Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis patients

Abstract

Leucocyte transendothelial migration is strictly regulated to prevent undesired inflammation and collateral damage of endothelial cells by activated neutrophils/monocytes. We hypothesized that in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV) patients' dysregulation of this process might underlie vascular inflammation. Peripheral blood mononuclear cells (PBMC) and neutrophils from AAV patients (n = 12) and healthy controls (HC, n = 12) were isolated. The influence of human umbilical vein endothelial cells (HUVEC) on neutrophil/monocytes function was tested by N-formyl-methionyl-leucyl-phenyl-alanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-mediated ROS production, degranulation and interleukin (IL)-8 production. In addition, the ability of lipopolysaccharide (LPS)-stimulated PBMC to produce tumour necrosis factor (TNF)-alpha in the presence or absence of HUVEC was tested. HUVEC inhibited ROS production dose-dependently by fMLP-stimulated neutrophils but did not influence degranulation. No differences between neutrophils from HC and AAV were found. However, in only one active patient was degranulation inhibited significantly by HUVEC only before cyclophosphamide treatment, but not 6 weeks later. Co-cultures of HUVEC with LPS-stimulated neutrophils/monocytes increased IL-8 production while TNF-alpha production was inhibited significantly. There was no apparent difference between AAV patients and HC in this respect. Our findings demonstrate that HUVEC are able to inhibit ROS and modulate cytokine production upon stimulation of neutrophils or monocytes. Our data do not support the hypothesis that endothelial cells inhibit ROS production of neutrophils from AAV patients inadequately. Impaired neutrophil degranulation may exist in active patients, but this finding needs to be confirmed.

Fig. 1

Influence of endothelial cells on neutrophil-reactive oxygen species (ROS) production and degranulation. (a) ROS production was measured in formyl-met-leu-phe (fMLP)-stimulated neutrophils to which endothelial cells were added at a ratio of 50 : 1 (neutrophils : endothelial cells) (filled squares). ROS production in the absence of endothelial cells was also measured (open squares). In addition, neutrophils were stimulated in the presence of 50 µM of 5′-(N-ethyl carboxamido)adenosine (NECA) (dashed symbol). **P<0·01, fMLP-stimulated neutrophils versus fMLP-stimulated neutrophils in the presence of endothelial cells and for fMLP-stimulated neutrophils versus fMLP-stimulated neutrophils in the presence of NECA. (b) Neutrophils were stimulated as in (a). Adenosine deaminase 10 units (dashed symbol) or an equal volume of medium (filled squares) was added to the combination of neutrophils and endothelial cells. fMLP-stimulated neutrophils served as control. *Adenosine deaminase versus medium, **fMLP-stimulated neutrophils versus fMLP-stimulated neutrophils in the presence of endothelial cells. (c) ROS production was measured in phorbol myristate acetate (PMA)-stimulated neutrophils essentially as described in (a). PMA stimulation alone (open squares), PMA stimulation in the presence of endothelial cells (filled squares), PMA stimulation in the presence of NECA (dashed symbol). (d) Degranulation of fMLP-stimulated neutrophils. Neutrophils were either stimulated with fMLP (10 µM) for 1 h at 37°C (hatched bars) or left untreated (open bars). During the incubation neutrophils were either in normal RPMI-1640 medium, in RPMI-1640 medium containing 100 µM NECA or endothelial cells were added to the neutrophils at a ratio of 50 : 1. After 1 h, the supernatants were collected and assessed for myeloperoxidase (MPO) release, as described in the Materials and methods. (e) Degranulation of PMA-stimulated neutrophils. The experiments were performed essentially as described in (d). For all experiments (a–e), the results of three independent experiments are expressed as mean relative light unit (RLU) ± standard deviation.

Fig. 2

Neutrophil-reactive oxygen species (ROS) production and degranulation in patients and controls. (a) ROS production in formyl-met-leu-phe (fMLP) (upper graph) and phorbol myristate acetate (PMA) (lower graph)-stimulated neutrophils. Stimulation occurred in the presence (filled circles) or absence (open circles) of endothelial cells (ratio 50 : 1). (b) Neutrophil degranulation of fMLP (upper graph) and PMA (lower graph)-stimulated neutrophils. The results are expressed as peak relative light unit (RLU) signal for each patient (n = 12) and controls (n = 12). The mean in each group is represented by the dashed symbol.

Fig. 3

Influence of endothelial cells on neutrophil-reactive oxygen species (ROS) production and degranulation in an active patient before and 6 weeks after initiation of cyclophosphamide. Upper graph shows the effect of endothelial cells on neutrophil ROS production. Neutrophils were stimulated with formyl-met-leu-phe (fMLP) in the absence (open symbols) or presence of endothelial cells (filled symbols). Lower graphs represent neutrophil degranulation. Neutrophils were stimulated with fMLP for 1 h in the absence (open bars) or presence of endothelial cells (filled bars). Hereafter the supernatants were collected and assessed for myeloperoxidase (MPO) release as described. The results are expressed as mean relative light unit (RLU) of triplicate determinations ± standard deviation.

Fig. 4

Influence of endothelial cells on lipopolysaccharide (LPS)-mediated tumour necrosis factor (TNF)-α and interleukin (IL)-8 production in co-cultures. (a) Peripheral blood mononuclear cells (PBMC) were either added to confluent monolayers of endothelial cells (filled symbols) or seeded in a similar concentration to 24-well plates in the absence of endothelial cells (open symbols). Hereafter the cells were stimulated with LPS 1 µg/ml for 24 h. Supernatants were collected and tested for TNF-α production by enzyme-linked immunosorbent assay (ELISA). Each patient and each control was tested in triplicate. LPS-stimulated human umbilical vein endothelial cells (HUVEC) was included in each experiment. The results are expressed as mean TNF-α production (pg/ml) for each patient (n = 12) and controls (n = 12). The mean in each group is represented by the dashed symbol. (b) Neutrophils were either added to confluent monolayers of endothelial cells (filled symbols) or were seeded in a similar concentration in 24-well plates in the absence of endothelial cells (open symbols). Hereafter the cells were stimulated with LPS 1 µg/ml for 24 h. Supernatants were collected and tested for IL-8 production by ELISA. LPS-stimulated HUVEC was included in each experiment to control for IL-8 production by HUVEC alone. IL-8 production of HUVEC alone was subtracted from the IL-8 production in co-cultures of HUVEC and neutrophils. The results are expressed as mean IL-8 production (pg/ml) for each patient (n = 12) and controls (n = 12). The mean in each group is represented by the dashed symbol.