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ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process
  1. N J Holden1,
  2. J M Williams2,
  3. M D Morgan1,
  4. A Challa1,
  5. J Gordon1,
  6. R J Pepper3,
  7. A D Salama3,
  8. L Harper1,
  9. C O S Savage1
  1. 1School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
  2. 2Wellcome Trust Clinical Research Facility, University Hospital Birmingham Foundation Trust, Birmingham, UK
  3. 3Centre for Nephrology, University College London, Royal Free Hospital, London, UK
  1. Correspondence to Professor C O S Savage, Renal Immunobiology Group, School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK; c.o.s.savage{at}bham.ac.uk

Abstract

Objectives To determine a role for antineutrophil cytoplasmic antibody (ANCA)-activated neutrophils in promoting B cell survival through the release of B lymphocyte stimulator (BLyS).

Methods Neutrophil BLyS expression was measured by flow cytometry. Concentrations of BLyS in cell supernatants and donor serum samples were measured by ELISA. Cell survival assays were carried out using an L3055 cell line and viability measured by flow cytometry.

Results Tumour necrosis factor α and formyl-Met-Leu-Phe (fMLP) treatment of non-primed neutrophils and treatment of primed neutrophils with anti-PR3 ANCA IgG resulted in a significant increase in surface expression of BLyS within 30 min which returned to basal levels by 2 h. Supernatants from ANCA-stimulated neutrophils were shown to contain increased levels of BLyS and to promote the survival of the centroblast cell line L3055. Serum BLyS concentrations are increased in patients with active ANCA-associated systemic vasculitis and these levels are increased further following 1–3 months of treatment with rituximab.

Conclusions ANCA specifically causes the release of BLyS from activated neutrophils which can support B cell survival in vitro. The presence of serum BLyS in active disease and its increase following B cell depletion suggest it is an important factor in disease pathogenesis and may facilitate disease relapse.

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Introduction

The role of the neutrophil in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis (ASV) is well established.1 However, the ability of the neutrophil to influence other leucocyte responses during vasculitic disease is less well known. B cells, while facilitating ANCA IgG production, may also promote inflammation during disease activity. Autoreactive B cells producing ANCA escape deletion both centrally and peripherally,2 3 and within granulomatous lesions may act as antigen presenting cells, producing high levels of inflammatory cytokines. If significant lymphoid structure development occurs, they may undergo selection and affinity maturation.4 Tertiary lymphoid tissue development and the presence of germinal centre markers in granulomatous lesions from patients with granulomatosis with polyangiitis (GPA; also known as Wegener's granulomatosis) has also been reported.5

Resident stromal cells are likely to promote B cell survival in the inflamed tissue, but cells arriving into areas of inflammation may also provide key signals that support this role. Neutrophils are a primary source of the B cell survival factor B lymphocyte stimulator (BLyS). BLyS can be released in response to granulocyte-macrophage colony-stimulating factor or interferon γ from an intracellular pool6 and through cleavage of a membrane bound form in response to tumour necrosis factor α (TNFα).7 Serum BLyS concentrations are increased in patients with active vasculitis.8 We currently report that ANCA IgG from patients with GPA significantly induces neutrophils to release active BLyS and can provide a survival signal to B cells in vitro. Furthermore, patients with active ASV have increased serum levels of BLyS and this is increased further following treatment with rituximab.

Materials and methods

Patient details and serum collection

Serum was collected from 14 patients (11 men, median age 58 years) with relapsing ASV (12 PR3- and 2 MPO-seropositive; Birmingham Vasculitis Score (BVAS) >2) before treatment with rituximab and 1–3 months after rituximab treatment when the patients were in clinical remission (BVAS <2) who had no detectable peripheral blood B cells by flow cytometry. Healthy donor serum was obtained from 16 donors (11 men, median age 61 years). The protocol was approved by the local ethics committee.

ANCA IgG isolation

Total IgG was isolated from anonymised plasmapheresis samples obtained from PR3-ANCA positive patients. All patients fulfilled Chapel Hill definitions and had active renal disease. IgG in serum was isolated on a protein G-sepharose column (GE Healthcare, Buckinghamshire, UK). The protocol was approved by the local ethics committee.

Neutrophil isolation

Blood from healthy donors was obtained by venipuncture into acid citrate dextrose and neutrophils were isolated by centrifugation over a Percoll discontinuous gradient (GE Healthcare) as described previously.9 Informed consent was received from all donors and the local ethics committee approved the protocol.

Neutrophil treatment and supernatant collection

Following isolation, neutrophils were resuspended with RPMI medium (Sigma, Gillingham Dorset, UK) left unprimed or primed with TNFα (2 ng/ml) for 15 min. Neutrophils (2.5×106) were treated for various durations with 200 mg/ml PR3-ANCA or normal IgG control, formyl-Met-Leu-Phe (fMLP, 1 µM) or TNFα (10 ng/ml). Following treatment the cells were centrifuged and supernatants collected and stored at −80°C until use.

