For measurement of CXCR1 and CXCR2 expression, 37 patients with quiescent AAV and 5 AAV patients with active disease were recruited from our out-patient clinic. Thirty healthy donors recruited from laboratory personnel were included as a normal control population. Characteristics of patients and controls are summarized in Table 1.

A diagnosis of granulomatosis with polyangiitis (GPA), Churg Strauss syndrome (CSS) or microscopic polyangiitis (MPA) was based on the Chapel Hill definitions 12. ANCA specificity for PR3 or myeloperoxidase (MPO) was determined by capture ELISA. Patients in remission were without treatment. Treatment of active patients is listed in Table 1. Disease severity was quantified using the Birmingham Vasculitis Activity Score (BVAS).

Sera from AAV patients and healthy volunteers were collected for cytokine profiling. For patients in remission, samples were drawn on the same day for CXCR1/2 measurement and cytokine testing. For patients who were in remission but had suffered from active disease before (n = 18), serum samples from active disease were retrieved from a stored serum bank. All sera were stored at -80°C before testing.

All subjects gave their informed consent and the study was approved by the hospital medical ethical committee.

Peridin chlorophyll protein (PerCP)/Cy5.5-conjugated anti-CD14 (HCD14; Biolegend, San Diego, CA, USA) was used to discriminate neutrophils from monocytes in whole blood staining. Phycoerythrin (PE)-conjugated anti-CD181 (CXCR1, 5A12; BD Bioscience, Alphen aan de Rijn, The Netherlands), allophycocyanin (APC)-conjugated anti-CD182 (CXCR2, 6C6; BD Bioscience, Alphen aan de Rijn, The Netherlands) and fluorescein isothiocyanate (FITC)-conjugated anti-CD177 (NB1, MEM166; abCAM, Cambridge, UK) were used to detect membrane expression of CXCR1, CXCR2 and CD177, respectively. Irrelevant antibodies of the same isotype were used as negative controls.

Human conditionally immortalized glomerular endothelial cells (CiGEnC) 13 were cultured in endothelial growth medium 2-microvascular (EGM2-MV; Cambrex-Lonza, Breda, The Netherlands) containing 5% FCS and growth factors, without vascular endothelial growth factor (VEGF), as supplement. CiGEnC up to passage 40 were propagated at 33°C to keep cells in a proliferative state. Experiments were carried out on cells that had grown into a confluent monolayer and had been incubated for 5 days at 37°C in order to acquire a non-proliferative/quiescent phenotype.

Neutrophils were isolated from peripheral blood according to routine procedures as described previously 14. Briefly, heparinized venous blood was centrifuged on Lymphoprep (Axis-Shield, Oslo, Norway). Contaminating erythrocytes were lysed with ice-cold ammonium chloride buffer. Afterwards, cells were washed with cold Hanks' balanced salt solution (HBSS) without Ca²⁺/Mg²⁺ (HBSS^-/-) and resuspended in HBSS with Ca²⁺/Mg²⁺ (HBSS^+/+; GIBCO/Life Technologies, Breda, The Netherlands) to obtain 10⁷ cells/ml.

Where indicated, cells were incubated with serial doses of interleukin-8 (IL-8; R&D Systems, Minneapolis, MN USA), angiopoietin-1 (ANGPT-1; R&D Systems, Minneapolis, MN USA), angiopoietin-2 (ANGPT-2; R&D Systems, Minneapolis, MN USA), recombinant human tumor necrosis factor-α (rhTNF-α; R&D Systems, Minneapolis, MN USA) or repertaxin (Sigma-Aldrich, ST. Louis, MO USA) at 37°C for 30 minutes. Non-stimulated cells were incubated with control medium under the same condition.

Membrane expression of CXCR1, CXCR2 and CD177 on neutrophils was assessed by flow cytometry. According to the manufacturer's instructions, 100 μl of whole blood from patients and controls or 10⁶ of purified neutrophils after in vitro stimulation were incubated with antibodies at room temperature, in the dark, for 15 minutes, followed by fixation and lysing of erythrocytes with 20 × volume of Fluorescence Activated Cell Sorting (FACS) lysing solution (BD Bioscience, San Jose, CA, USA). Fluorescence intensity was measured by FACS Calibur (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA) and calibrated using CellQuest™ software (Becton Dickinson). Results were analyzed using the Win-List software package (Verity Software House, Topsham, ME, USA). Mean fluorescence intensity (MFI) of 5.10⁴ counted cells was measured in each sample. Neutrophils were gated as forward scatter^high (FSC^high)-side scatter^high (SSC^high)-CD14^low cells. Expression levels were presented as MFI corrected for nonspecific binding of isotype control antibodies on neutrophils from the same donor.

