Reagents
Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China). Fluorochrome dihydrorhodamine (DHR), phorbol myristate acetate (PMA), and normal human immunoglobulin (Ig)G were purchased from Sigma (St Louis, USA). For indirect enzyme-linked immunosorbent assay (ELISA), mouse anti-human CD177 antibody was purchased from Sino Biological Inc. and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Abcam (Cambridge, UK). For flow cytometry analysis, phycoerythrin (PE)-conjugated mouse monoclonal antibody against human CD177 and the isotype control mouse IgG1 were purchased from BioLegend (San Diego, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody against human PR3 and the isotype control mouse IgG1 were purchased from Abcam. For magnetic neutrophil sorting, anti-PE microbeads and separation columns were purchased from Miltenyi Biotech (Bergisch-Gladbach, Germany). For Western blot, antibodies against SHP-1 (C14H6) and phosphor-SHP-1 (Tyr564) were purchased from Cell Signaling Technology (Boston, MA, USA), and mouse anti-human GAPDH antibody was purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).
Cell culture
Primary human renal GEnCs (ScienCell Research Laboratories, San Diego, CA, USA) were cultured in endothelial cell basal medium (ECM) (ScienCell) with the addition of 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% endothelial cell growth factor for the formation of a confluent endothelial cell monolayer. The flasks for cell subculture were bio-coated with human plasma fibronectin (Millipore, Billerica, USA) beforehand according to the manufacturer’s recommendations. For synchronization of the cell cycle, GEnC monolayers were starved in basal medium without serum and endothelial cell growth factor for 12 h without bio-coating. All experiments were performed using GEnCs at passage 3–5. All cultures were incubated at 37 °C in 5% CO2.
Interaction between CD177 and PECAM-1
The interaction between CD177 and PECAM-1 was detected by ELISA with recombinant soluble PECAM-1 (sPECAM-1) at 2 μg/ml as the solid-phase antigen. sPECAM-1 in a coating buffer (0.05 M bicarbonate buffer, pH 9.6) was used to coat the wells of half of a polystyrene microtiter plate (Nunc-Immuno plate; Nunc, Roskilde, Denmark) and was incubated overnight at 4 °C. The other half of the plate was coated with coating buffer alone to establish antigen-free wells. The wells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and incubated with CD177 at various concentrations for 1 h at 37 °C. After washing three times with PBS-tween 20 (PBS-T), the wells were incubated with mouse anti-human CD177 antibody (1:1000; Sino Biological) for 1 h at 37 °C, and HRP-conjugated goat anti-mouse IgG (1:1000) was used as the secondary antibody. Tetramethylbenzidine (TMB; Sigma, St. Louis, MO) was used as the substrate and the reaction was stopped by the addition of sulfuric acid 0.5 mol/L (Carl Roth GmbH, Germany). Optical densities of formed complexes were measured at 450 nm using a microplate reader (Bio-Rad, Tokyo, Japan).
Isolation and priming of neutrophils
Neutrophils were isolated as described previously [18]. In brief, venous human blood for neutrophil isolation was obtained from healthy donors by venipuncture and anticoagulated with ethylenediaminetetraacetic acid (EDTA). Neutrophils were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). Erythrocytes were lysed with ice-cold red cell lysis buffer (Tiangen Biotech, Beijing, China), and then neutrophils were washed in PBS without Ca2+/Mg2+ (PBS−/−; Chemical reagents, Beijing, China) and suspended in PBS−/− to a concentration of 1 × 106 cells/ml and used for further analysis. The trypan blue staining technique was used as an index of the proportion of viable cells in a cell population. Where indicated, cells were primed with 2 ng/ml recombinant TNF-α (Sigma, USA) at 37 °C for 15 min, and untreated cells were incubated with control medium under the same conditions.
Magnetic neutrophil sorting
CD177-negative neutrophils were separated with negative selection by magnetic cell sorting (MACS) separation columns (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to the manufacture’s manual, as described previously [19]. All steps were carried out on ice. Freshly isolated neutrophils were stained with PE-conjugated monoclonal antibody to CD177 (MEM166). Subsequently, the cells were labeled with anti-PE microbeads (Miltenyi Biotech) and loaded on MACS LD columns (Miltenyi Biotech). The flow-through containing the nonlabeled CD177-negative neutrophils was collected. The purity of CD177-negative neutrophils was 86.4 ± 8.5% as assessed by flow cytometry.
