Autoantibodies Recognizing Secondary NEcrotic Cells Promote Neutrophilic Phagocytosis and Identify Patients With Systemic Lupus Erythematosus

Abstract

Deficient clearance of apoptotic cells reportedly contributes to the etiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Based on this knowledge, we developed a highly specific and sensitive test for the detection of SLE autoantibodies (AAb) utilizing secondary NEcrotic cell (SNEC)-derived material as a substrate. The goal of the present study was to validate the use of SNEC as an appropriate antigen for the diagnosis of SLE in large cohort of patients. We confirmed the presence of apoptotically modified autoantigens on SNEC (dsDNA, high mobility group box 1 protein, apoptosis-associated chromatin modifications, e.g., histones H3-K27-me3; H2A/H4 AcK8,12,16; and H2B-AcK12). Anti-SNEC AAb were measured in the serum of 155 patients with SLE, 89 normal healthy donors (NHD), and 169 patients with other autoimmune connective tissue diseases employing SNEC-based indirect enzyme-linked immunosorbent assay (SNEC ELISA). We compared the test performance of SNEC ELISA with the routine diagnostic tests dsDNA Farr radioimmunoassay (RIA) and nucleosome-based ELISA (anti-dsDNA-NcX-ELISA). SNEC ELISA distinguished patients with SLE with a specificity of 98.9% and a sensitivity of 70.6% from NHD clearly surpassing RIA and anti-dsDNA-NcX-ELISA. In contrast to the other tests, SNEC ELISA significantly discriminated patients with SLE from patients with rheumatoid arthritis, primary anti-phospholipid syndrome, spondyloarthropathy, psoriatic arthritis, and systemic sclerosis. A positive test result in SNEC ELISA significantly correlated with serological variables and reflected the uptake of opsonized SNEC by neutrophils. This stresses the relevance of SNECs in the pathogenesis of SLE. We conclude that SNEC ELISA allows for the sensitive detection of pathologically relevant AAb, enabling its diagnostic usage. A positive SNEC test reflects the opsonization of cell remnants by AAb, the neutrophil recruitment to tissues, and the enhancement of local and systemic inflammatory responses.

Keywords: autoantibodies; autoimmunity; connective tissue diseases; inflammation; secondary necrosis; systemic lupus erythematosus.

Figure 1

Immobilized secondary NEcrotic cells (SNECs) expose naïve and modified nuclear autoantigens. (A) Recognition of immobilized autoantigens on SNEC by specific antibodies. Coated material contained histone H3 (#34), DNA (#42), nucleosomes (BT131), and the apoptotically modified histones H3 K27-me3 (BT164), H2A/H4 AcK8,12,16, (KM-2), and H2B-AcK15 (LG11-2). Respective isotype control antibodies (iso) displayed background signals. (B) Immunofluorescence of SLE-borne IgG autoantibodies (AAb) binding immobilized SNEC counter-stained for DNA by propidium iodide (PI). NHD, normal healthy donor; SLE, systemic lupus erythematosus; magnification 20×. (C) Immunofluorescence of histone H3 and modified histone H3 K27-me3 on immobilized SNEC counter-stained for DNA by PI; magnification 40×. (D) Immunofluorescence staining of histone H3 and modified histone H3 K27-me3 on HEp-2 cells. (E) Immunofluorescence of binding pattern of IgG autoantibodies present in the serum of patients with SLE to immobilized SNEC stained for DNA by PI; FOV, field of view. (F) Morphometric evaluation of (E), intensity of DNA staining versus the intensity of IgG staining.