Numbers of adherent or migrated neutrophils in adhesion or migration assay were quantified by myeloperoxidase (MPO) activity assay 15. Briefly, cells were lysed with 0.5% Triton-X-100 at 4°C for 20 minutes and acidified by adding 1 M citrate buffer (pH = 4.2, 50 μl/1 ml of cell lysate) just prior to the reaction. Aliquots (75 μl) from each sample were transferred into wells of a flat-bottomed 96-well plate. 2.2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) at a concentration of 0.05% in 100 mM citrate with 0.03% H₂O₂ was loaded in an equal volume as substrate for MPO. After the reaction, OD₄₀₅ was measured using an ELISA plate reader. Numbers of neutrophils in each well were determined as based on standards generated from known concentrations of neutrophils from the same isolation.

Neutrophil adhesion to endothelial monolayers was quantified by adhesion assay as described previously with modifications 16. CiGEnCs were seeded in 24-well plates and allowed to proliferate to confluence. Cell monolayers were carefully washed with HBSS^+/+ and stimulated with 10 ng/ml TNF-α at 37°C for 4 hours. Cells were then washed three times before 0.5 × 10⁶ neutrophils were loaded. Where indicated, neutrophils were pretreated with serial doses of IL-8, TNF-α or repertaxin which is a noncompetitive allosteric inhibitor of CXCR1 and CXCR2 17. After incubation of endothelial cells with neutrophils at 37°C for 30 minutes, loosely adherent or non-adherent neutrophils were washed off with HBSS^-/- and the endothelial monolayers plus adherent neutrophils were lysed in 0.5% Triton-X-100. All experiments were run in duplicate. The numbers of neutrophils were quantified, as described above, by MPO activity assay.

Transwell inserts (Corning, NY, USA), with 3.0 μm pores in 12-well plates, were precoated with CiGEnCs. Confluence of the endothelial monolayers was determined by continued monitoring of trans-endothelial electrical resistance (TEER) 13 which plateaued at 20 to 25 Ω on the 5^th day after seeding. Histological staining of the membrane showed that the formation of monolayers on inserts required 6 × 10⁴ cells and 5 days of culture. Therefore, all the Transwell inserts were coated with CiGEnCs following this standard procedure. Isolated neutrophils from healthy donors were preincubated with repertaxin, IL-8 or TNF-α, and 1.10⁶ cells were loaded in the upper chamber and 10 ng/ml of IL-8, which showed optimal efficiency as a chemoattactant in titration in preliminary experiments (data not shown), was placed in the lower chamber. Wells without IL-8 in the lower chamber or neutrophils without any treatment in the upper chamber were included as controls. All experiments were run in duplicate. After co-incubation of neutrophils with the inserts at 37°C for 2 hours, the plates with inserts within the wells were centrifuged for 5 minutes at 50 g and 4°C to dislodge adherent neutrophils on the lower surface of the inserts. The cells in the lower chamber were collected and quantified by MPO activity assay.

Sera collected from AAV patients in remission (n = 37), patients with active disease (n = 18) and healthy blood donors (n = 30) were used for measurement of IL-8, TNF-α, ANGPT-1 and ANGPT-2. IL-8 and TNF-α levels were measured by ELISA (R&D Systems, Minneapolis, MN, USA) as described previously 18. After sample incubation and binding of the biotinylated detecting antibodies, color reaction was performed with streptavidin-poly-HRP (Sanquin, Amsterdam, The Netherlands) and tetramethyl-benzidin (TMB, Roth, Karlsruhe, Germany). Levels of angiopoietin-1 and angiopoietin-2 in sera were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Normal levels of these cytokines in serum were defined as the mean ± 2 SD of healthy controls.

Results of CXCR1 and CXCR2 expression are presented as medians. Serum levels of IL-8, TNF-α, ANGPT-1 and ANGPT-2 and membrane expression of adhesion molecules are presented as means. Data were analyzed by the Mann-Whitney test and Spearman's rank correlation test using GraphPad Prism 4.03 (GraphPad Software, San Diego, CA, USA). P values less than 0.05 were considered significant.

We first evaluated expression levels of CXCR1 and CXCR2 on neutrophils in AAV patients (remission, n = 37; active, n = 5) in comparison with healthy blood donors (n = 30). Both receptors were highly expressed on the membrane of neutrophils. There was no difference in CXCR1 and CXCR2 expression in patients with different AAV diseases or between patients with MPO- or PR3- ANCA specificity (data not shown). Neutrophils from patients in remission (median 1153, range 640 to approximately 1,986) showed comparable expression of CXCR1 as those from HC (1,163, 790 to approximately 1,790, P = 0.3), while neutrophil CXCR1 levels were significantly decreased in patients with active disease (533, 416 to approximately 1,071) compared to either HC (P = 0.003) or patients in remission (P = 0.0023) (Figure 1A). CXCR2 expression was significantly down-regulated in AAV patients in remission (453, 201 to approximately 817) compared to HC (589, 290 to approximately 941, P = 0.0004) and further decreased in active patients (323, 242 to approximately 564) as compared to HC (P = 0.0086) (Figure 1B). A significant correlation was observed between the expression of CXCR1 and CXCR2 on neutrophils of AAV patients, regardless of disease activity (Figure 1C, spearman r = 0.87, P < 0.0001).

