Amelioration of Lupus Serum-Induced Skin Inflammation in CD64-Deficient Mice

Abstract

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by high autoantibodies levels and multiorgan tissue damage. The current study investigated the role of CD64 in SLE patients and animal models. According to a flow cytometry study, SLE patients showed an increase in CD64 expression in circulating monocytes. There was a correlation between CD64 and SLEDAI, blood urea nitrogen levels, and anti-Sm antibodies. In skin lesions of lupus MRL/lpr mice, there was high IgG deposition and CD64 expression. In vitro, cytokines IL-10 and IFN-γ upregulated CD64 expression in monocytes/macrophages that was inhibited by glucocorticoids. In CD64-deficient mice, skin inflammation induced by lupus serum was reduced. Furthermore, activation of spleen tyrosine kinase (Syk), Akt, and extracellular signal-regulated kinase (Erk) was inhibited in CD64-deficient monocytes. The results suggest that CD64 could be a biomarker for observing SLE progression, as well as a mechanistic checkpoint in lupus pathogenesis.

Keywords: CD64; flow cytometry; inflammation; monocytes/macrophages; systemic lupus erythematosus.

Figure 1

CD64 expression in monocytes increased in SLE. (A) Representative flow cytometry of CD64, CD32 and CD16 expression on CD14⁺ monocytes of peripheral blood mononuclear cells from SLE patients and healthy controls. (B) Flow cytometry analysis of surface expression of CD64, CD32 and CD16 on circulating monocytes in healthy controls and SLE patients. Detection of CD64 expression comprises 18 healthy controls (male:female=2:16, age 40.33±3.03) and 45 SLE patients (male:female=5:40, age 40.78±2.21). CD32 data comprises 12 healthy controls (male:female=2:10, age 43.25±4.12) and 39 SLE patients (male:female=3:36 , age 41.31±2.35). CD16 data comprises 14 healthy controls (male:female=2:12, age 42.79±3.61) and 44 SLE patients (male:female=3:41, age 40.31±2.14). Bars represent the average mean fluorescent intensity (MFI) of CD64, CD32 or CD16 on monocytes. Error bars represent standard deviation. *p < 0.05; ns, not significant.

Figure 2

CD64 expression in monocytes correlated with SLEDAI, anti-Sm antibodies and blood urea nitrogen levels in SLE. SLE patients were classified into low and high CD64 groups, and the analysis of their SLEDAI (A), blood urea nitrogen (B), anti-Sm antibodies (C) and anti-dsDNA antibodies (D) were presented. 21 of 22 low CD64 patients were tested anti-dsDNA and anti-Sm; 21 of 23 high CD64 patients were tested anti-Sm; 22 of 23 high CD64 patients were tested anti-dsDNA. *p < 0.05; n.s., not significant.

Figure 3

CD64 markedly increased in skin damages in MRL/lpr mice. (A) Representative photograph of H&E staining skin inflammation of female 30-week-old MRL/lpr mice and normal C57BL/6 mice. (B) Representative photograph of IgG and CD64 deposition stained by immunohistochemistry in skins of female 30-week-old MRL/lpr mice and normal C57BL/6 mice.

Figure 4

IL-10 and IFN-γ upregulated CD64 expression. (A) Flow cytometry detected CD64 expression in monocytes stimulated with various doses of IL-10 or 10 μM dexamethasone (DXM) for 20 h. Relative expression of CD64, showed as the fold group 0 ng/mL in MFI of monocytes. (B) Flow cytometry detected CD64 expression in monocytes stimulated with various doses of IFN-γ or 10 μM dexamethasone (DXM) for 20 h. Relative expression of CD64, displayed as the fold group 0 ng/mL in MFI of monocytes. *p < 0.05, **p < 0.01.

Figure 5

Deficiency of CD64 in mice alleviated skin inflammation induced by SLE serum. (A) Flow cytometry analysis of CD64 and CD11b in splenic cells. n=5 per group. **p < 0.01. (B) Representative histopathology of skin inflammation from CD64 deficient (^-/-) mice and wild (^+/+) mice with intradermal injection of 100 μL lupus serum. Black arrows refer to inflammatory cells. (C) Immunohistochemistry of phosphorylated Syk (p-Syk) in the skins from CD64-deficient and wild mice with intradermal injection of lupus serum. Black arrows refer to deposited p-Syk.

Figure 6

SLE serum promoted inflammation through CD64/Syk/Akt/Erk signaling pathway in macrophages. Bone marrow-derived macrophages (BMMs) were isolated from CD64 wild (^+/+) and CD64 deficient (^-/-) mice. Western blot identified protein levels in BMMs stimulated with 20μL SLE serum or normal saline for 2 h. (A) Representative picture of phosphorylated Syk (p-Syk) and total Syk, phosphorylated Akt (p-Akt) and total Akt, phosphorylated Erk (p-Erk) and total Erk, and mTOR protein levels measured by Western blot. (B) Western blot identified phosphorylated IκBα (p- IκBα) and total IκBα, phosphorylated p65 (p-p65) and total p65, phosphorylated JNK (p-JNK) and total JNK in BMMs. (C–F) Bar graphs depicting the changes in the relative expression of p-Syk (C), p-Akt (D), p-Erk (E) and mTOR (F). **p < 0.01, ****p < 0.0001.