L3055 culture

L3055 cells (established from a patient with sporadic Burkitt's lymphoma, EBVneg) were maintained in early passage as previously described.10 For survival and proliferation assays, L3055 (5×106) cells were cultured for 48 h as above with 2.5% fetal calf serum with the addition of neutrophil cell supernatants as described above (diluted to 10% v/v in medium). To determine if BLyS in the supernatant was acting as a survival factor, a paired supernatant sample was incubated with BLyS neutralising antibody (10 ng/ml) (R and D Systems, Abingdon, UK) 1 h prior to culture. All antibodies including ANCA IgG were removed from the supernatants with the use of protein G beads (Pierce, Chester, UK) prior to culture.

Flow cytometry

Neutrophils were analysed for the surface expression of BLyS using a phycoerythrin-conjugated murine monoclonal antibody at 2 µg/ml (E-Bioscience, San Diego, USA) and an IgG isotype control (BD Pharmingen, Oxford, UK). Following culture, L3055 cells were labelled with FITC-conjugated annexin V and 7-aminoactinomycin D (7-AAD) (BD Pharmingen) according to the manufacturer's instructions. All samples were acquired on a FACScalibur flow cytometer using Cell Quest software (Becton Dickinson, Oxford, UK) with 1×104 cells acquired per tube. List mode data were analysed using WinDMI (2.9) software.

ELISA

Soluble BLyS in cell supernatants and patient and control donor serum samples was detected by sandwich ELISA (R&D Systems, Minneapolis, USA).

Results

Neutrophils express BLyS on the membrane surface and shed the protein upon stimulation with TNFα.7 To assess whether our neutrophil preparations yielded similar results, we carried out a time course assay for BLyS expression following TNFα and fMLP treatment of neutrophils (figure 1A). Neutrophils treated with either TNFα or fMLP exhibited a significant increase in surface expression within 15 min, which was lost from the cell surface following 2 h of treatment. We next investigated whether PR3-ANCA IgG treatment increased BLyS expression in similar time course assays (figure 1B). Treatment for 15–30 min with PR3-ANCA IgG significantly increased BLyS expression compared with normal IgG-treated cells and was then significantly reduced following 2 h of treatment. Soluble BLyS was measured in supernatants from cells treated for 2 h (figure 1C). BLyS was detected in all cellular supernatants but significant increases in BLyS levels above control samples was only observed following treatment with PR3-ANCA IgG.

Figure 1

BLyS expression and release in stimulated neutrophils in vitro. (A) Neutrophils (2.5×106) were treated with TNFα (10 ng/ml) or fMLP (1 µM) in a time course assay and BLyS surface expression was measured by flow cytometry (n=7). (B) Neutrophils (2.5×106) primed with TNFα for 15 min and treated with normal IgG or PR3-ANCA IgG in a time course assay were also assessed for BLyS expression by flow cytometry (n=7). PR3-ANCA IgG responses are a mean of two. Data for panels (A) and (B) have been normalised as a percentage of BLyS expression at time zero control. Supernatants from cells treated for 2 h as above were saved and analysed for the presence of BLyS. (C) Supernatants from unprimed neutrophils treated with TNFα or fMLP as above (n=5) and primed cells treated with normal or PR3-ANCA IgG (n=7) were analysed. *p<0.05 by paired Wilcoxon signed rank test between treatment groups and one-way Kruskal–Wallis test with Dunn's multiple comparison test within treatment groups. ANCA, antineutrophil cytoplasmic antibody; BLyS, B lymphocyte stimulator; fMLP, formyl-Met-Leu-Phe; TNFα, tumour necrosis factor α.

We next investigated whether neutrophils treated with PR3-ANCA IgG were able to promote B cell survival in vitro (figure 2A,B). Supernatants (10%) from neutrophils treated with PR3-ANCA, normal IgG or control untreated cells were added to L3055 cell cultures in low serum conditions. After 48 h culture in low serum conditions, L3055 cells exhibited high levels of cell death measured by the binding of annexin V and the nuclear stain 7-AAD. Supernatants from neutrophils treated with PR3-ANCA but not normal IgG induced a significant increase in B cell viability. Adding recombinant BLyS (1 ng/ml) into the L3055 culture also promoted survival in vitro. To assess the role of BLyS in this recovery, paired supernatants were incubated with a BLyS neutralising antibody prior to culture. BLyS neutralisation abrogated the increased L3055 cell survival induced by supernatants from neutrophils treated with PR3-ANCA IgG.