IL-8 and TNF-α are proinflammatory cytokines reported to be elevated in the serum of AAV patients 192021. Serum levels of ANGPT-2 have also recently been reported to be increased in AAV and to correlate with disease activity 22. IL-8 is also produced by endothelial cells stimulated with ANGPT-1 23. Therefore, we first tested the influences of these cytokines on CXCR1 and CXCR2 expression on neutrophils of HC. As a result, both IL-8 and TNF-α down-regulated CXCR1 and CXCR2 expression on isolated neutrophils in a dose-dependent manner. IL-8, at a concentration of 5 ng/ml, reduced CXCR2 levels to 61% compared to controls incubated with normal medium, while IL-8 down-regulated membrane expression of CXCR1 to 71% at 20 ng/ml (Figure 2A left figure). TNF-α significantly reduced CXCR1/CXCR2 expression already at a dose of 1 ng/ml (Figure 2B left figure). When tested in parallel, CD177 (NB1) expression was slightly increased by IL-8 and significantly up-regulated by TNF-α, as reported in earlier studies 1424. The effects were less obvious when neutrophils of quiescent AAV patients were used. A significant reduction in expression of CXCR1 and CXCR2 was observed only at a concentration of 20 ng/ml of IL-8 (Figure 2A, right figure). Incubation with TNF-α decreased their expression but not to the same extent as seen for HC neutrophils (Figure 2B, right figure). Possibly AAV neutrophils are already pre-primed in the circulation and, therefore, respond less to in vitro treatment with cytokines. ANGPT-1 and ANGPT-2 did not affect CXCR1/CXCR2 or CD177 expression on isolated neutrophils (Figure 2C and 2D).

Levels of the cytokines mentioned above, which may have direct or indirect effects on CXCR1/CXCR2 expression, were evaluated in our patient cohort in comparison with HC.

Defining the normal range as mean ± 2SD of the IL-8 concentration in 30 healthy controls, 5 out of 37 (14%) patients in remission and 10 out of 18 (56%) active patients showed increased IL-8 levels. Levels of IL-8 were not significantly different between patients with quiescent AAV (median 2.81 pg/ml, range 0.0 to approximately 19.64 pg/ml) and healthy controls (2.30 pg/ml, 0 to approximately 15.43 pg/ml). However, a negative correlation was present between CXCR1/CXCR2 expression on neutrophils and levels of IL-8 in patients in remission (Figure 3). A negative correlation was not observed between IL-8 and CXCR1/2 in active AAV, probably due to the small sample size. Nevertheless, in concert with further decreased CXCR1/2 expression, IL-8 levels increased remarkably during active disease (8.67 pg/ml, 3.96 to approximately 51.55 pg/ml), and were significantly higher than levels in HC and patients in remission (Figure 4A).

As for levels of ANGPT-1, no significant difference was observed between HC and patients in remission. The median level of ANGPT-1 in active patients was lower than in HC (Figure 4B). Levels of ANGPT-2 were not increased in patients in remission nor in patients with active disease (Figure 4C). TNF-α was not detectable in sera of either HC or patient groups.

To further clarify possible consequences of CXCR1 and CXCR2 down-regulation on neutrophils, we investigated neutrophil adhesion and transmigration through a glomerular endothelial monolayer. Neutrophils were pretreated with repertaxin, mimicking decreased efficiency of IL-8 receptors, or stimulated with IL-8/TNF-α, both of which lead to decline of CXCR1 and CXCR2 expression.

Glomerular endothelial cell layers were stimulated with 10 ng/ml of TNF-α for 4 hours prior to the adhesion or migration assay to mimic activated glomerular endothelial cells as occur in AAV. Preincubation with repertaxin inhibited neutrophil migration in a dose-dependent way. Migration was reduced to 76% by 100 nM of repertaxin, significantly lower than control incubated neutrophils. IL-8 preincubation reduced the number of migrated neutrophils to 39%. Although incubation with TNF-α resulted in a significantly reduced expression of CXCR1/CXCR2 on the membrane of neutrophils, migration was not significantly influenced, and numbers of migrated neutrophils were comparable with control incubation (Figure 5B).

Neutrophils preincubated with 2 ng/ml TNF-α showed enhanced adhesion to glomerular endothelium compared to control incubation. IL-8 (up to 20 ng/ml), however, did not show a remarkable effect on neutrophil adhesion. Blocking CXCR1 and CXCR2 with repertaxin also increased the number of adherent neutrophils dose-dependently, from 135% with 10 nM of repertaxin up to more than 300% at 0.5 μM as compared to cells without stimulation (Figure 5A).