Neutrophils from vasculitis patients exhibit an increased propensity for activation by anti-neutrophil cytoplasmic antibodies

Abstract

Anti-neutrophil cytoplasmic antibodies (ANCA) are thought to be pathogenic in ANCA-associated vasculitis (AAV) by stimulating polymorphonuclear leucocytes (PMNs) to degranulate and produce reactive oxygen species (ROS). The aim of this study was to investigate if PMNs from AAV patients are stimulated more readily by ANCA compared with PMNs from healthy controls (HCs). Differences in ANCA characteristics that can account for different stimulation potential were also studied. PMNs from five AAV patients and five HCs were stimulated with 10 different immunoglobulins (Ig)Gs, purified from PR3-ANCA-positive patients, and ROS production, degranulation and neutrophil extracellular trap (NET) formation was measured. ANCA levels, affinity and clinical data of the AAV donors were recorded. The results show that PMNs from AAV patients produce more intracellular ROS (P = 0·019), but degranulate to a similar extent as PMNs from HCs. ROS production correlated with NET formation. Factors that may influence the ability of ANCA to activate PMNs include affinity and specificity for N-terminal epitopes. In conclusion, our results indicate that PMNs from AAV patients in remission behave quite similarly to HC PMNs, with the exception of a greater intracellular ROS production. This could contribute to more extensive NET formation and thus an increased exposure of the ANCA autoantigens to the immune system.

Keywords: ANCA-associated vasculitis; autoimmunity; degranulation; reactive oxygen species.

Figure 1

Reactive oxygen species (ROS) activation of polymorphonuclear leucocytes (PMNs). (a) PMNs from anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients (n = 5) and healthy controls (HCs) (n = 5) stimulated with 10 AAV immunoglobulin (Ig)G at 200 μg/ml. The bars represent mean values of 10 stimulations read after 20 min incubation. Phosphate-buffered saline (PBS) is the negative control. AAV PMNs gave rise to more intracellular ROS production (measured with luminol + scavengers) than PMNs from healthy controls (HCs) (P = 0·019). Extracellular ROS was measured with isoluminol. (b) Representative graph showing ROS production over time in PMNs (from an AAV patient, filled circles, from a HC open circles) stimulated with one representative AAV IgG. Filled lines represent extracellular ROS and dotted lines intracellular ROS. (c,d) The characteristic of each IgG is shown with a mean value of five experiments in each donor group (HC and AAV PMNs). PMA is excluded from extracellular ROS graphs for clarity, as these values were much higher than for IgG stimulations. There were no significant differences between HC and AAV PMNs in any individual IgG. RLU = relative luminescence units. Statistics were calculated using the Mann–Whitney U-test. Error bars represent standard deviations.

Figure 2

Degranulation. (a) Degranulation of five different granula markers after stimulation of polymorphonuclear leucocytes (PMNs) from anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients (n = 5) and healthy controls (ctrl) [HCs (n = 5)] with 10 AAV immunoglobulin (Ig)G. No significant differences were detected; 37 ctrl represents PMNs from both HC and AAV PMN donors that were stimulated with buffer only at 37°C. Release is the amount of protein in supernatant compared with total amount of protein. (b). AAV IgG compared to HC IgG in ability to stimulate HC PMNs to degranulate. The same 10 AAV IgG as in (a) were tested on five different HC PMNs (median of five is reported) compared to seven HC IgG (for albumin) or five HC IgG [for gelatinase, lactoferrin, proteinase 3 (PR3) and myeloperoxidase (MPO)]. Median of several experiments with the same IgG is reported in cases where the same IgG had been tested on several HC PMNs. All different granular markers were released to a higher degree when stimulated with AAV IgG compared to HC IgG: albumin (P = 0·0097), gelatinase (P = 0·0027), lactoferrin (P = 0·0007), PR3 (P = 0·0047), MPO (P = 0·0007). Statistics were calculated using the Mann–Whitney U-test. Error bars represent standard deviations.

Figure 3

(a–e) Degranulation characteristics of the 10 different anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) immunoglobulin (Ig)G preparations. Each bar represents a mean value of five experiments in each donor group [HC and AAV polymorphonuclear leucocytes (PMNs)]. No significant difference was seen between HC and AAV PMNs for any IgG. PMA was used as a positive control (ctrl); 37 ctrl represents PMNs that were stimulated with buffer only at 37°C. Statistics were calculated using the Mann–Whitney U-test. Error bars represent standard deviations.

Figure 4

Neutrophil extracellular trap (NET) formation. (a) NET formation measured as the degree of extracellular DNA after stimulation with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) immunoglobulin (Ig)G in a fluorometric assay. One or two experiments per IgG were performed and in cases of two, a mean value of these is shown. IgG number 4 is missing from NET formation experiments. (b,c) Intracellular ROS (luminol + scavengers) and extracellular reactive oxygen species (ROS) (isoluminol) correlates with NET formation; r_s = 0·733, P = 0·031 and r_s = 0·750, P = 0·026, respectively. Spearman's correlation coefficient r_s was calculated.

Figure 5

(a) Activation score do not correlate with capture proteinase 3 (PR3)-anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) levels. (b,c) Gelatinase and lactoferrin degranulation correlates with the human–mouse chimeric PR3 proteins HHm and Hm added together; r_s = 0·649, P = 0·049 and r_s = 0·661, P = 0·044, respectively. (d) Activation score correlates with PR3 affinity: r_s = 0·758, P = 0·015. Spearman's correlation coefficient r_s was calculated.