A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus

Abstract

The precise role of B cells in systemic autoimmunity is incompletely understood. Although B cells are necessary for expression of disease (Chan, O., and M.J. Shlomchik. 1998. J. Immunol. 160:51-59, and Shlomchik, M.J., M.P. Madaio, D. Ni, M. Trounstine, and D. Huszar. 1994. J. Exp. Med. 180:1295-1306), it is unclear whether autoantibody production, antigen presentation, and/or other B cell functions are required for the complete pathologic phenotype. To address this issue, two experimental approaches were used. In the first, the individual contributions of circulating antibodies and B cells were analyzed using MRL/MpJ-Faslpr (MRL/lpr) mice that expressed a mutant transgene encoding surface immunoglobulin (Ig), but which did not permit the secretion of circulating Ig. These mice developed nephritis, characterized by cellular infiltration within the kidney, indicating that B cells themselves, without soluble autoantibody production, exert a pathogenic role. The results indicate that, independent of serum autoantibody, functional B cells expressing surface Ig are essential for disease expression, either by serving as antigen-presenting cells for antigen-specific, autoreactive T cells, or by contributing directly to local inflammation.

Figure 1

Tg schematic. The mIgM heavy chain Tg is depicted. The DNA directing secretion (μs) and the transcription termination site (pA_s) have been deleted. V_H186.2 is the rearranged heavy chain V(D)J segment. E indicates the heavy chain intronic enhancer. Much of the switch region has been deleted; a small region of residual switch region is indicated.

Figure 2

The Tg restores splenic B cells in J_HD mice. Spleen cells from J_HD (A), mIgM (B), and MRL/lpr (C) mice were analyzed via flow cytometry. Live (propidium iodide–negative) cells were analyzed for CD19 (PE) and B220 (FITC) expression. B cells are B220⁺/CD19⁺ (upper right quadrant). Percentages of B cells among live splenocytes are shown. In MRL/lpr mice, B220⁺/CD19⁻ cells are T cells.

Figure 3

Spontaneous splenic CD4⁺ T cell activation is restored by the mIgM Tg. Three-color FACS^® analysis was conducted on splenocytes. Representative FACS^® contour plots are shown for J_HD (A), MRL/lpr (B), and mIgM (C) mice. CD4^high gated cells were analyzed for CD44 (FITC) and CD62L (PE) expression. Percentages of total CD4⁺ T cells (D) were obtained for the naive (CD44^lowCD62L^high), activated (CD44^highCD62L^high), and memory (CD44^highCD62L^low) subsets as depicted in parts A–C. Cell numbers (E) were calculated by multiplying total cell percentages of each activation subset by the total spleen cell number for each mouse. Columns show the averages of percentages (D) or cell numbers (E) of a cohort of mice. Error bars represent one standard deviation. Sample sizes for cell percentages are: J_HD (n = 17), MRL/lpr (n = 26), mIgM (n = 6) mice. Sample sizes for cell numbers are: J_HD (n = 9), MRL/lpr (n = 25), mIgM (n = 5). All mice were 24–32 wk of age. Asterisks indicate significant differences of P < 0.05 with the B cell–deficient group, as determined by the two-tailed Mann-Whitney U test.

Figure 4

Lupus nephritis occurs in the absence of circulating Ig. Kidneys were fixed in formalin, and sections were stained with hematoxylin and eosin: (A) 23 wk mIgM, (B) 30 wk mIgM, (C and D) 42 wk mIgM, (E) 31 wk MRL/lpr, (F) 30 wk MRL/lpr, and (G and H) 45 wk J_HD. Cellular interstitial and perivascular infiltrates are observed in mIgM (A–D) and MRL/lpr (E and F), but not in J_HD mice, even up to 45 wk of age (G and H). Black arrows point to glomerular lesions in the mIgM mice (A and C).

Figure 5

Kidney disease scores and mortality in MRL/lpr mice. Kidney samples were examined via light microscopy and scored blindly for disease on a semiquantitative scale from 0 (normal) to 4 (maximum). Scores for the glomeruli (A), interstitium (B), and vessels (C) are shown above. Each point represents an individual animal. Black horizontal bars indicate the median score. All animals were 24–30 wk of age. Comparisons to J_HD mice were conducted, and statistical significance is indicated in each graph. Sample sizes for the mortality graph (D) are: MRL/lpr (n = 203), J_HD (n = 267), and mIgM (n = 71). The J_HD mice had greater survival than either MRL/lpr (P < 0.0001) or mIgM (P < 0.0001) mice. Survival curves were generated as described in Materials and Methods using the Kaplan-Meier method and examined for statistical significance using the Mantel-Cox logrank tests.

Figure 6

Serum Ig levels. Sera were obtained from mIgM, MRL/lpr, and J_HD mice. Ig levels as determined by ELISA for isotypes IgM, IgG1, and IgG2a are shown on the logarithmic y axis in A. Anti-dsDNA, anti-IgG2a rheumatoid factor, and antichromatin Ig are shown in B. Each point represents one mouse. Horizontal bars indicate the median Ig level.

Figure 7

Lack of renal antibody deposition in mIgM mice. Mice were assayed for Ig deposition, as determined by anti–mouse κ-FITC: (A) MRL/lpr, (B) J_HD, and (C) mIgM. Deposition was observed in MRL/lpr mice (A), whereas none was detected in J_HD (B) and mIgM (C) mice. All animals were 29–36 wk of age.