Figure 2

SLE-derived AAb are specifically and sensitively detected by secondary NEcrotic cell (SNEC) ELISA. (A) Reactivity of sera from normal healthy donors (NHD) and patients with SLE, secondary anti-phospholipid syndrome (sAPS, gray dots in the SLE cohort), primary anti-phospholipid syndrome (PAPS), spondyloarthropathy (SpA), psoriatic arthritis (PsA), and systemic sclerosis (SSc) against SNEC were assessed by ELISA. Using receiver operating characteristic (ROC) analysis, the cut-off for positive values was calculated at 246 mean optical density (dashed red line). Frequencies of SNEC positivity are displayed above the respective cohort in percent (%). (B) The overall test performance of the SNEC ELISA (left), radioimmunoassay (RIA) dsDNA Farr assay (center), and anti-dsDNA-NcX-ELISA (right) was evaluated employing ROC analysis. The respective diagnostic parameters (specificity, sensitivity, negative predictive value, positive predictive value, and diagnostic odds ratio) were calculated and are displayed below. (C) Organ involvement was analyzed by comparison of odds ratios for SNEC ELISA, anti-dsDNA RIA, anti-dsDNA-NcX ELISA, and anti-nuclear antigen on HEp-2 titers. The depicted odd ratios are within the 95% confidence interval.

Figure 3

Anti-secondary NEcrotic cell (SNEC) IgG correlates with serological disease variables but not with the disease activity. The correlation of SNEC ELISA levels with serological and clinical variables was evaluated by Bravais-Pearson correlation coefficients and corrected after Bonferroni. SNEC positivity was compared to (A) anti-nuclear antigen on HEp-2 cell titers, (B) extractable nuclear antigen positivity (Nucleosomes, Histones, Ro52), (C) Complement C3 levels (cut off C3 levels at 90 mg/ml indicated as blue dotted line), (D) European Consensus Lupus Activity Measurement Manifestation Score and others (see also Tables 1, 2 and S1, S2 in Supplementary Material). The SNEC ELISA cut-off at 246 mean optical density is depicted as a red dotted line.

Figure 4

Secondary NEcrotic cell (SNEC) positivity correlates with uptake of SNEC into blood-borne neutrophils. (A) Correlation of SNEC uptake by neutrophils assessed by flow cytometry with anti-SNEC IgG. Values are displayed as ranks. Spearman’s rank correlation coefficient was calculated using the values of patients with SLE. (B) Representative microphotographs of z-stack series showing that PI-stained SNEC (red) was taken up by neutrophils with a segmented nucleus (CD15POS; displayed in green; Hoechst 33342POS; displayed in blue). White arrows indicate SNEC taken up by neutrophils. SNEC phagocytosed by a CD15NEG mononuclear phagocyte is indicated by a red arrow. (C) Uptake of PI-stained SNEC by CD16POS HLA-DRNEG granulocytes in whole blood assessed by flow cytometry in the presence (+SNEC, non-adsorbed) or absence (+SNEC, adsorbed) of anti-SNEC autoantibodies. CD16POS HLA-DRNEG granulocytes in whole blood without addition of SNEC served as negative control (−SNEC). Shown is the uptake of SNEC in the presence of a representative adsorbed and non-adsorbed SLE serum. (D) Percentage of SNEC uptake into CD16POSHLA-DRNEG granulocytes in the presence of 10 anti-SNEC antibody adsorbed SLE sera relative to the corresponding non-adsorbed sera. (E) Percentage of residual anti-SNEC IgG in adsorbed relative to the corresponding non-adsorbed sera assessed by SNEC ELISA.

Figure 5

Inflammation in SLE is driven by FcγR-mediated uptake into phagocytes of secondary NEcrotic cell (SNEC) opsonized by autoantibodies (AAb). In healthy individuals (NHD), apoptotic cells are cleared rapidly and silently by professional phagocytes involving a plethora receptors and bridging molecules such as α_Vβ₃ integrin, Tryo3–Axl–Mer (TAM), milk fat globule-EGF factor 8 protein (MFG-E8), growth arrest-specific protein 6 (Gas6), and Proteins S. This marked redundancy avoids disintegration of apoptotic cells and the generation of SNEC. In patients with systemic lupus erythematosus (SLE), the concurrence of AAb and SNEC results in the opsonization and Fcγ Receptor (FcγR)-mediated uptake of SNEC into phagocytes, especially neutrophils. This perpetuates inflammatory responses and causes tissue damage. Lupus erythematosus (LE) cell.