Aldosterone abrogates nuclear factor kappaB-mediated tumor necrosis factor alpha production in human neutrophils via the mineralocorticoid receptor.

Mineralocorticoid receptor (MR) activation by aldosterone controls salt homeostasis and inflammation in several tissues and cell types. Whether or not a functional MR exists in polymorphonuclear neutrophils is unknown. We investigated the hypothesis that aldosterone modulates inflammatory neutrophil responses via the MR. By flow cytometry, Western blot analysis, and microscopy, we found that neutrophils possess MR. Preincubation with aldosterone (10(-11) to 10(-6) M) dose-dependently inhibited nuclear factor kappaB activation in interleukin (IL)-8- and granulocyte/macrophage colony-stimulating factor-treated neutrophils on fibronectin by IkappaBalpha Western blotting, electrophoretic mobility shift assay, and RT-PCR for IkappaBalpha mRNA. Aldosterone had no effect on tumor necrosis factor alpha- and lipopolysaccharide-mediated nuclear factor kappaB activation or on IL-8- and granulocyte/macrophage colony-stimulating factor-induced extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or phosphatidylinositol 3-kinase/Akt activation. Spironolactone prevented nuclear factor kappaB inhibition, indicating an MR-specific aldosterone effect. By RT-PCR, we found that neutrophils have 11beta-hydroxysteroid dehydrogenase. Tumor necrosis factor alpha, which is controlled by nuclear factor kappaB, increased in the cell supernatant with IL-8 treatment. Aldosterone completely prevented this effect. RT-PCR showed a strong tumor necrosis factor alpha mRNA increase with IL-8 that was blocked by aldosterone, excluding the possibility that the tumor necrosis factor alpha increase was merely a consequence of secretion. Finally, conditioned medium from IL-8-treated neutrophils increased intercellular adhesion molecule-1 expression on endothelial cells and subsequently the adhesion of IL-8-treated neutrophils to endothelial cells. These effects were reduced when conditioned medium from aldosterone-pretreated neutrophils was used, and spironolactone blocked the aldosterone effect. Our data indicate that a functional MR exists in neutrophils mediating antiinflammatory effects that are at work when neutrophils interact with endothelial cells. These data could be relevant to MR-blockade treatment protocols.

NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils.

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.