Preparation of PR3-ANCA-positive IgGs
PR3-ANCA-positive IgGs were prepared from plasma exchange liquid of patients with active PR3-ANCA-positive primary small vessel vasculitis using a High-Trap-protein G column on an AKTA-FPLC system (GE Biosciences, South San Francisco, CA, USA). The preparation of IgGs was performed according to methods described previously [20]. In brief, plasma exchange liquid was filtered through a 0.2-μm syringe filter (Schleicher & Schuell, Duesseldorf, Germany) and applied to a High-Trap-protein G column on an AKTA-FPLC system (GE Biosciences). The column was treated with equal volume of 20 mmol/l Tris-HCl buffer, pH 7.2 (binding buffer), and IgG was eluted with 0.1 mol/l glycine-HCl buffer, pH 2.7 (elution buffer). After the antibodies emerged from the column, the pH value of the eluent was adjusted to pH 7.0 using 2 mol/l Tris-HCl (pH 9.0) immediately. The protein concentration of the antibodies was measured using the Nanodrop-1000 (Pierce, Rockford, IL, USA), and the level of PR3-ANCA IgG was measured by an ELISA kit (EUROIMMUN, Lubeck, Germany). We obtained written informed consent from the participants involved in our study. The research was in compliance with the Declaration of Helsinki and was approved by the clinical research ethics committee of the Peking University First Hospital.
Detection of mPR3 expression on neutrophils and PR3 in supernatant after incubation with PECAM-1
Expression of mPR3 on neutrophils was detected by flow cytometry. Primed neutrophils were incubated with PECAM-1 at serial concentrations (Fig. 1b). There was a dose-dependent response of PECAM-1 in inducing mPR3 downregulation. In further experiments, neutrophils were incubated with PECAM-1 at a concentration of 30 μg/ml or buffer control for 2 h at 37 °C. Since PECAM-1 and JAM-1 are both members of the immunoglobulin superfamily of adhesion molecules with many similarities in their expression profiles and functions [21, 22], JAM-1 was used as the control. Levels of PR3 in the supernatant were tested using commercially available ELISA kits (Elabscience, Wuhan, China). The assay was conducted according to the manufacturer’s instructions. All further steps of mPR3 detection on neutrophils were performed on ice, and washing steps were performed using PBS. TruStain FcR Solution (BioLegend, San Diego, CA, USA) was used in all samples prior to the addition of antibodies to block nonspecific binding. Next, cells were stained with a saturating dose of FITC-conjugated mouse monoclonal antibody directed against human PR3 (Abcam, Cambridge, UK) or with isotype antibody for 20 min in the dark. Fluorescence intensity of FITC was analyzed using flow cytometry. Samples were analyzed using a FACScan (BD, Biosciences, USA). Neutrophils were gated in forward/sideward scatter (FSC/SSC) and data were collected from 10,000 cells per sample. Data were analyzed using FlowJo software (TreeStar, Ashland, Oregon, USA).
Evaluation of neutrophil respiratory burst by DHR assay
We assessed the generation of ROS using DHR as described previously [23]. This method is based on the fact that reactive oxygen radicals cause an oxidation of the nonfluorescent DHR to the green fluorescent rhodamine. In brief, isolated neutrophils suspended in Hanks’ balanced salt solution (HBSS) were incubated with 0.05 mM DHR123 (Sigma-Aldrich, Louis, USA) for 30 min at 37 °C. Sodium azide (NaN3; 2 mM) was added to prevent intracellular breakdown of H2O2 by catalase. The neutrophils were then primed with TNF-α (2 ng/ml) for 15 min at 37 °C. After incubating with PECAM-1 (30 μg/ml) or JAM-1 (30 μg/ml) as described above, patient-derived ANCA-positive IgGs (5 RU/ml) or normal IgG were added. After incubation at 37 °C for 1 h, the reaction was stopped by the addition of 1 ml ice-cold 1% BSA in HBSS. Samples were kept on ice and analyzed using a FACScan. Neutrophils were gated in FSC/SSC and mean fluorescence intensity (MFI; representing the level of neutrophil activation) were collected from 10,000 cells per sample. Data were analyzed using FlowJo software (TreeStar, Ashland, Oregon, USA).