Figure 2

Ability of neutrophil supernatants to promote B cell survival in vitro. Supernatants from primed neutrophils left untreated or treated with normal IgG or PR3-ANCA IgG (200 µg/ml) for 2 h were collected and put into L3055 culture to assess their ability to promote B cell survival. (A) Recombinant BLyS protein (1 ng/ml) was also added to the cells as a positive control. L3055 (5×106) cells were cultured in low fetal calf serum (2.5%) to increase apoptosis in the presence of supernatants (10%). To distinguish a role for BLyS in culture, paired supernatants were also incubated with a neutralising antibody to BLyS (10 ng/ml) prior to culture. Cell viability was measured by staining cells after culture with annexin V and 7-AAD and negatively stained cells were deemed viable. Data (n=6) are presented as the percentage increase in cell viability above L3055 cells cultured in medium alone. (B) Representative dot plots for annexin V/7-AAD staining are shown with the percentage of double negative (viable cells) in the bottom left quadrant calculated. **p<0.01, *p<0.05 by paired Wilcoxon signed rank test. 7-AAD, 7-aminoactinomycin D; ANCA, antineutrophil cytoplasmic antibody; BLyS, B lymphocyte stimulator.

Before treatment with rituximab, patients with active ASV had significantly higher levels of BLyS than an age-matched control cohort (figure 3A). Serum BLyS levels were also assessed 1–3 months following the initial dose of rituximab and were significantly increased above active disease pretreatment levels. Serum BLyS concentrations remained high and were not significantly altered before and after dosing in samples collected from seven patients who underwent a second cycle of rituximab treatment following disease relapse. In patients who received two cycles of rituximab, the presence or absence of B cells was compared with BLyS levels before and after receiving rituximab. Patients who had detectable peripheral B cells prior to receiving rituximab on the first occasion had significantly lower serum concentrations of BLyS than patients with no detectable B cells. These patients also exhibited a more pronounced increase in serum concentrations of BLyS following treatment (figure 3B).

Figure 3

(A) Serum B lymphocyte stimulator (BLyS) levels in patients with associated systemic vasculitis (ASV) treated with the monoclonal antibody drug, rituximab. Levels of BLyS in the serum of patients with active ASV prior to treatment with rituximab were measured by ELISA and compared with an age- and sex-matched healthy control cohort. Serum BLyS levels were also measured following 1–3 months of treatment with rituximab and the onset of clinical remission. Serum samples were collected from seven patients following disease flare prior to a second cycle of rituximab treatment and again 3 months after treatment. The horizontal line indicates the median value. (B) The seven patients receiving two cycles of rituximab treatment were further grouped depending on the detection of peripheral blood B cells by flow cytometry prior to rituximab treatment. Data are grouped into donors with positive B cell detection and those with no detectable B cells prior to the first and second cycle of rituximab treatment. The horizontal line indicates the median value. ***p<0.001, *p<0.05 by paired Wilcoxon signed rank test within donor groups and Mann–Witney test between different donor groups.

Discussion

In this study we show that ANCA IgG directed against membrane PR3 on the neutrophil can cause the upregulation and release of bioactive BLyS that is capable of promoting B cell survival in vitro. We also confirm other studies demonstrating that BLyS is increased in the serum of patients with active ASV and provide evidence that, following treatment with rituximab, BLyS levels increase further despite patients being in clinical remission.

Neutrophils express a membrane form of BLyS which can be cleaved by the serine protease furin in response to TNFα.7 We extend these observations to show that PR3-ANCA IgG can activate the TNFα-primed neutrophil to release BLyS further. In our study, neutrophils increased membrane expression of BLyS up to 30 min after treatment; this differs from a previous study and is likely to represent differences in neutrophil isolation procedures. However, BLyS was lost from the cell surface following prolonged treatment and could be measured in cell supernatants. BLyS released from ANCA-activated neutrophils promoted the increased survival of a B cell line under low serum culture conditions. Thus, ANCA-activated neutrophils arriving into inflammatory sites may directly promote the survival of B cells already in situ, thereby supporting ongoing inflammation.

We confirm previous studies that patients with active ASV have increased serum levels of BLyS. In these patients, serum BLyS levels are further increased following treatment with rituximab. Rituximab is specific for human CD20 and results in the depletion of peripheral B cells11 and, although it appears to be an effective treatment for GPA,12 a number of studies have suggested that rituximab treatment may fail to deplete B cells in the target organ.13 14 Autoreactive B cells have a heightened BLyS dependency and excessive BLyS production rescues autoreactive B cells from peripheral deletion, allowing their migration to follicular and marginal zones.15 16 Thus, any treatment that deletes B cells in ASV, including rituximab, may result in the increased release of BLyS and may provide safe sanctuary sites for autoreactive B cells thereby facilitating subsequent disease relapse. It remains to be determined whether long-term use of rituximab or other B cell-depleting therapies for maintenance therapy may paradoxically increase the likelihood of relapse in ASV.

References

Footnotes

  • Funding This work was funded by a Stuart Strange Vasculitis Trust postdoctoral fellowship.

  • Competing interests COSS is currently employed by Glaxo Smith Kline.

  • Ethics approval This study was conducted with the approval of the South Birmingham ethics committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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