Autoantibodies against integral membrane proteins are usually pathogenic. Although anti-endothelial cell antibodies (AECAs) are considered to be critical, especially for vascular lesions in collagen diseases, most molecules identified as autoantigens for AECAs are localized within the cell and not expressed on the cell surface. For identification of autoantigens, proteomics and expression library analyses have been performed for many years with some success. To specifically target cell-surface molecules in identification of autoantigens, we constructed a serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). Here, we present an overview of recent research in AECAs and their target molecules and discuss the principle and the application of SARF. Using SARF, we successfully identified three different membrane proteins: fibronectin leucine-rich transmembrane protein 2 (FLRT2) from patients with systemic lupus erythematosus (SLE), intercellular adhesion molecule 1 (ICAM-1) from a patient with rheumatoid arthritis, and Pk (Gb3/CD77) from an SLE patient with hemolytic anemia, as targets for AECAs. SARF is useful for specific identification of autoantigens expressed on the cell surface, and identification of such interactions of the cell-surface autoantigens and pathogenic autoantibodies may enable the development of more specific intervention strategies in autoimmune diseases.
Inappropriate humoral and cellular immune responses mediate the tissue damage in autoimmune diseases, and the outcome of an autoimmune disease is influenced mainly by the tissue distribution of target self antigens [
Autoantibodies cause damage through a number of mechanisms, including the formation of immune complexes, cytolysis or phagocytosis of target cells, and interference with cellular physiology [
In 1971, Lindqvist and Osterland first described autoantibodies to vascular endothelium based on indirect immunofluorescence (IIF) experiments [
The presence of AECAs has been reported in patients with a wide variety of diseases, including collagen diseases (Table
Prevalence of anti-endothelial cell antibodies.
Disease | % of positive sera |
---|---|
Systemic lupus erythematosus | 15–85 |
Rheumatoid arthritis | 0–87 |
Mixed connective tissue disease | 33–45 |
Systemic sclerosis | 15–84 |
Polymyositis/dermatomyositis | 44–64 |
Antiphospholipid syndrome | 0–64 |
Sjögren’s syndrome | 24-25 |
Polyarteritis nodosa | 50–56 |
Microscopic polyangiitis | 2–60 |
Granulomatosis with polyangiitis | 19–80 |
Eosinophilic granulomatosis |
50–69 |
Takayasu arteritis | 54–95 |
Giant-cell arteritis | 33–50 |
Behçet’s disease | 14–80 |
Kawasaki disease | 65 |
AECAs are detected even in healthy subjects [
Methods for detection of AECAs have not been standardized, and a number of methods have been reported, including IIF, cell-based-enzyme linked immunosorbent assay (ELISA), flow cytometry, radioimmunoassay, western blotting (WB), and immunoprecipitation [
Human umbilical vein endothelial cells (HUVECs) are commonly used as a substrate, but antigen patterns of ECs differ among other ECs, passage numbers, and culture conditions [
An experimental animal model for pathogenicity of AECAs was reported by Damianovich et al. [
AECAs have been shown to be correlated with disease activities, and have the potential to induce vascular lesions because their targets are expressed on ECs that are readily accessible to these circulating antibodies. AECAs are also considered to play roles in the development of pathological lesions by a number of methods as described below [
The first is the cytotoxicity of ECs through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). CDC activity of AECAs was reported in patients with SLE, Takayasu arteritis, hemolytic-uremic syndrome, and Kawasaki disease [
The second is the induction of coagulation. AECAs may exhibit procoagulant effects by the production of tissue factor in SLE and the release of heparin sulfate in systemic sclerosis (SSc) [
The third is the induction of apoptosis. AECAs may induce EC apoptosis through CD95 or cross-reaction with anti-phospholipid antibodies [
The fourth is the activation of ECs. AECAs were reported to induce the secretion of interleukin (IL)-1
Alard et al. reported that recognition of cell-surface adenosine triphosphate (ATP) synthase in the low pH microenvironment contributes to intracellular acidification of ECs, which may induce cell death and trigger inflammation [
As described above, there is a great deal of evidence that AECAs play pathogenic roles in collagen diseases. Identification of targets of AECAs is required because (a) antigen-specific detection systems are important for establishing diagnostic tools and standardization of AECAs measurement, (b) identification will enable thorough analysis of the pathogenicity of AECAs, and (c) AECA-autoantigen interactions may be good targets for specific therapeutic approaches against highly pathogenic autoantibodies.