Measurement of neutrophil degranulation by lactoferrin quantification
Lactoferrin, an iron-binding multifunctional glycoprotein, is an abundant component of the specific granules of neutrophils [24, 25]. Lactoferrin is considered as a biomarker of neutrophil degranulation [26,27,28]. Neutrophils were primed with TNF-α (2 ng/ml) at 37 °C for 15 min, and then were incubated with PECAM-1 or JAM-1 (30 μg/ml) at 37 °C for 2 h followed by stimulation with patient-derived ANCA-positive IgGs or normal IgG for 1 h. Lactoferrin in the supernatant was tested by ELISA using a commercial kit (Abcam, Cambridge, UK). The ELISA procedure for measuring lactoferrin was performed according to the manufacturer’s instructions, as described previously [27].
SHP-1 phosphorylation detected by Western blot
To detect phosphor-SHP-1, isolated neutrophils were primed with TNF-α (2 ng/ml) or PBS at 37 °C for 15 min, and then were incubated with or without PECAM-1 (30 μg/ml) for 1 h. The cells were then incubated on ice in cell lysis buffer (Beyotime Biotechnology, Beijing, China) supplemented with proteinase inhibitors (Sigma, St. Louis, MO, USA) and phosphatase inhibitors (Roche, Mannheim, Germany) for 30 min. The insoluble material was pelleted, and samples were boiled with reduced loading buffer and run in 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Protein was transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA), and detected by rabbit anti-human SHP-1 antibody (1:1000), rabbit anti-human phosphor-SHP-1 antibody (1:1000), and mouse anti-human GAPDH antibody (1:500) overnight at 4 °C. Finally, the strips were incubated with HRP-conjugated goat anti-mouse (1:5000) or goat anti-rabbit secondary antibodies (1:5000) for 1 h at room temperature with gentle agitation and then revealed on autoradiographic film using the ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).
GEnC activation and injury indicated by soluble intercellular cell adhesion molecule-1
Soluble intercellular cell adhesion molecule-1 (sICAM-1) is considered as one of the markers of endothelial cell activation and injury [29]. To explore the role of PECAM-1 in PR3-ANCA-induced endothelial cell injury, the isolated neutrophils were incubated with PECAM-1, JAM-1, or control buffer at 37 °C for 1 h after priming with TNF-α. After incubation, neutrophils were added to GEnC monolayers with patient-derived PR3-ANCA-positive IgGs or normal IgG. After incubation for 4 h at 37 °C, the cell culture supernatant was collected for the ICAM-1 assays. Samples were tested using the human ICAM-1/CD54 ELISA kit (R&D, Abingdon, UK). The assay was conducted according to the manufacturer’s instructions as described previously [20]. In brief, samples were added to the microtiter plate coated with capture antibody and incubated for 2 h at room temperature, followed by detection antibody incubation for another 2 h. Then HRP-conjugated streptavidin was added. After 20 min of incubation avoiding direct light, the plate was washed, and substrate solution was added to the wells. After adding the stop solution, the absorption measurements were obtained at 450 nm (with a correction of 570 nm to eliminate optical imperfections in the plate) using a microtiter plate reader (Bio-Rad iMark™ Microplate Reader). All samples and standards were performed in duplicate.
Statistical analysis
The Shapiro-Wilk test was used to examine whether the data were normally distributed. Quantitative data are expressed as mean ± SD for data that was normally distributed or the median and range for data that was not normally distributed. Differences in quantitative parameters between groups were assessed using one-way analysis of variance (ANOVA) for data that was normally distributed or the Mann-Whitney U test for data that was not normally distributed, as appropriate. Differences were considered significant if p < 0.05. Analysis was performed with SPSS statistical software package (version 13.0, Chicago, IL, USA).
