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.2016 May 15;196(10):4030-9.
doi: 10.4049/jimmunol.1500418. Epub 2016 Apr 8.

Apoptotic Debris Accumulates on Hematopoietic Cells and Promotes Disease in Murine and Human Systemic Lupus Erythematosus

Affiliations

Apoptotic Debris Accumulates on Hematopoietic Cells and Promotes Disease in Murine and Human Systemic Lupus Erythematosus

SunAh Kang et al. J Immunol..

Abstract

Apoptotic debris, autoantibody, and IgG-immune complexes (ICs) have long been implicated in the inflammation associated with systemic lupus erythematosus (SLE); however, it remains unclear whether they initiate immune-mediated events that promote disease. In this study, we show that PBMCs from SLE patients experiencing active disease, and hematopoietic cells from lupus-prone MRL/lpr and NZM2410 mice accumulate markedly elevated levels of surface-bound nuclear self-antigens. On dendritic cells (DCs) and macrophages (MFs), the self-antigens are part of IgG-ICs that promote FcγRI-mediated signal transduction. Accumulation of IgG-ICs is evident on ex vivo myeloid cells from MRL/lpr mice by 10 wk of age and steadily increases prior to lupus nephritis. IgG and FcγRI play a critical role in disease pathology. Passive transfer of pathogenic IgG into IgG-deficient MRL/lpr mice promotes the accumulation of IgG-ICs prior to significant B cell expansion, BAFF secretion, and lupus nephritis. In contrast, diminishing the burden IgG-ICs in MRL/lpr mice through deficiency in FcγRI markedly improves these lupus pathologies. Taken together, our findings reveal a previously unappreciated role for the cell surface accumulation of IgG-ICs in human and murine lupus.

Copyright © 2016 by The American Association of Immunologists, Inc.

