Summary
A genetic variant of the killer immunoglobulin‐like receptor 3DL1 (KIR3DL1) has been found in patients with systemic lupus erythematosus (SLE). Herein, we investigated the presence of autoantibodies to KIR3DL1 in a cohort of patients with SLE. We tested sera from 28 patients with SLE, 11 patients with rheumatoid arthritis (RA) and 17 healthy control subjects for anti‐KIR3DL1 activity by an enzyme‐linked immunosorbent assay (ELISA) using recombinant KIR3DL1‐enhanced green fluorescent protein (EGFP) and EGFP proteins. Anti‐KIR3DL1 antibodies were detected in 22 (79%) of the 28 patients with SLE, whereas they were present in only three (27%) of the 11 patients with RA examined. Notably, 10 (91%) of the 11 samples from patients with SLE prior to therapy had anti‐KIR3DL1 antibodies. None of the samples from healthy donors were positive for the antibodies. Here, we report the presence of anti‐KIR3DL1 antibodies in the sera of patients with SLE for the first time. Anti‐KIR3DL1 autoantibodies may be involved in the pathogenesis of autoimmune diseases.
Keywords: autoantibody, killer cell immunogloblin‐like receptor (KIR)‐3DL1, systemic lupus erythematosus
Introduction
Systemic lupus erythematosus (SLE) is characterized by immunological abnormalities of immune cell subsets and by the production of autoantibodies. More than 100 autoantibodies are found in patients with SLE, including anti‐nuclear, anti‐phospholipid and anti‐red blood cell antibodies 1. Many different symptoms in patients with SLE alternate between sporadic inflammatory and dormant phases, making diagnosis difficult. SLE symptoms include cutaneous rashes, arthritis, muscle pain, serositis and nephritis. The remission rate is quite low, and most patients suffer from persistent inflammation 2. Diagnosis of SLE has been based on the American College of Rheumatology (ACR) classification criteria 3. However, these are based mainly on cutaneous lupus, and overlook other organ system symptoms 4. In addition, the serological diagnostic criteria combine a limited number of antibodies [anti‐double‐stranded DNA (anti‐dsDNA), anti‐phospholipid, anti‐Sm, anti‐nucleosome, anti‐histone and anti‐SSA antibodies] that do not provide enough specificity for rapid and accurate diagnosis 4, 5.
Autoimmune diseases develop through the release of autoantigens from target organs, homing of immune cells to the target organ and destruction of tissues 6, 7. Decreased numbers of natural killer (NK) cells and the decrease of NK cell activity are observed in many autoimmune diseases 8, 9, 10. In patients with SLE, NK cells are associated with the pathological state through the increase of interferon (IFN)‐γ, accompanied by decreases in their cell number and function 11, 12. The disruption of the immunological mechanisms in NK cells is induced by a blockade of inhibitory signals and the resulting overproduction of cytokines by NK cells. Two factors help to block the inhibitory signal: (i) the activating signal is increased to a greater extent than the inhibitory signal (ii) and the autoantibodies bind to NK cells. NK cells produce various cytokines, including IFN‐γ, tumor necrosis factor (TNF)‐α and interleukin (IL)‐10. These cytokines activate other immune cells, inducing cellular injury or inflammation 13. Clinical studies have shown correlations between frequencies or activities of NK cells and disease onset. However, the precise mechanism, including how NK cells trigger autoimmune diseases, remains to be clarified.
Autoantibodies to NK cells have been found in the sera of patients with SLE 14, and inhibit the cells’ regular function 15. Anti‐killer immunoglobulin‐like receptor 2DL1 (KIR2DL1) and KIR2DL3 autoantibodies have been found in patients with SLE, rheumatoid arthritis (RA) and Behçet’s disease 16, and the receptor expression level of KIR2DL1, determined by flow cytometry analysis in patients with SLE, was found to be lower than that in healthy controls; that of KIR3DL1 was similar in both types of individuals 12. However, the carrier frequencies of KIR3DL1 have been associated with various autoimmune diseases, including SLE 12, 17, 18, 19, 20, 21, motivating us to study the prevalence of anti‐KIR3DL1 autoantibodies in patients with SLE. Here, we provide the first report, to our knowledge, that anti‐KIR3DL1 autoantibodies are highly prevalent in the serum IgG fraction of patients with SLE.
