Enhanced auto-antibody production and Mott cell formation in FcμR-deficient autoimmune mice

Abstract

The IgM-Fc receptor (FcμR) is involved in IgM homeostasis as evidenced by increased pre-immune serum IgM and natural auto-antibodies of both IgM and IgG isotypes in Fcmr-deficient C57BL/6 (B6) mice. To determine the impact of Fcmr-ablation on autoimmunity, we introduced the Fcmr null mutation onto the Fas-deficient autoimmune-prone B6.MRL Fas (lpr/lpr) mouse background (B6/lpr). Both IgM and IgG auto-antibodies against dsDNA or chromatin appeared earlier in FcμR(-) B6/lpr than FcμR(+) B6/lpr mice, but this difference became less pronounced with age. Splenic B2 cells, which were 2-fold elevated in FcμR(+) B6/lpr mice, were reduced to normal B6 levels in FcμR(-) B6/lpr mice, whereas splenic B1 cells were comparable in both groups of B6/lpr mice. By contrast, marginal zone (MZ) B cells were markedly reduced in FcμR(-) B6/lpr mice compared with either FcμR(+) B6/lpr or wild type (WT) B6 mice. This reduction appeared to result from rapid differentiation of MZ B cells into plasma cells in the absence of FcμR, as IgM antibody to a Smith (Sm) antigen, to which MZ B cells are known to preferentially respond, was greatly increased in both groups (B6/lpr and B6) of FcμR(-) mice compared with FcμR(+) B6/lpr or B6 mice. Mott cells, aberrant plasma cells with intra-cytoplasmic inclusions, were also increased in the absence of FcμR. Despite these abnormalities, the severity of renal pathology and function and survival were all indistinguishable between FcμR(-) and FcμR(+) B6/lpr mice. Collectively, these findings suggest that FcμR plays important roles in the regulation of auto-antibody production, Mott cell formation and the differentiation of MZ B cells into plasma cells in B6.MRL Fas (lpr/lpr) mice.

Keywords: FcμR; IgM; Mott cells; auto-antibody; marginal zone B cells.

Fig. 1.

Lack of FcμR expression in Fcmr ^−/− B6/lpr mice. (A) Genotyping of offspring of Fcmr ^−/− C57BL/6 and B6.MRL Fas ^lpr/lpr mice. Tail DNA was subjected to genomic PCR using diagnostic sets of primers to determine the genotype of Fcmr and Fas in individual mice. Representative genotyping data are shown. The normal (WT) and targeted (KO) alleles of Fcmr give rise to 570 and 730-bp PCR products, respectively. The WT and mutant (Lpr) alleles of Fas are 179 and 217bp, respectively. (B) RT–PCR analysis. Total RNAs isolated from spleen of Fcmr ^+/+ (lanes 1 and 4) or Fcmr ^−/− (lanes 2 and 5) B6/lpr mice and control WT mice (lanes 3 and 6) were subjected to RT–PCR and amplified products of FcμR (lanes 1–3) and control PIR-A (lanes 4–6) were electrophoresed in 0.7% agarose and stained with ethidium bromide. Lane 7 is a PCR control without a first-strand cDNA template. Note the lack of full-length FcμR transcripts (1283bp) in Fcmr ^−/− B6/lpr mice and presence of PIR-A transcripts (253bp). (C) Flow cytometric analysis of cell surface FcμR. Splenocytes from the indicated mice were first incubated with FcγR-blocking mAbs and then with biotinylated, anti-FcμR (lines) or isotype-matched control mAb (shaded) and FITC-anti-CD19 mAb. The bound biotinylated mAbs were detected by addition of PE-streptavidin. CD19⁺ B cells were gated and examined for their expression of FcμR.

Fig. 2.

Serum levels of IgM and IgG auto-antibodies and of total IgM, IgG and IgA. Sera were collected from WT B6 (n = 5–24; closed circles), FcμR(+) B6/lpr (n = 13–23; closed inverted triangles) and FcμR(−) B6/lpr (n = 16–28; closed diamonds) female mice at the age of 10 weeks (top), 20 weeks (middle) and 50 weeks (bottom), and were subjected to ELISA for assessment of titers of antibody against dsDNA (A). Concentrations (μg/ml) of total serum IgM, IgG and IgA are also depicted (B). Each symbol represents data from an individual mouse. *P < 0.05, **P < 0.01 and ***P < 0.001, respectively.

Fig. 3.