The prevalence of AECAs varies according to the type of ECs used for detection [
Target antigens of AECAs have been investigated intensively, but they are heterogeneous, and the following classification of target antigens was proposed: membrane component, ligand-receptor complex, and molecule adhering to the plasma membrane [
Reported target antigens of anti-endothelial cell antibodies.
Disease | Target antigen | Pathogenicity |
| ||
Systemic lupus erythematosus | DNA-DNA-histone | |
Ribosomal P protein PO | ||
Ribosomal protein L6 | ||
Elongation factor 1-alpha | ||
Adenylyl cyclase-associated protein | ||
Profilin 2 | ||
Plasminogen activator inhibitor | ||
Fibronectin | ||
Heparan sulfate | ||
|
||
Heat-shock protein 60 (Hsp 60) | Apoptosis | |
Heat-shock protein 70 (Hsp 70) | ||
Fibronectin leucine-rich transmembrane protein 2 (FLRT2) | Complement-dependent cytotoxicity | |
| ||
Mixed connective tissue disease | Voltage-dependent anion-selective channel 1 (VDAC-1) | |
| ||
Systemic sclerosis | Topoisomerase I | |
Centromere protein B (CENP-B) | ||
| ||
Vasculitis | Proteinase 3 | |
Myeloperoxidase | ||
Peroxiredoxin 2 | Cytokine secretion | |
Adenosine triphosphate (ATP) synthase | Intracellular acidification | |
| ||
Microscopic polyangiitis | Human lysosomal-associated membrane protein 2 | |
| ||
Behçet's disease | Alpha-enolase | |
C-terminus of Ral-binding protein 1 (RLIP76) | Apoptosis | |
| ||
Kawasaki disease | Tropomyosin | |
T-plastin | ||
| ||
Transplantation | Vimentin | |
Keratin-like protein | ||
| ||
Thrombotic thrombocytopenic purpura | Glycoprotein CD36 | |
| ||
Heparin-induced thrombocytopenia | Platelet factor 4 (PF4) | |
Heparin sulfate |
Several molecules can bind to ECs and are called “planted antigens” for AECA presumably via charge-mediated mechanisms, a DNA-histone bridge, or a specific receptor. Myeloperoxidase, DNA, and
As methods for identification of target antigens of AECAs, immunoprecipitation and WB of glycoproteins from the EC membrane with AECA-positive sera have been used [
Alternative methods have been developed, such as proteomics analysis using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time of flight mass spectrometry [
Proteomics analysis identified vimentin, Hsp60, voltage-dependent anion-selective channel 1 (VDAC-1), peroxiredoxin 2, and ATP synthase as targets for AECAs [
To overcome this problem, we constructed a novel expression cloning system for specific identification of cell-surface antigens [
Serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). (a) Generation of human umbilical vein endothelial cell (HUVEC) cDNA-expressing cells. (b) Sorting of cells expressing cell-surface autoantigens.
Our strategy to identify AECA target molecules involves use of a retroviral vector system and flow cytometry [
AECAs can bind only to cell-surface molecules in flow cytometry. Therefore, sorting of IgG-binding cells can concentrate and isolate cells expressing target molecules for AECAs on the cell surface. After staining of HUVEC cDNA-expressing YB2/0 cells with AECA IgG and secondary antibody, cells with strong fluorescent signals are sorted by flow cytometry. This step of sorting is repeated for several rounds to concentrate AECA IgG-binding cells. After concentration, several cell clones can be established from the AECA IgG-binding cell population by the limiting dilution method.
After polymerase chain reaction (PCR) amplification and cloning of HUVEC cDNA inserted into the genomic DNA of cloned cells, DNA sequencing can be performed followed by BLAST analysis, which enables the identification of the inserted cDNA. In this step, microarray analysis is an alternative method to identify the inserted cDNA. Next, an expression vector of the identified cDNA is generated and transfected into a cell line that does not express the identified protein. Finally, it is necessary to confirm that AECA IgG shows binding activity to 7-amino-actinomycin D-(7-AAD-) negative identified protein-expressing cells. If the binding activity is confirmed, it can be concluded that the identified protein is a novel autoantigen.
