Antineutrophil cytoplasmic antibodies induce reactive oxygen-dependent dysregulation of primed neutrophil apoptosis and clearance by macrophages

Abstract

This study assessed whether anti-neutrophil cytoplasmic antibodies (ANCAs) interfere with the safe deletion of neutrophils by apoptosis and phagocytic clearance. Tumor necrosis factor (TNF)-primed neutrophils were incubated with normal IgG (N IgG) or ANCA IgG for up to 36 hours. Compared with N IgG, ANCAs accelerated constitutive apoptosis of TNF-alpha primed neutrophils, as assessed by morphology and confirmed by DNA laddering pattern on gel electrophoresis, and accelerated progression to secondary necrosis. The accelerated apoptosis induced by ANCA was dependent on reactive oxygen species generation, as primed neutrophils from patients with chronic granulomatous disease failed to show an effect of ANCAs on apoptosis. However, there was no change in the rate at which neutrophils exhibited annexin V binding, indicating that externalization of phosphatidylserine was not accelerated by ANCAs. Furthermore, when ANCA-treated primed neutrophils were interacted with human or murine peritoneal macrophages after 12 hours there was significantly less phagocytosis by human macrophages and no difference in phagocytosis by murine peritoneal-derived macrophages when compared with N IgG-treated controls. In conclusion, ANCAs accelerate apoptosis and secondary necrosis in TNF-primed neutrophils by a mechanism dependent on the generation of reactive oxygen species, with uncoupling of nuclear and surface membrane changes, resulting in a "reduced window of opportunity" for phagocytic recognition and engulfment before disintegration.

Figure 1.

Fluorescence microscopy of aging PMNs, which were stained with a mixture of ethidium bromide and acridine orange. Original magnification, ×100. Top: Freshly isolated cells (A) appear large with multilobed nuclei, staining orange. Bottom: Early apoptotic morphology is observed as yellow staining of shrunken nuclei (B), whereas late apoptotic morphology was seen as cellular ghosts staining dark green with an absence of nuclear material (C).

Figure 2.

Fluorescence microscopy of primed PMNs that were cultured with TNF only, normal IgG, or ANCA (either anti-MPO or anti-PR3) (200 μg/ml) and stained with a mixture of ethidium bromide and acridine orange. There were accelerated changes of early apoptosis in those cells incubated with ANCA at 12 hours (A) and changes in late apoptosis at 18, 24, and 36 hours compared with normal IgG (*P < 0.05) (B). Data are expressed as a percentage of the total number of cells that showed apoptosis. Data are means ± SD of six experiments.

Figure 3.

Cell viability was assessed by trypan blue staining. TNF-primed PMNs alone or TNF-primed PMNs incubated with normal IgG or ANCA (either anti-MPO or anti-PR3) (*P < 0.05) were assessed for cell viability at various time points. Data are expressed as a percentage of the total number of cells. Data are means ± SD of six experiments.

Figure 4.

Coculture of TNF-α primed PMN with TNF only, normal IgG, or ANCA (either anti-MPO or anti-PR3) (200 μg/ml). Primed PMNs incubated with ANCA showed no increase in phosphatidylserine externalization, as assessed by FACS analysis of binding of FITC-conjugated annexin V (*P < 0.05). Data are expressed as a percentage of the total number of cells that showed apoptosis. Data are means ± SD of six experiments.

Figure 5.

DNA fragmentation of PMNs cultured for 18 hours. Lane 1: markers; lane 2: unprimed neutrophils alone; lane 3: primed PMNs incubated with MPO-ANCA; lane 4: primed PMNs incubated with N IgG; lane 5: primed PMNs incubated with PR3-ANCA; lane 6: primed PMNs alone.

Figure 6.

Primed neutrophils were aged in culture for 12 hours with N IgG or ANCA, and apoptosis was assessed by morphology and expressed as a percentage of the total number of cells that showed apoptosis (A). Macrophage interaction experiments were performed using human monocyte-derived macrophages and data expressed by the phagocytic index (B). Data are means ± SD of eight experiments. There were significantly more apoptotic cells when they were incubated with ANCA than when they were incubated with normal IgG (A). However, significantly fewer of those cells incubated with ANCA were phagocytosed compared with those cells incubated with normal IgG (B) (*P < 0.05).

Figure 7.

Primed neutrophils aged in culture for 12 hours with N IgG or ANCA. Apoptosis was assessed by morphology and expressed as a percentage of the total number of cells that were apoptotic (A). Macrophage interaction experiments were performed using murine peritoneal-derived macrophages, and data were expressed by the phagocytic index (B). Data are means ± SD of three experiments. There were significantly more apoptotic cells when they were incubated with ANCA than when they were incubated with normal IgG (A). However, there was no difference in the number of cells phagocytosed when they were incubated with ANCA or normal IgG (B) (*P < 0.05).

Figure 8.

PMNs isolated from patients with chronic granulomatous disease were primed with TNF and cultured alone or with normal IgG or ANCA (anti-MPO or anti-PR3). Apoptosis was assessed by morphology, viewed by fluorescent microscopy, and stained with ethidium bromide and acridine orange. Apoptosis was delayed as expected, and there was no difference between those cells that were primed with TNF and incubated with ANCA compared with TNF-primed PMNs incubated with normal IgG (*P < 0.05). Data are expressed as a percentage of the total number of cells that showed apoptosis. Data are means ± SD of three experiments.