Sources of human sera
Two hundred thirty-three patients with collagen diseases (196 female and 37 male patients) were enrolled in the study. The mean age was 42.5 years, with a range of 18 to 72 years. The patients comprised 95 with SLE and 138 with other collagen diseases. All of the patients were diagnosed according to the respective classification criteria [19
]. Thirty-five age- and sex-matched healthy donors were enrolled as a control group. Sera were collected and stored at -20°C until use. All subjects gave written consent after the purpose and potential risks involved in the study were explained. The study protocol complied with the principles of the Declaration of Helsinki and was approved by the Ethical Committee of Tohoku University Graduate School of Medicine.
Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), human lung microvascular endothelial cells (HMVEC-Ls), and EGM-2 medium were purchased from Lonza (Basel, Switzerland). Human renal glomerular endothelial cells (HRGECs) and endothelial cell medium were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were grown in 5% CO2 at 37°C on polystyrene flasks (BD Biosciences, Bedford, MA, USA). These ECs were used at sooner than the fifth passage. HEK293T cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), Plat-E and Plat-GP packaging cells were purchased from Cell Biolabs (San Diego, CA, USA) and cultured in Dulbecco modified Eagle medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). Rat myeloma cells, YB2/0, were purchased from ATCC and cultured in RPMI1640 medium (Sigma) containing 10% FBS.
IgG fractions were purified from sera by using HiTRAP Protein G HP columns (Amersham Biosciences, Roosendaal, The Netherlands). The concentration of purified IgG was determined by measuring the OD at 280 nm (OD280). Purified IgG was stored at -20°C until use.
Binding activities of antibodies to the surface of ECs and FLRT2 molecules were measured by using FACSCalibur and FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) [17
], and the data were analyzed with FlowJo Software (Tree Star, Ashland, OR, USA). In brief, attached cells were dissociated from plates by using Cell Dissociation Solution (Sigma) and washed with phosphate-buffered saline (PBS). Aliquots of 1 × 105
cells/tube were incubated in blocking buffer (PBS containing 1% bovine serum albumin and 50 mg/ml goat gamma globulin fraction (Sigma)) with primary antibodies at 4°C for 30 minutes. After washing, cells were incubated with secondary antibodies and 7-amino-actinomycin D (7-AAD) (BD Biosciences) at 4°C for 30 minutes and analyzed with flow cytometry.
Primary antibodies included 1:10 diluted human serum, 0.5 mg/ml of purified human IgG, and 10 μg/ml goat anti-human FLRT1/FLRT2/FLRT3 antibody (R&D Systems, Minneapolis, MN, USA). Secondary antibodies included 1:50 diluted fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated goat anti-human IgG (Abcam, Cambridge, UK), PE-conjugated donkey anti-goat IgG (Abcam), PE-conjugated mouse anti-human IgG1/IgG2/IgG3/IgG4 antibody (Beckman Coulter, Fullerton, CA, USA), and DyLight 650-conjugated anti human IgM antibody (Abcam). For staining of the intracellular FLRT2 domain, IntraStain (Dako, Glostrup, Denmark) and anti-human FLRT2 antibody (K-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.
For measurement of AECA activity, the relative mean fluorescence intensity (MFI) ratio was calculated as follows: (sample MFI - control MFI)/control MFI × 100 [33
]. Relative MFI ratio of mean + 3 standard deviations (SD) among the control group was defined as the cutoff value for AECAs. For measurement of anti-FLRT2 activity against the cell-surface domain, the relative MFI ratio was calculated as follows: (MFI against FLRT2-expressing cells - MFI against non-FLRT2-expressing cells)/MFI against non-FLRT2-expressing cells × 100. In each set of experiments, relative MFI ratios of titrated reference serum with high anti-FLRT2 activity were calculated, and a standard curve was generated. The relative MFI ratio was converted to arbitrary units (AUs) according to the standard curve. AU of mean + 3 SD in the control group was defined as the cutoff value for the anti-FLRT2 antibody. Recombinant human FLRT2 (R&D Systems) was added at the indicated dose in inhibition tests. The percentage inhibition was calculated as follows: % inhibition = (AECA titer of sample serum - AECA titer of sample serum with inhibitor)/AECA titer of sample serum × 100.
HUVEC cDNA library
Total RNA was generated from HUVECs by using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and poly(A)+ RNA was purified with an mRNA Purification Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Double-stranded cDNA was synthesized by using a cDNA library construction kit (Takara Bio, Shiga, Japan). DNA fragments > 1,000 bp in length were ligated into the pMX vector (kindly donated by Toshio Kitamura, University of Tokyo, Tokyo, Japan).
