Local renal autoantibody production in lupus nephritis

Abstract

Autoantibodies are central to the pathogenesis of several autoimmune diseases including systemic lupus erythematosus. Plasma cells secrete these autoantibodies, but the anatomical sites of these cells are not well defined. Here, we found that although dsDNA-specific plasma cells in NZB/W mice were present in spleen and bone marrow, a large number were in the kidneys and their number correlated with the serum dsDNA-IgG titer. We observed renal plasma cells only in mice with nephritis, where they located mainly to the tubulointerstitium of the cortex and outer medulla. These cells had the phenotypic characteristics of fully differentiated plasma cells and, similar to long-lived bone marrow plasma cells, they were not in cell cycle. In patients with lupus nephritis, plasma cells were often present in the medulla in those with the most severe disease, especially combined proliferative and membranous lupus nephritis. The identification of the kidney as a major site of autoreactive plasma cells has implications for our understanding of the pathogenesis of lupus nephritis and for strategies to deplete autoreactive plasma cells, a long-standing therapeutic aim.

Figure 1.

Autoreactive plasma cells are found in the inflamed kidneys of NZB/W mice. (A) Total IgG antibody–forming cells (AFCs) present in the spleen, kidneys, and bone marrow of NZB/W and sex- and age-matched C57BL/6 mice were detected by ELISPOT. One independent experiment representative of two is shown (n = 5 mice per group). (B) dsDNA-specific IgG–secreting cells in NZB/W and C57BL/6 mice detected with a modified ELISPOT assay. Pooled results of three experiments are shown (n = 11 mice per group). Absolute numbers were multiplied by 2 for the kidney and by 7.9 for the bone marrow to account for the two kidneys and the whole bone marrow. (C) Correlation between serum dsDNA IgG and autoreactive plasma cells in NZB/W kidneys. NZB/W mice were divided into three groups depending on the number of dsDNA-specific AFCs in the different organs: (Low) <2 times, (Intermediate) 2 to 5 times, (High) >5 times above background level detected in C57BL/6 mice). Low, intermediate, and high numbers of AFCs are represented by circles, triangles, and squares, respectively. dsDNA-specific serum IgG titers were determined by ELISA (relative units, R.U.). Error bars represent SEM. P values were determined using the Mann-Whitney unpaired test with a risk of 5% except in (C) where a Spearman correlation test was used.

Figure 2.

Renal plasma cells are fully differentiated and long-lived. (A) PCs of NZB/W mice were detected by flow cytometry as CD138+ and B220lo/− in spleen, bone marrow, and kidney. (B) Expression of surface markers on spleen, bone marrow, and kidney PCs gated as CD138+B220lo/− or CD138+/CD19lo/−. Mean fluorescence intensity is indicated for each histogram. Staining for CD38, CD43, and CXCR4 are in red and isotype controls are in blue. One representative dot plot of at least three mice is shown. (C) Percentage of Ki67+ and Ki67− plasma cells in the different organs in NZB/W mice. Frozen sections of spleen (top left), bone marrow (top middle), and kidney (top right) of NZB/W mice were stained for IgG-secreting PCs (green), B cells (B220; red), and the cell cycle marker Ki67 (blue). Scale bars = 20 μm. Spleen, n = 225 cells per four mice; kidney, n = 215 cells per three mice; bone marrow, n = 245 cells per five mice.

Figure 3.

Most NZB/W renal plasma cells are scattered in the tubulointerstitium. (A) PCs were detected by their characteristic shape and nucleus in formalin-fixed sections stained with hematoxylin and eosin. PCs (red arrows) were present in lymphocyte infiltrates (left) and scattered in the tubulointerstitium of the outer medulla (center) and cortex (right). Magnification, ×40. (B) Localization of PCs in the tubulointerstitium was confirmed by staining PCs with an anti-mouse IgG antibody (red) and the tubular basal membrane with a rabbit anti-mouse laminin (green). Left panel: representative view of the outer medulla. Scale bar = 20 μm. Right panels: Magnified view of PCs. Scale bar = 10 μm. (C) PCs are in the same area as infiltrating myeloid cells. PCs were stained with anti-mouse IgG antibody (green), myeloid cells with anti-CD11b (red), and tubular basement membranes with rabbit anti-mouse laminin (blue). Left panel: Representative view of the tubulointerstitium. Scale bar = 20 μm. Right panels: Magnification view of plasma cells in contact with CD11b+ myeloid cells. Scale bar = 10 μm.

Figure 4.

Plasma cell infiltration in the medulla of kidneys of patients with SLE correlates with inflammation and disease severity. (A) Combined class IV diffuse (A and C) and class V membranous lupus nephritis with segmental endocapillary proliferation and prominent glomerular basement membranes due to widespread glomerular, but not tubular, immune deposits with tubulointerstitial fibrosis and inflammation (Jones' silver stain, ×20). (B) Occasional medullary inflammatory cells in class IV diffuse lupus nephritis (Jones' silver stain, ×40). (C) Fibrosis and interstitial medullary inflammation in combined class IV diffuse and class V membranous lupus nephritis (×20). (D) Occasional cortical interstitial plasma cells in combined class IV diffuse and class V membranous LN (anti-CD138). (E) Scattered medullary interstitial plasma cells in class IV diffuse lupus nephritis (anti-CD138): (A, C, D) from same case; (B and E) from same case. (F) Class IV LN kidney biopsies were stained with an anti-human IgG detection kit (top panels) or with an anti-human IgM antibody conjugated to alkaline phosphatase (bottom panels) and counterstained with eosin. PCs are stained by the anti-IgG (brown, red arrows) and are positive for eosin (blue). (G) H&E scoring of the amount of inflammatory cells in the interstitium depending on the class of LN. 0 = no inflammatory cells, 1 = rare inflammatory cells, 2 = moderate infiltration, and 3 = severe infiltration. ●, LN class II; ■, class III; ▴, class IV; ▾, class V; ♦, class III+V; and ○, class IV+V.