Methods
Patients and controls
We obtained random serum samples from 28 patients with SLE and 11 patients with RA who, during or prior to therapy, are treated at the hospital of the National Center for Global Health and Medicine (NCGM). SLE was diagnosed according to the classification criteria of the ACR 3. We also obtained control serum samples from 17 healthy donors. The participants signed informed consent forms, allowing us to use the samples for research purposes. We aliquoted each serum sample to avoid repeated thawing and stocked them at ‒20°C until assayed. The Institutional Review Board of the NCGM, Tokyo, approved the study (NCGM‐G‐001292‐00).
Expression and purification of recombinant KIR3DL1‐enhanced green fluorescent protein (EGFP) and EGFP proteins
We subcloned cDNA fragments of EGFP and KIR3DL1‐EGFP into the pEU plasmid. We inserted the Strep‐tagged sequence that binds to the biotin‐binding site of streptavidin, consisting of eight amino acids (WSHPQFEK), just after the C‐terminus. We expressed the recombinant KIR3DL1‐enhanced green fluorescent protein (EGFP) (rKIR3DL1‐EGFP) and EGFP (rEGFP) proteins from the constructs in a wheatgerm expression system, as reported previously 22. The resulting lysates were applied to a column charged with Strep‐Tactin resin (Novagen, San Diego, CA, USA) in a binding buffer [150 mM NaCl, 100 mM Tris‐HCl and 1 mM ethylenediamine tetraacetic acids (EDTA), pH 8.0]. The Strep‐tagged recombinant proteins were eluted with a buffer (150 mM NaCl, 100 mM Tris‐HCl, 1 mM EDTA and 2·5 mM desthiobiotin, pH 8.0). We determined the protein concentrations from the intensity of Coomassie Brilliant Blue (CBB) band signals and confirmed the expression of the full‐length recombinant proteins by Western blotting using rabbit anti‐GFP (Medical and Biological Laboratories, Aichi, Japan) and rabbit anti‐KIR3DL1 (Proteintech, Rosemont, IL, USA) polyclonal antibodies (Supporting information, Fig. S1). We dialyzed rKIR3DL1‐EGFP and rEGFP before use.
Detection of anti‐KIR3DL1 autoantibodies in patients with SLE
We assessed the reactivity of sera to KIR3DL1 by a serial dilution test in an enzyme‐linked immunosorbent assay (ELISA). We coated each well of a 96‐well streptavidin‐coated plate (Thermo Scientific, Waltham, MA, USA) with 10 μg/ml rKIR3DL1‐EGFP or rEGFP. After reacting with the coated recombinant proteins for 12 h at 4°C, the wells were washed five times in Tris‐buffered saline with 0·1% Tween 20 (TBS‐T). We then used 5% skimmed milk in TBS‐T to block the wells. We diluted the serum samples in TBS‐T at 1 : 1000 and incubated them for 1 h at room temperature with the coated recombinant proteins. Next, the bound antibodies were incubated with horseradish peroxidase‐conjugated goat anti‐human immunoglobulin (Ig)G (Jackson ImmunoResearch, West Grove, PA, USA) at a 1 : 2000 dilution for 30 min at room temperature, and then we added the substrate 3,3′,5,5′‐tetramethylbenzidine (TMB) to obtain the reactions. The reaction was terminated by 1 N HCl and quantified using a microplate photometer at 450 nm. All samples were independently processed for three independent experiments. We normalized the optical density (OD) values for each sample using the OD values for the positive control. Finally, we determined an ELISA score for each sample as the OD values for KIR3DL1‐EGFP – OD values for EGFP. The cut‐off values for positive and negative assignments were determined as mean values + 3 × standard deviation (s.d.) compared with controls. Rabbit anti‐KIR3DL1 polyclonal antibody was used as positive control in all experiments. We defined serum samples as positive when OD values were higher than 0·084.
Statistical analysis
We determined statistical significance using the Mann–Whitney U‐test on the basis of three independent experiments.