Immunofluorescent assessment of cellular compartments in Fcmr ^−/− and control B6/lpr mice. (A) Representative immunofluorescence profiles. Splenocytes (top three rows) and LN cells (bottom row) isolated from WT B6 (left column), FcμR(+) B6/lpr (middle column) and FcμR(−) B6/lpr (right column) female mice at 20 weeks of age were first incubated with FcγR-blocking reagents and then with fluorochrome-labeled mAbs specific for the indicated surface markers. Stained cells with the light scatter characteristics of lymphocytes/macrophages were analyzed using an Accuri C6 flow cytometer. In the second and third rows, the CD19⁺ B cells were gated and examined for their expression of the indicated surface markers. Numbers indicate percentages of cells. Note (i) a sizable population of CD11b⁺/CD5⁻ B cells, (ii) marked expansion of CD3⁺/B220⁺ atypical T cells in both FcμR(+) and FcμR(−) B6/lpr mice and (iii) a clear reduction of CD21⁺/CD23⁻ MZ B cells in FcμR(−) B6/lpr mice. (B) Total cell numbers in each population in spleen (top two rows) and BM (bottom row) from WT B6 (closed circles), FcμR(+) B6/lpr (closed inverted triangles) and FcμR(−) B6/lpr (closed diamonds) mice (five in each group). Each symbol represents data from an individual mouse. The horizontal bar indicates the arithmetic mean. *P < 0.05 and **P < 0.01, respectively. Note (i) normal levels of splenic B and B2 cells and marked reduction of MZ B cells in FcμR(−) B6/lpr mice, (ii) comparable levels of B1b and B1a cells as well as plasma cells in both FcμR(+) and FcμR(−) B6/lpr mice and (iii) significant reduction of BM mature B cells and normal levels of BM plasma cells in FcμR(−) B6/lpr mice.

Fig. 4.

High titer of anti-Sm antibody and enhanced Mott cell formation in the absence of FcμR. (A) Assessment of anti-Sm antibody. Serum levels of anti-Sm antibodies in FcμR (+) B6 (closed circles), FcμR(−) B6 (n = 17–27; closed triangles), FcμR(+) B6/lpr (closed inverted triangles) and FcμR(−) B6/lpr (closed diamonds) female mice were similarly assessed by ELISA as described in the Fig. 2 legend. (B) Mott cells. Left: Representative PAS-stained spleen sections from FcμR(−) B6/lpr mice of 20 weeks of age depicting strongly PAS⁺ Mott cells (upper) and representative immunofluorescent staining of Mott cells with FITC anti-μ antibodies with short (lower left) and long (lower right) exposure. Right: Number of Mott cells per mm² of spleen and LNs from WT B6 (closed circles), FcμR(−) B6 (closed triangles), FcμR(+) B6/lpr (closed inverted triangles) and FcμR(−) B6/lpr (closed diamonds) mice (n = 3–6 in each group). *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 5.

Pathological changes of kidney. (A) Representative PAS-stained kidney sections from FcμR(+) (left panel) and FcμR(−) (right panel) B6/lpr mice of 50 weeks of age at two different magnifications. Scale bars indicate 50 μm (upper panel) and 20 μm (lower panel). Note mesangial cell proliferation and increase mesangial matrix with homogeneous PAS+ substances in both groups of mice. (B) Representative immunofluorescence images of glomerular deposition of IgM (top), IgG (middle) and C3 (bottom) from FcμR(+) (left panel) and FcμR(−) (right panel) B6/lpr mice. Note the presence of immune deposits primarily in the mesangium of both groups of mice. (C) Mean fluorescence intensity (MFI) of deposition of IgM, IgG, IgA and C3 in glomeruli from FcμR(+) (closed inverted triangles) and FcμR(−) (closed diamonds) B6/lpr mice. Results are shown as arbitrary units.

Fig. 6.

Kidney function and survival. (A) Results show the BUN levels (mg/dl) in WT B6 (n = 5–13; closed circles), FcμR(+) B6/lpr (n = 8–16; closed inverted triangles) and FcμR(−) B6/lpr (n = 15–21; closed diamonds) mice at 20 (top) and 50 (bottom) weeks of ages. Each symbol represents data from an individual mouse. The shaded area represents the range within 2 SDs of the mean of WT B6 mice. (B) Survival of FcμR(+) B6/lpr (n = 17; unfilled circles) and FcμR(−) B6/lpr (n = 20; filled circles) mice up to 50 weeks of age.