Atrial natriuretic peptide down-regulates neutrophil recruitment on inflamed endothelium by reducing cell deformability and resistance to detachment force.

Abstract

2 BACKGROUND: In Japan, Recombinant atrial natriuretic peptide (ANP) is administered in 3 patients with acute heart failure in Japan to improve renal function and hemodynamics, but its anti-inflammatory effect on activated leukocytes may also contribute to its therapeutic efficacy OBJECTIVE: Examine unconventional role of ANP in neutrophil adhesion to inflamed endothelium. METHODS: Human neutrophils were perfused over endothelial monolayers in a microfluidic 10 lab-chip assay. Cell rheology was assessed by micropipette aspiration to assess changes in 11 cortical tension and viscosity. Fluorescence microscopy was applied to measure adhesive contact 12 area and β 2 -integrin focal bond formation. 13 14 RESULTS: ANP inhibited neutrophil rolling and firm adhesion without influencing the 15 upregulation of cellular adhesion molecules on endothelium or the regulation of high affinity 16 CD18 and shedding of L-selectin during neutrophil activation. Conversion Exposed to fluid 17 shear, to shear resistant integrin mediated arrest was disrupted with ANP treatment, which 18 elicited formation of long tethers and diminished cell spreading and contact. This correlated with 19 an ~40% increase in neutrophil viscosity and a reduction in the adhesive footprint. CONCLUSIONS: A decrease in cell deformation and PMN neutrophil flattening with ANP 22 results in fewer integrin bond clusters, which translates to higher tensile forces and impaired 23 adhesion strengthening and cell detachment. prior to measurement. ANP did not alter the up-regulation of β 2 -integrin or 21 the shedding of L-selectin when compared to untreated control (n=3). PMN F-actin was measured using Phalloidin with and without IL-8 stimulation after simultaneous fixing and 23 permeabilization. Data shows replicates for n=3 donors with no significant effect of ANP 24 observed.