Results
We developed an ELISA system using a Strep‐tagged KIR3DL1 protein eluted by a Strep‐tagged/Strep‐Tactin protein purification system 23. In order to resolve the problem of KIR3DL1 being an insoluble protein 24, we fused the EGFP tag to its C‐terminus (KIR3DL1‐EGFP). In addition, we synthesized the Strep‐tagged rKIR3DL1‐EGFP using a wheatgerm protein synthesis system, which should be suitable for the synthesis of insoluble proteins 25. In this study, we analyzed the KIR3DL1 reactivity of each serum sample. The rKIR3DL1‐EGFP obtained by the above system was used in an ELISA analysis to detect anti‐KIR3DL1 antibodies in sera of patients with SLE.
We analyzed the presence of anti‐KIR3DL1 antibodies in randomly selected patients, 29 with SLE (Table 1), 11 with RA and 17 healthy controls. Anti‐KIR3DL1 autoantibodies were positive in the sera of 22 (78·6%) of the 28 patients with SLE (Fig. 1a). We found that 10 of the 11 (90·9%) patients who had not initiated therapy and 12 of the 17 (70·6%) patients under therapy showed reactivity to KIR3DL1 (Fig. 1a,b). The mean ELISA scores of serum anti‐KIR3DL1 antibodies in patients with SLE and RA were significantly different at 0·30 and 0·068, respectively, whereas that of healthy subjects was 0·024 (Fig. 1a). The ELISA score of patients being treated was significantly higher than that of normal controls (Fig. 1b).
Table 1.
Condition of patients with systemic lupus erythematosus (SLE) analysed in this study
| ID | Gender | ELISA score | Prior to/during therapy |
|---|---|---|---|
| 1 | F | 0·09 | Prior to therapy |
| 2 | F | 0·42 | Prior to therapy |
| 3 | F | 0·52 | Prior to therapy |
| 4 | F | 1·83 | Prior to therapy |
| 5 | F | 0·41 | Prior to therapy |
| 6 | F | 0·18 | Prior to therapy |
| 7 | F | 0·07 | Prior to therapy |
| 8 | F | 0·36 | Prior to therapy |
| 9 | M | 0·33 | Prior to therapy |
| 10 | F | 1·36 | Prior to therapy |
| 11 | F | 0·10 | Prior to therapy |
| 12 | F | 0·03 | During therapy |
| 13 | F | 0·07 | During therapy |
| 14 | F | 0·05 | During therapy |
| 15 | F | 0·34 | During therapy |
| 16 | F | 0·09 | During therapy |
| 17 | F | 0·36 | During therapy |
| 18 | F | 0·14 | During therapy |
| 19 | F | 0·08 | During therapy |
| 20 | F | 0·27 | During therapy |
| 21 | F | 0·25 | During therapy |
| 22 | F | 0·25 | During therapy |
| 23 | F | 0·19 | During therapy |
| 24 | F | 0·19 | During therapy |
| 25 | F | 0·10 | During therapy |
| 26 | F | 0·16 | During therapy |
| 27 | F | 0·02 | During therapy |
| 28 | F | 0·19 | During therapy |
ELISA = enzyme‐linked immunosorbent assay.
Figure 1.
Enzyme‐linked immunosorbent assay (ELISA) detection of anti‐killer immunoglobulin‐like receptor 3DL1 (KIR3DL1) antibodies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) (a) and in patients with SLE prior to or during therapy (b). Values represent the mean of three independent experiments. Statistical analysis was performed using the Mann–Whitney U‐test. * P < 0·05; ** P < 0·005.
Discussion
In this study, we found autoantibodies against KIR3DL1 in the sera of 79% of patients with SLE. In particular, 91% of patients with SLE who had not initiated therapy were positive for anti‐KIR3DL1 autoantibodies. Conversely, 71% of patients with SLE being treated and 27% of patients with RA were autoantibody‐positive. All 17 healthy controls were negative for autoantibodies (Fig. 1). These results indicate that the anti‐KIR3DL1 antibodies are disease‐specific and the titers reflect the condition of patients with SLE (prior to or during therapy). The SLE Disease Activity Index (SLEDAI) is generally used to assess the disease activity of SLE 26. SLEDAI scores for patients with SLE prior to therapy were significantly higher than those for patients with SLE being treated (Table 2). However, the ELISA score for anti‐KIR3DL1 was not found to be associated with SLEDAI (Fig. 2), indicating that the anti‐KIR3DL1 autoantibody is not related to factors that affect the SLEDAI score, such as oxidative stress 27. Instead, the production of anti‐KIR3DL1 autoantibodies could be regulated by B cell activity, as in the case of dsDNA levels that are decreased by SLE therapy (Table 2) and reflect B cell numbers and phenotype 28.
