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. 2006 Aug 1;108(3):974-82.
doi: 10.1182/blood-2005-12-006858.

Autoreactive MZ and B-1 B-cell activation by Faslpr is coincident with an increased frequency of apoptotic lymphocytes and a defect in macrophage clearance

Ye Qian  1 , Kara L Conway, Xiangdong Lu, Heather M Seitz, Glenn K Matsushima, Stephen H Clarke
Affiliations

Autoreactive MZ and B-1 B-cell activation by Faslpr is coincident with an increased frequency of apoptotic lymphocytes and a defect in macrophage clearance

Ye Qian et al. Blood. .

Abstract

Murine autoreactive anti-Smith (Sm) B cells are negatively regulated by anergy and developmental arrest, but are also positively selected into the marginal zone (MZ) and B-1 B-cell populations. Despite positive selection, anti-Sm production occurs only in autoimmune-prone mice. To investigate autoreactive B-cell activation, an anti-Sm transgene was combined with the lpr mutation, a mutation of the proapoptotic gene Fas (Fas(lpr)), on both autoimmune (MRL) and nonautoimmune backgrounds. Fas(lpr) induces a progressive and autoantigen-specific loss of anti-Sm MZ and B-1 B cells in young adult Fas(lpr) and MRL/Fas(lpr) mice that does not require that Fas(lpr) be B-cell intrinsic. This loss is accompanied by a bypass of the early pre-plasma cell (PC) tolerance checkpoint. Although the MRL bkg does not lead to a progressive loss of anti-Sm MZ or B-1 B cells, it induces a robust bypass of the early pre-PC tolerance checkpoint. Fas(lpr) mice have a high frequency of apoptotic lymphocytes in secondary lymphoid tissues and a macrophage defect in apoptotic cell phagocytosis. Since Sm is exposed on the surface of apoptotic cells, we propose that anti-Sm MZ and B-1 B-cell activation is the result of a Fas(lpr)-induced defect in apoptotic cell clearance.