We reported the membrane protein FLRT2 as a novel autoantigen of AECAs in patients with SLE based on results obtained using SARF [
As AECAs can be detected in patients with collagen diseases, especially SLE, RA, and Takayasu arteritis [
Identification of intercellular adhesion molecule 1 (ICAM-1) as a target antigen of anti-endothelial cell antibodies (AECAs). (a) Nonpermeabilized HUVECs were stained with 0.5 mg/mL of IgG of control or X10-3 from a patient with rheumatoid arthritis followed by secondary antibody and analyzed by flow cytometry. (b) HUVEC cDNA-expressing cells were stained with 0.5 mg/mL of X10-3 IgG followed by secondary antibody, and cells in the positive fraction were sorted (black box). (c) Unsorted and 4th sorted cells (left) and unsorted and cloned cells from 4th sorted cells, C5 (right), were stained with 0.5 mg/mL of X10-3 IgG followed by secondary antibody and analyzed by flow cytometry. (d) ICAM-1 cDNA fragments inserted into the genomic DNA of C5 were amplified, and PCR products were electrophoresed on an 0.8% agarose gel. (e) Unsorted and C5 were stained with isotype control or anti-ICAM-1 antibody, followed by secondary antibody and analyzed by flow cytometry. (f) Expression vector, empty-IRES-GFP, or ICAM-1-IRES-GFP were transfected into YB 2/0 cells, and these cells were stained with 0.5 mg/mL of control IgG or X10-3 IgG, followed by secondary antibody and analyzed by flow cytometry.
ICAM-1 was also confirmed to transduce signals “outside in” [
Using serum from an SLE patient who showed hemolytic anemia, SARF revealed that cDNA inserted into the cloned cells that were sorted with this AECA-IgG was alpha 1,4-galactosyltransferase (A4GALT). This AECA showed significant binding activity to 7-AAD-negative A4GALT-overexpressing YB2/0 cells. The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal Gal
Gb3 is the Pk blood group antigen and has been designated CD77 [
Identification of A4GALT indicated the usefulness of SARF, which can be used to identify genes that encode not only the membrane protein itself, but also the transferase(s) responsible for modifying the membrane protein.
As described above, this system is very useful for identification of cell-surface autoantigens. Although this system seems to present difficulties in sorting cells at very low frequency, we could isolate and clone autoantigen-expressing cells by repeated sorting.
As AECAs are a heterogeneous group of autoantibodies that target ECs, it is predicted that there are different autoantigens. Thus, it is important to determine the clinical significance and potential pathogenicity of identified autoantibodies. If an autoantibody is specific for a disease or pathophysiology, it could be used as a marker for diagnosis or classification according to the underlying pathophysiology. At the same time, the pathogenic potential of the autoantibody should also be examined. Along with in vitro studies mentioned previously, experimental animal models of identified autoantibody should be constructed to determine the pathogenetic reactions in vivo.
AECAs are considered to be critical, especially for vascular lesions in collagen diseases, but most are directed against molecules localized within the cell and not expressed on the cell surface. In addition to conventional immunoprecipitation and WB, proteomics and expression library analyses have been performed to identify the targets for AECAs with some success. SARF was developed to identify autoantigens expressed on the EC surface with greater sensitivity. Using SARF, we successfully identified three different membrane proteins as targets for AECAs: FLRT2 from patients with SLE, ICAM-1 from a patient with RA, and Pk (Gb3/CD77) from an SLE patient with hemolytic anemia. Using this technology, it may be possible to determine cell-surface autoantigens of AECAs and achieve a comprehensive understanding of AECA-mediated vascular injury. Furthermore, SARF can be used when autoantibodies against cell-surface molecules are considered to take part in autoimmune diseases. The identification of such pathogenic autoantibodies may enable the development of more specific intervention strategies in autoimmune diseases.
The authors declare that they have no conflict of interests.
The authors thank the staff of the Department of Hematology and Rheumatology, Tohoku University, for help and discussion. This work was supported in part by Network Medicine Global-COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and Biomedical Research Core of Tohoku University Graduate School of Medicine.