Table 2.
Comparison of clinical characteristics between patients with SLE prior to and during therapy
| Variables | Patients with SLE | P‐value a | |
|---|---|---|---|
| Prior to therapy | During therapy | ||
| (n = 11) | (n = 17) | ||
| Age, mean (s.d.), years | 46 (16) | 43 (11) | 0.74 |
| Gender, n (%), female | 10 (91) | 17 (100) | 0.70 |
| ELISA score, median (IQR) | 0·52 (0·07–1·83) | 0·16 (0·02–0·36) | 0·027* |
| SLEDAI, median (IQR) | 15·9 (8–29) | 8·9 (0–19) | 0·0035* |
| Malar erythema, n (%) | 3 (27) | 3 (18) | 0·69 |
| Arthritis, n (%) | 1 (9) | 4 (24) | 0·54 |
| Serositis, n (%) | 3 (27) | 0 (0) | 0·24 |
| Nephritis, n (%) | 8 (73) | 4 (24) | 0·032* |
| Headache, n (%) | 2 (18) | 2 (12) | 0·79 |
| Peripheral Neuropathy, n (%) | 0 (0) | 0 (0) | – |
| APS, n (%) | 2 (18) | 3 (18) | 1·00 |
| Prednisolone, median (IQR), mg/day | – | 15·3 (5–40) | – |
| Concurrent immunosuppressant, n (%) | – | 5 (29) | – |
| Leucocytes, median (IQR) ×103/μl | 5·8 (3·0–11·8) | 7·4 (4·2–11·8) | 0·11 |
| Haemoglobin, median (IQR), g/dl | 10·7 (5·5–13·0) | 11·3 (5·5–13·9) | 0·65 |
| Platelets, median (IQR) ×104/μl | 19·2 (4·2–47·2) | 22·3 (7·6–53·4) | 0·42 |
| C3, median (IQR), mg/dl | 66·6 (35·6–109·5) | 71·9 (35·3–124) | 0·62 |
| Anti‐dsDNA antibody, median (IQR), IU/ml | 124·2 (9·8–400·0) | 31·7 (2·2–308·3) | 0·0060* |
| Antibody positivity, n (%) | |||
| Anti‐SSA | 5 (56) | 4 (57) | 1·00 |
| Anti‐Sm | 4 (44) | 6 (67) | 0·45 |
| Anti‐U1 RNP | 3 (43) | 3 (38) | 0·90 |
Mann–Whitney U‐test P‐value.
P < 0·05 indicates significant differences between patients with systemic lupus erythematosus (SLE) are prior to and during therapy. RNP = ribonucleoprotein; IQR = interquartile range; APS = anti‐phospholipid syndrome; SLEDAI = systemic lupus erythematosus Disease Activity Index; ELISA = enzyme‐linked immunosorbent assay; s.d. = standard deviation.
Figure 2.
Lack of correlation between systemic lupus erythematosus (SLE) Disease Activity Index (SLEDAI) and enzyme‐linked immunosorbent assay (ELISA) scores in patients with SLE.
The KIR3DL1 expressed on the surface of NK cells is an inhibitory receptor, and KIR3DL1 recognizes HLA‐B and HLA‐A alleles of a Bw4 epitope on target cells 29. When KIR3DL1 antigens bind Bw4, inhibitory signals including Src homology region 2 domain‐containing phosphatase 1 (SHP‐1) inhibit any activating signals, and NK cells do not lyse target cells (even if the ligand for activating receptor is expressed on the target cells) 24, 30, 31. Although the possibility that the KIR genotype, in combination with the HLA ligand, contribute to the pathogenesis have been proposed previously 12, 17, 18, 19, 20, 21, in this study we revealed the existence of autoantibody to KIR3DL1 for the first time. Our results suggest that anti‐KIR3DL1 autoantibodies in the sera of patients with SLE binding KIR3DL1 may impair the inhibitory function of KIR3DL1+ NK cells and result in the lysis of the Bw4 expressing target cells, altering the normal immune system function. Future studies should address this possibility to explain how anti‐KIR3DL1 autoantibodies in the sera of patients with SLE affect the normal KIR3DL1+ NK function.