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Figures

Figure 1.
Figure 1.
Anti-Sm antibody ASC production in autoimmune mice. (A) Serum anti-Sm levels are shown for mice 2 to 4 months of age. (B) The number ± SEM of anti-Sm ASCs in the bone marrow (BM), spleen, MLN, and LP as determined by ELISpot assay is shown (**P < .01 in comparison with 2-12H mice; n ≥ 3).
Figure 2.
Figure 2.
Anti-Sm B-cell development in spleen of 2-12H Tg, 2-12H MRL, 2-12H/Faslpr, and 2-12H MRL/Faslpr mice. (A) Anti-Sm B cells are present in the spleen of 2-12H Tg, 2-12H MRL, 2-12H/Faslpr, and 2-12H MRL/Faslpr mice. All histograms are gated on CD19+ B cells, and anti-Sm B cells are boxed. All mice are 2 to 4 months of age. (B) Numbers of splenic B cells and anti-Sm B cells are presented. Means ± SEM of 5 6-month-old mice of the indicated strain are plotted. Asterisks indicate a statistically significant difference (*P < .05 and **P < .01) from cell numbers of their nonautoimmune counterparts. Cell numbers at 3 months are not statistically different from those shown for 6 months (data not shown). (C) B-cell subset analysis of splenic anti-Sm B cells from 1-month-old and 3-month-old mice based on CD21 and CD23 expression. Anti-Sm B cells are gated as indicated in panel A. The CD21hi, CD23lo cells are MZ B cells, and the CD21int, CD23hi cells are FO B cells. The percent of total anti-Sm B cells for each subset is given. (D) Total number of anti-Sm MZ B cells from 1- to 6-month-old mice of each strain is given. Gating for anti-Sm B cells is as shown in panel A. Mean ± SE is plotted (n = 6). (E) Sm staining of MZ and FO B cells is shown. Gating on total MZ and FO B cells was based on CD21 and CD23 expression as indicated in panel B. The shaded regions are Sm staining of non-Tg B cells; the solid lines are Sm staining by B cells of their Tg counterparts.
Figure 3.
Figure 3.
ELISpot analysis of anti-Sm ASCs in 2-12H, 2-12H/Faslpr, and 2-12HMRL/Faslpr mice. (A) Spleen cells were stained for expression of CD19, CD138, and Sm. The CD138 expression of CD19+, Sm+ cells is shown. Boxes indicate the CD138int and CD138hi populations. The percent of each population is provided. Data are representative of 3 or more mice of each strain. The percentages of CD138int B cells are as follows: 17% ± 2.4% for 2-12H, 14% ± 3.1% for 2-12H/Faslpr, 7.0% ± 1.1% for 2-12H MRL, and 7.7% ± 0.50% for 2-12H MRL/Faslpr. The percentages of CD138hi B cells are as follows: 0.13% ± 0.07% for 2-12H, 0.09% ± 0.01% for 2-12H/Faslpr, 1.4% ± 0.46% for 2-12H MRL, and 1.9% ± 0.66% for 2-12H MRL/Faslpr. (B) Size, IgM expression, and CXCR4 expression are shown for anti-Sm CD138int and CD138hi B cells from mice of the indicated strains. The gates are illustrated in panel A. Data are representative of 3 or more mice. (C) Spleen cells were stained for CD19, CD138, CD21, and CD23. The gating scheme used to sort the FO, MZ, and CD138+ cells is indicated using 2-12H spleen cells. The top histogram shows the identification of CD19+, CD138+ cells sorted for ELISpot analysis. The CD19+ CD138– cells were further fractionated by CD21 and CD23 expression to identify FO and MZ B cells as shown. (D) The number of small and large anti-Sm ELISpots was determined for each of the indicated cell populations. The definition of large and small ELISpots is given in “Materials and methods.” The average size of small ELISpots characteristic of MZ and FO B cells is 2 × 10–3 ± 4 × 10–4 mm2, and the average size of the large ELISpots characteristic of CD138+ pre-PCs is 1.7 × 10–2 ± 4 × 10–4 mm2. Examples of the small FO/MZ-like spots and large CD138+ pre-PC ELISpots are shown. The number of FO anti-Sm ELISpots per 105 cells in 2-12H and 2-12H/Faslpr mice is 23.3 ± 2.36 and 3.51 ± 2.48, respectively (P < .001). The number of MZ anti-Sm ELISpots per 105 cells in 2-12H and 2-12H/Faslpr mice is 183 ± 19.2 and 22.5 ± 10.9, respectively (P = .002). The number of CD138+ pre-PC anti-Sm ELISpots per 105 cells from 2-12H, 2-12H/Faslpr, and 2-12H MRL/Faslpr mice is 26.0 ± 5.6, 1540 ± 135, and 7600 ± 265, respectively (P < .01 for all comparisons). ND indicates none detected.
Figure 4.
Figure 4.
Peritoneal anti-Sm B-1 cells in 2-12H Tg, 2-12H MRL, 2-12H/Faslpr, and 2-12H MRL/Faslpr mice. (A) Phenotypic analysis of peritoneal B cells from 2-12H Tg, 2-12H MRL, 2-12H/Faslpr, and 2-12H MRL/Faslpr mice. Peritoneal cells from 1-month-old and 3-month-old mice were stained with CD19, IgM, CD11b, and Sm. Anti-Sm B-1 cells are boxed. All histograms are gated on CD19+ B cells. (B) Analysis of total B-1–cell numbers ± SEM (top and bottom graphs) and anti-Sm B-cell numbers ± SEM (middle) for mice between 1 and 6 months of age is shown.
Figure 5.
Figure 5.
Anti-Sm B-1–cell activation in Faslpr mice after peritoneal cell transfer. (A) 2-12H/Faslpr peritoneal cells were stained for CD19 and CD11b, and the CD19+ CD11b+ B-1 cells and CD19+ CD11b– B-2 cells were sorted and transferred to Faslpr recipient mice. Two weeks after transfer, anti-Sm ASCs in the bone marrow, spleen, MLN, and LP were quantified (± SEM) by ELISpot. ND indicates none detected. (B) Unsorted peritoneal cells from 2-12H/Faslpr mice were transferred to wild-type (wt) or Faslpr recipient mice. ELISA was used to measure (± SEM) serum IgMa+ anti-Sm (inset), and ELISpot was used to quantify (± SEM) anti-Sm ASCs from BM, spleen, MLN, and LP. (C) The same as panel B except that 2-12H peritoneal cells were transferred to wt or Faslpr recipients. Asterisks indicate the differences (P < .05) between wt and Faslpr recipients.
Figure 6.
Figure 6.
The effect of Faslpr on PtC-specific B cells. (A) Comparison of 6-1 and 6-1/Faslpr peritoneal B cells for liposome binding and CD43 and CD5 expression. Histograms are gated on CD19+ B cells. PtC-specific B-1 cells (CD43+ and CD5+) are boxed, and the percent of total CD19+ B cells is indicated. (B) The number, ± SEM, of PtC-specific B-1 cells present in the peritoneal cavity of 6-1 and 6-1/Faslpr between 1 and 6 months is graphed. PtC-specific B-1 cells were gated as shown in panel A. (C) Number, ± SEM, of IgMa ASCs in wt and Faslpr recipient mice 2 weeks after transfer of 6-1 peritoneal cells.
Figure 7.
Figure 7.
Apoptotic cells in wt and Faslpr mice. (A) Flow cytometry analysis for the presence of apoptotic cells in the spleens and MLNs of wt and Faslpr mice by FITC-VAD-FMK staining is shown. Representative histograms for wt and Faslpr spleen cells are shown (top). Each histogram is gated on lymphocytes and the percent VAD-FMK+ is provided. The graph below shows the average ± SEM of percent apoptotic lymphocytes in the spleen and MLN of mice at 2 months and 3 months of age (n = 3). **A significant difference with wt (P < .01). (B) TUNEL-positive cell distributions in spleen and MLN of both wt and Faslpr mice. Both low (× 10) and high (× 40) magnifications are shown. Boxes indicate the areas of higher magnification. WP indicates white pulp; RP, red pulp; GC, germinal center; and F, follicle. (C) Macrophages from wt, Faslpr, MRL/Faslpr, and Merkd mice were incubated with apoptotic thymocytes for 60 minutes, and the percent of phagocytized thymocytes was determined by fluorescent microscopy. Controls are the percentage of live thymocytes (nonapoptotic) phagocytized. The graph is representative of 4 experiments and shows the average ± SEM of 3 mice per strain at both 2 months and 3 months of age. *P < .05, compared with wt.
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