Screening of cDNA library
The HUVEC cDNA library in pMX was retrovirally transfected into the YB2/0 rat myeloma cell line [34
]. Aliquots of 1 × 107
YB2/0 cells expressing the HUVEC cDNA library were incubated with 0.5 mg/ml of IgG with high AECA activity at 4°C for 30 minutes. After washing, cells were incubated with FITC-conjugated goat anti-human IgG and 7-AAD at 4°C for 30 minutes. The cells showing a high level of FITC fluorescence signal were sorted with FACS Vantage (Becton Dickinson). Sorted cells were kept in culture until the cell number increased sufficiently for the next round of sorting. Subcloning of cells bound to IgG with AECA activity was performed by the limiting dilution method.
Genomic DNAs of clones were purified by using the Wizard SV Genomic DNA Purification system (Promega Corporation, Madison, WI, USA). DNA fragments from the HUVEC cDNA library were amplified by polymerase chain reaction (PCR) by using TaKaRa LA Taq (Takara Bio) with primers corresponding to the 5' and 3' ends of the multiple cloning site of pMX (5'-GGTGGACCATCCTCTAGACTG, 3'-CCTTTTTCTGGAGACTAAAT, respectively). The PCR products were cloned into the pCR-TOPO vector (Invitrogen), and DNA sequences were analyzed with the BLAST program.
Expression of FLRT2 in HEK293T cells
The full-length FLRT2 fragment was amplified by PCR from genomic DNA of FLRT2-expressing YB2/0 clone sorted as described earlier, by using Phusion High-Fidelity DNA Polymerase (Finnzymes, Keilaranta, Espoo, Finland). Primer sequences were as follows: 5'-CCCACCACATTGTATTTTATTTCC, 3'-CTTGATAACGCTGGGCCTCT. The FLRT2 fragment was inserted into the pMX-IRES-GFP vector (Cell Biolabs). An FLRT2 expression vector with deletion of the unique region was made by using an In-Fusion HD Cloning Kit (Clontech Laboratories, Madison, WI, USA) with two PCR segments constructed to omit the unique region (363 to 419 amino acids) and inserted into the pMX-IRES-GFP vector. pMX-FLRT2-IRES-GFP was transfected directly into HEK293T cells with FuGENE HD (Roche Diagnostics, Basel, Switzerland) or retrovirally transfected into HEK293T cells. Full-length FLRT1 and FLRT3 fragments were amplified as described earlier and inserted into the pMX-IRES-GFP vector.
Cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). The lysate was mixed with 5 × sodium dodecyl sulfate (SDS) sample buffer and separated by electrophoresis on an 8% polyacrylamide gel. The proteins were then transferred onto Immobilon Transfer Membranes (Millipore, Billerica, MA, USA). The membranes were treated with 0.1 μg/ml of goat anti-FLRT2 antibody and IRDye680-conjugated donkey anti-goat IgG (LI-COR Biosciences, Lincoln, NE, USA), and fluorescence intensity was determined with the Odyssey Infrared Imaging System (LI-COR).
CDC was assessed by the tetrazolium salt reduction method by using WST-1 (Roche Diagnostics) [35
]. In brief, cells were seeded in 96-well culture plates at a concentration of 4 × 104
cells per well and cultured overnight. Cells were incubated with 100 μl of diluted IgG for 30 minutes followed by addition of 50 μl of rabbit complement (Cedarlane Laboratories, Burlington, ON, Canada) at the indicated concentrations for 2 hours at 37°C. Then 15 μl of WST-1 was added, and cells were incubated for an additional 4 hours. Absorbance at 450 nm (A450
) was measured and expressed as relative fluorescence units (RFUs), reflecting the number of viable cells. Triton X-100 (1%) and heat-inactivated complement were added to the wells to measure background or maximal absorbance of WST-1, respectively. Recombinant FLRT2 was added in the inhibition tests. The percentage cytotoxicity for each sample was calculated by using the formula:
ADCC was determined by using the LDH Cytotoxicity Detection Kit (Takara Bio) and the manufacturer's protocol [36
The percentage cytotoxicity was calculated as follows:
Detection of adhesion molecule expression
HUVECs were cultured overnight in 96-well culture plates and incubated with IgG (640 μg/ml) for 6 hours at 37°C. Harvested cells were stained with PE-conjugated anti-CD62E antibody (BioLegend, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-CD106 antibody (BioLegend), and Pacific blue-conjugated anti-CD54 antibody (BioLegend), and were analyzed with flow cytometry.
Detection of EC apoptosis
HUVECs were seeded in 48-well culture plates and incubated with test IgG (640 μg/ml) for 24 hours, and apoptosis in the harvested cells was measured with annexin V and 7-AAD (Apoptosis Detection Kit; BD Biosciences) with flow cytometry. Annexin V-positive/7-AAD-negative cells were measured as apoptotic cells.
Data were analyzed by using the two-tailed Student t test or Mann-Whitney U test for continuous variables. Pairwise comparisons were assessed by using the Wilcoxon signed-rank test. Spearman rank correlation was used to explore the associations between anti-FLRT2 titer and clinical parameters. All analyses were performed by using Prism software (GraphPad Software, La Jolla, CA, USA). In all analyses, P < 0.05 was taken to indicate statistical significance.