Ninety‐five per cent of Japanese are genetically KIR3DL1‐positive 20. Although the protein expression level of KIR3DL1 was reported to be similar between patients with SLE and healthy controls 12, carrier frequencies of KIR3DL1 have been associated with SLE 12, 17, 18, 19, 20, 21. Thus, further analysis should be conducted to determine the correlation between anti‐KIR3DL1 autoantibody levels and KIR3DL1 genotype. In addition, it is possible that anti‐KIR3DL1 autoantibodies could cross‐react to the other KIR haplotype products, such as KIR3DL2, KIR3DL3 and KIR3DS1. Future studies should reveal the cross‐reactivity of anti‐KIR3DL1 autoantibodies, because the detection of anti‐KIR3DL1 autoantibodies could be applied to the diagnosis of SLE without the consideration of KIR haplotypes.
It has been reported that anti‐KIR2DL1 and anti‐KIR2DL3 autoantibodies are prevalent in 20–30% of patients with SLE 16. We found a much higher prevalence in our cohort of patients (approximately 90% in patients with SLE during the initial stages) than for anti‐KIR2DL1 and anti‐KIR2DL3 autoantibodies in previous reports. Among the autoantibodies used in clinical practice, the prevalence of autoantibodies in patients with SLE is 70–98% for anti‐dsDNA, 20–40% for anti‐Sm, 61–85% for anti‐nucleosome, 30–40% for anti‐phospholipid, 70% for anti‐histone and 30% for anti‐SSA antibodies 5 (Table 3). Thus, detection of the anti‐KIR3DL1 autoantibody, in combination with other markers, could be applied as a diagnostic marker of SLE, especially during the onset.
Table 3.
Prevalence of autoantibody in patients with SLE
| Autoantibody | Prevalence (%) | Comments | References |
|---|---|---|---|
| Anti‐dsdna | 70–98 | 5 | |
| Anti‐Sm | 20–40 | 5 | |
| Anti‐nucleosome | 61–85 | 5 | |
| Anti‐phospholipid | 30–40 | 5 | |
| Anti‐histone | 70 | 96–100% in SLE patients induced by drugs | 5 |
| Anti‐SSA | 30 | 90% in neonatal lupus erythematosus | 5 |
| Anti‐KIR2DL1, 3 | 20–30 | 16 | |
| Anti‐KIR3DL1 | 80 | 90% in patients with SLE prior to therapy | This study |
KIR3DL1 = killer immunoglobulin‐like receptor 3DL1; SLE = systemic lupus erythematosus.
Disclosure
All authors declare having no conflicts of interest for the current work.
Supporting information
Fig. S1. Purification of rKIR3DL1‐EGFP (a) and rEGFP (b). Left panel shows CBB stained SDS‐PAGE gel for elution fractions of recombinant proteins. Right panel shows Western blotting detection of anti‐KIR3DL1 antibody (a) or anti‐GFP antibody (b). Arrows indicate the position of rKIR3DL1‐EGFP (a; 67kDa) and rEGFP (b; 30kDa), respectively. M, Protein molecular weight (kDa) marker.
Acknowledgement
This work was supported by grants‐in‐aid for research from the National Center for Global Health and Medicine (22A‐104) and the Department of Intractable Diseases from the Ministry of Health, Labor, and Welfare of Japan.
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Associated Data
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Supplementary Materials
Fig. S1. Purification of rKIR3DL1‐EGFP (a) and rEGFP (b). Left panel shows CBB stained SDS‐PAGE gel for elution fractions of recombinant proteins. Right panel shows Western blotting detection of anti‐KIR3DL1 antibody (a) or anti‐GFP antibody (b). Arrows indicate the position of rKIR3DL1‐EGFP (a; 67kDa) and rEGFP (b; 30kDa), respectively. M, Protein molecular weight (kDa) marker.