and histones) that can also trap microorganisms. We studied the effects of ALDO following digestion of the NETs by DNAse, and observed that 30–35% of the total cellular MPO was NET-associated. We also observed that incubation with 10⁻⁸ M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3), a responses that was blocked by pre-incubation of cells with 1 uM CA (P<0.03, n=3). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). Thus our results suggest that activation of MR by ALDO leads to degranulation and NET production in neutrophils that may contribute to the inflammatory responses associated with MR activation in vivo. Furthermore, the association between degranulation and NET release implicates PDI as a novel regulator of MPO generated NET production.