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Figures

Figure 1
Figure 1. Nuclear self-antigens are displayed on the surface of hematopoietic cells
(A) Splenic DCs, MFs, B cells, T cells, and CD45neg cells from B6 mice were stained with anti-Sm (2.12.3, black line) or isotype control antibody (gray line) and analyzed by flow cytometry. Representative histograms from >5 experiments (n = >20 mice). (B) Splenic DCs or MFs untreated or treated with trypsin and stained for Sm (2.12.3, red). (C) Splenocytes untreated or treated with DNase (100 μg/ml) were stained for surface DNA (33H11, red) or nucleosome (PL2-3, red). Representative images from 6 experiments (n = 7 mice, 10–15 cells per mouse). Scale bar = 3.5μm. (D) Splenic DCs, MFs, B cells, T cells, and CD45neg cells from MRL/lpr mice (16–28 weeks old) were stained for Sm and analyzed by flow cytometry. Representative data from >5 experiments (n = >20 mice). (E) Splenocytes (circle) or blood cells (triangle) from B6 (black) or MRL/lpr (white) at different ages were stained for Sm and analyzed by flow cytometry. (n = 4–5 mice per age group, 2 experiments). (F) Surface Sm levels were quantitated on splenocytes from different mouse models. (n = 3–14 mice). In (E) results are mean ± SEM. In (F), bars represent median. *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant by Mann-Whitney test. MFI = Median fluorescence intensity.
Figure 2
Figure 2. Peripheral blood mononuclear cells from SLE patients accumulate surface nuclear self-antigens
(A) Whole blood cells from healthy controls (HC) or SLE patients (SLE) with SLEDAI score > 6 were analyzed for surface DNA (33H11) or Sm (2.12.3) by flow cytometry. (B) Representative histograms are shown for each cell type (isotype antibody: gray, anti-DNA: black). n = 8–9 from >3 separate experiments. (C) Peripheral blood T cells from HC (upper panels) or SLE patients (lower panels) were stained for CD3 (blue) and DNA (red). Scale bar = 3 μm. (n = 3, 10 cells per sample). In (A), bars represent median. *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant by Mann-Whitney test. MFI = median fluorescence intensity.
Figure 3
Figure 3. Nuclear self-antigens bind FcγRs as IgG-ICs
(A) Surface Sm was stained on splenic DCs and MFs from B6 mice deficient of individual FcγR (FcγRI, IIB, III, or IV) or Fc-common-gamma-chain (γ). (n = 4–14 mice, 5 experiments). (B) Surface IgG levels on splenic DCs and MFs from B6 and MRL/lpr mice at different ages were analyzed by flow cytometry. (n = 2–6 mice per age group, 2 experiments). (C) Purified splenic DCs were stained for surface Sm (magenta) and IgG (green). Representative images from >3 experiments. Scale bar = 2.5μm. (n = 5–7 mice, 5–15 cells per mouse). (D) Colocalization of Sm with IgG on DCs and MFs was analyzed using Mander’s Coefficient and ImageJ. Each circle represents a cell (n = 7–15 cells from 2–3 mice, 4 experiments). Expression levels of phosphorylated (E) Syk, (F) Akt-Threonine308 (Akt-T), and (G) S6 in splenic DCs and MFs from B6 and MRL/lpr mice were analyzed by flow cytometry. (n = 5–15 mice, 2–3 experiments). Levels of FcγRI(H) surface, or(I) gene expression, on splenic MFs and DCs from B6, MRL/lpr, and FcγRI−/−MRL/lpr mice were analyzed by flow cytometry or qPCR (relative expression over FcγRI−/−MRL/lpr mice,I). (n = 3–7 mice, 2 experiments). In (A,B, andD–I) bars represent median. *p<0.05, **p<0.01, ***p<0.001 by Kruskal-Wallis test (A) or Mann-Whitney test (B andD–I).
Figure 4
Figure 4. Lack of FcγRI in MRL/lpr mice reduces the levels of surface IgG-IC and lupus-related pathologies
(A) Numbers of splenic B cells, T cells, DCs, and MFs from age matched B6, MRL/lpr, or FcγRI−/−MRL/lpr mice (20 weeks old) were enumerated by flow cytometry analysis. (n = 5–7 mice, 2 experiments). (B) Surface Sm levels on splenic DCs, MFs, B cells, and T cells. (C) IgG levels on splenic DCs and MFs from B6, MRL/lpr, or FcγRI−/−MRL/lpr mice (>20 weeks old) were analyzed by flow cytometry. (n = 5–8 mice, 3 experiments). The expression of intracellular phosphorylated (D) Syk, (E) Akt-Threonine308, and (F) S6 levels in splenic DCs and MFs were analyzed by flow cytometry. The data for B6 and MRL/lpr includes data from Figure 3E–G. (n = 5–15, 3 experiments). (G) Anti-nucleosome IgG, (H) anti-dsDNA IgG, or (I) BAFF levels in the sera collected from B6, MRL/lpr, or FcγRI−/−MRL/lpr (>20 weeks old) were measured by ELISA. (n = 4–8 mice from 2 experiments forG andH, n=6–15 mice from 5 experiments forI). (J) Number of B cells, T cells, DCs, and MFs infiltrating the kidneys were enumerated by flow cytometry. (n = 5–11 mice, 2 experiments). (K) Levels of glomerular inflammation were scored using H&E stained kidney sections. (n = 5–6, 2 experiments). (L) Urine samples were analyzed for protein levels. Bars represent median. *p<0.05, **p<0.01, ***p<0.001, n.s. = not significant, by Mann-Whitney test (A andD–L) or by Kruskal-Wallis test (B,C).
Figure 5
Figure 5. Anti-nucleosome IgG induces accumulation of IgG-ICs prior to appearance of lupus-related pathologies
(A) AID−/−MRL/lpr mice were treated (i.v.) with PL2-3 (500 μg/mouse) or control antibodies once a week for 2 or 5 weeks. Untreated, age matched B6 and MRL/lpr mice were used as controls. (B) Surface bound IgG (green) on purified splenic MFs from PBS (upper left) or PL2-3 (lower left) treated mice for 2 weeks. Representative images from 3 experiments (10–15 cells/mouse). Surface IgG on splenic DCs (upper right) and MFs (lower right) analyzed by flow cytometry. (C) The expression of intracellular phosphorylated Syk (pSyk) levels in splenic DCs and MFs analyzed by flow cytometry. The data for B6 and MRL/lpr control mice includes data from Figure 3E. (D) Splenic B cells enumerated by flow cytometry. (E) Levels of anti-nucleosome, (F) anti-dsDNA, or (G) total IgM in sera were analyzed by ELISA. (H) BAFF secreting splenic DCs or MFs enumerated by ELISPOT. (I) H&E stained kidney sections. Arrows indicate fibrocellular crescents. Representative images from >3 experiments. Scale bar = 1μm. Scores of (J) glomerular and (K) tubulointerstital inflammation of the kidneys. (L) Proteinuria scores. In (B–L), n = 2–5 mice per treatment per experiment, >3 experiments. In (B–H,J–L) bars represent median. *p<0.05, **p<0.01, ***p<0.001, n.s.= not significant by Kruskal-Wallis test (B) or Mann-Whitney test (C–H, andJ–L).
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