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. 2011 Jan 31;13(1):R9.
doi: 10.1186/ar3230.

Anti-class a scavenger receptor autoantibodies from systemic lupus erythematosus patients impair phagocytic clearance of apoptotic cells by macrophages in vitro

Xiao-wei Chen  1 , Yan Shen, Chuan-yin Sun, Feng-xia Wu, Yi Chen, Cheng-de Yang
Affiliations

Anti-class a scavenger receptor autoantibodies from systemic lupus erythematosus patients impair phagocytic clearance of apoptotic cells by macrophages in vitro

Xiao-wei Chen et al. Arthritis Res Ther. .

Abstract

Introduction: Inadequate clearance of apoptotic cells by macrophages is one of the reasons for the breakdown of self-tolerance. Class A scavenger receptors, macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A), which are expressed on macrophages, play important roles in the uptake of apoptotic cells. A previous study reported the presence of the anti-MARCO antibody in lupus-prone mice and systemic lupus erythematosus (SLE) patients. The purpose of this study was to investigate the prevalence of anti-class A scavenger receptor antibodies in patients with various autoimmune diseases, in particular SLE, and the functional implication of those autoantibodies in the phagocytic clearance of apoptotic cells by macrophages.

Methods: Purified recombinant scavenger receptor cysteine-rich (SRCR) polypeptide (ligand-binding domain of MARCO) and recombinant SR-A were used as antigens. By using enzyme-linked immunosorbent assay, the anti-SRCR and anti-SR-A antibodies were detected in the sera of untreated patients with SLE (n = 65), rheumatoid arthritis (n = 65), primary Sjögren syndrome (n = 25), and healthy blood donors (n = 85). The effect of IgG purified from SLE patients or healthy controls on the phagocytosis of apoptotic cells by macrophages was measured by the flow cytometry assay.

Results: Anti-SRCR antibodies were present in patients with SLE (18.5%) and rheumatoid arthritis (3.1%), but not in those with primary Sjögren syndrome. Anti-SR-A antibodies were present in patients with SLE (33.8%), rheumatoid arthritis (13.8%), and primary Sjögren syndrome (12.0%). IgG from SLE patients positive for anti-SRCR or anti-SR-A antibodies showed a higher inhibition rate on binding of apoptotic cells to macrophages than IgG from healthy controls (both P < 0.05). IgG from SLE patients positive for both anti-SRCR and anti-SR-A antibodies showed a significantly higher inhibition rate on ingestion of apoptotic by macrophages than IgG from healthy controls (P < 0.05).

Conclusions: Our results indicated that autoantibodies to class A scavenger receptors might contribute to the breakdown of self-tolerance by impairing the clearance of apoptotic debris and play a role in the pathogenesis of autoimmune disease, especially in SLE.

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Figures

Figure 1
Figure 1
Levels of anti-class A scavenger receptors IgG in patients and healthy individuals. The optical density (OD) value for each individual is represented as a single point. Dashed lines indicate the OD values that exceed the mean control value by more than three standard deviations (SDs). Horizontal bars represent the mean values for each group. CONTROL, healthy donors; pSS, primary Sjögren syndrome; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus. A. Levels of anti-SRCR IgG for patients. B. Levels of anti-SR-A IgG for patients.
Figure 2
Figure 2
THP-1 derived macrophage phagocytosis of apoptotic Jurkat cells. A. Apoptosis induction was confirmed by the detection of percentage of Annexin V and 7-AAD staining by flow cytometric assessment. More than 50% cells were Annexin V +7-AAD-(early apoptotic cells) and less than 5% cells were Annexin V +7-AAD+ (late apoptotic and necrotic cells) after ultraviolet B irradiation. This analysis was repeated prior to each phagocytosis assay. One representative image is shown. B. Flow cytometry based phagocytosis assay. To discriminate between binding and internalization of apoptotic Jurkat cells, CD3 staining of CFSE+ macrophages was performed. Macrophages that had ingested apoptotic Jurkat cells were CD3- (upper left quadrant), while macrophages with Jurkat cells bound to their surface were CD3+ (upper right quadrant). Macrophages were pretreated with IgG from SLE patients or healthy controls before being incubated with apoptotic cells, as described in Materials and Methods. Representative flow cytometry images for phagocytosis by macrophages pretreated with IgG from control (left panel) and SLE patients with anti-class A scavenger receptor antibodies (right panel) are shown.
Figure 3
Figure 3
Phagocytosis of apoptotic cells by macrophages that were pretreated with purified IgG. A, B. Macrophages were pre-treated with IgG from SLE patients who were anti-SR-A positive (SP), anti-SRCR (anti-MARCO) positive (MP), anti-SR-A and anti-SRCR double-positive (DP), anti-SRCR and anti-SR-A double negative (DN), or IgG from healthy controls who are anti-SR-A and anti-SRCR double negative (CONTROL) before being cultured with apoptotic cells. Purified IgG were incubated with SRCR and SRA (antigen adsorbed) or not (non-antigen adsorbed) before added to interact with macrophage as described in Materials and Methods. Pre-incubation with DP reduced the percentage of macrophages ingesting apoptotic cells, resulted in a significantly higher inhibition rate of ingestion. Pre-incubation with SP, MP, and DP reduced the percentage of macrophages with apoptotic cells binding to their surface, as inhibition rates significantly increased. The inhibition rates significantly decreased when anti-SRCR and anti-SRA antibodies had been adsorbed off (all P < 0.05). An * indicates a statistically significant difference P < 0.05 by Mann-Whitney U test or paired t-test. Each bar represents the mean + SEM (n = 4). Results are representative of three experiments. C. Dose response of the IgG mediated inhibition of phagocytosis. Decreased percentage of macrophages ingesting apoptotic cells correlated with increased concentration of incubating IgG from one SLE patient positive for both anti-SRCR and anti-SR-A (P) (r = -0.943, P = 0.005). Concentration of IgG from one healthy control (control) did not correlate with the percentage of macrophages ingesting apoptotic cells (P = 0.208). Bar represents the mean ± SEM (n = 2). Results are representative of three experiments.
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