Modulation of peripheral B cell tolerance by CD72 in a murine model - PubMed (original) (raw)
Modulation of peripheral B cell tolerance by CD72 in a murine model
Daniel Hsieh-Hsin Li et al. Arthritis Rheum. 2008 Oct.
Abstract
Objective: B cells play a dominant role in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus. It is not well understood how B cell signaling contributes to autoantibody production. The goal of this study was to elucidate the role of CD72 in modulating B cell receptor (BCR)-mediated tolerogenic signaling and peripheral B cell tolerance.
Methods: A mouse model utilizing hen egg lysozyme (HEL) "anergic" B cells was studied. CD72-deficient mice carrying the BCR-specific IgHEL and/or soluble HEL (sHEL) transgenes were generated by breeding IgHEL-transgenic MD4 mice and/or sHEL-transgenic ML5 mice with congenic, CD72-deficient C57BL/6J mice. Normal and anergic B cells were isolated for analyses of B cell signaling. Aged wild-type and CD72-deficient mice were also examined for autoimmune phenomena.
Results: In the absence of CD72, anergic B cells inappropriately proliferated and survived in response to stimulation with self antigen. Biochemical analyses indicated that in anergic B cells, CD72 dominantly down-regulated BCR signaling to limit the antigen-induced elevation in [Ca2+]i and the activation of NFATc1, NF-kappaB, MAPK, and Akt. Mechanistically, CD72 was associated with, and regulated, the molecular adaptor Cbl-b in anergic B cells, suggesting that Cbl-b may play a role in mediating the negative effects of CD72 on BCR signaling. Moreover, in aged CD72-deficient mice, spontaneous production of antinuclear and anti-double-stranded DNA autoantibodies and features of lupus-like autoimmune disease were observed.
Conclusion: CD72 is required to maintain B cell anergy and functions as a regulator of peripheral B cell tolerance. Thus, altered CD72 expression may play a role during the development of systemic lupus erythematosus.
Figures
Figure 1
Analyses of B cell proliferation and B cell death in vitro in _IgHEL_-transgenic (Tg) and double-transgenic (dTg) CD72−/− mice compared with _IgHEL_-Tg and dTg control mice. A, Serum levels of anti–hen egg lysozyme (anti-HEL) antibodies (n ≥ 15 per group). B, Total numbers of splenic B cells (n = 5 per group). Horizontal bar indicates the mean. * = P < 0.04; ** = P < 0.001. C, Anti-HEL autoantibody production in mature splenic dTg B cells after stimulation with HEL antigen with or without anti-CD40. Bars show the mean and SEM of duplicate cultures. D, Assessment of B cell proliferation, using 3H-thymidine incorporation, after stimulation with HEL (0.5 _μ_g/ml) with or without interleukin-4 (IL-4) or anti-CD40 (10 ng/ml each). E, Proliferation of dTg B cells after antigen stimulation with various concentrations of HEL (0.5 _μ_g/ml, 5 _μ_g/ml, or 10 _μ_g/ml) with or without IL-4. Bars in D and E show the mean and SD of triplicate cultures. F, Flow cytometric analysis of bromodeoxyuridine-positive (BrdU+) cells after stimulation with HEL with or without IL-4 or anti-CD40. Values over bars are the percentage of BrdU+ cells. G, Flow cytometric analysis of cell death using annexin V (AnnV) staining. Values over bars are the percentage of annexin V–positive cells. H, Cytoplasmic expression of cyclin D2, p27kip, Bcl-xL, Myc, and CD72. Actin was used to show equal loading. I, Cell surface expression of CD72 after stimulation with HEL in vitro. Bars show the mean ± SD representative results from 1 of 2 independent experiments in triplicate cultures, with values expressed as the mean fluorescence intensity (MFI).
Figure 2
Regulation of B cell receptor signaling by CD72 in B cells from _IgHEL_-Tg and dTg CD72−/− mice compared with _IgHEL_-Tg and dTg control mice. A, Calcium flux in splenic B cells after stimulation with HEL antigen. Results are expressed as Ca2+, calculated as the ratio of indo-1 blue to violet. B, Dephosphorylation of NFATc1 after stimulation with HEL. Hsp90 was used to show equal loading. Lanes marked with the asterisk indicate incubation with HEL plus anti-CD40 (1 _μ_g/ml). Arrowheads and brackets indicate the phosphorylated (P) and dephosphorylated NFATc1 isoforms, respectively. C, NFATc1 nuclear binding activity after stimulation with HEL. B cells treated with cyclosporin A (CsA) (100 ng/ml) for 30 minutes were used as a negative control. Broken line indicates the cutoff for positivity. Bars show the mean and SD optical density at 450 nm (OD450). D, NF-_κ_B activity after stimulation with HEL. Bars show the mean and SD relative light units (RLU). ** = P < 0.01. E, ERK activity after stimulation with HEL. Results were obtained by in vitro kinase assay using Elk as the substrate. Total Elk was used to show equal loading. F, Akt phosphorylation and Akt kinase activity. Results were obtained by in vitro kinase assay using glycogen synthase kinase 3 (GSK3) as the substrate. Total GSK3 was used to show equal loading. Results in E and F are representative of at least 2 independent experiments. See Figure 1 for other definitions.
Figure 3
Regulation of Cbl-b phosphorylation by CD72 in B cells from IgHEL_-Tg and dTg CD72−/− mice compared with IgHEL_-Tg and dTg control mice, and effects of short-term inhibition of CD72 on anergic B cell proliferation. A, Tyrosine phosphorylation of CD72 after stimulation with HEL antigen. Results are expressed as pY levels. CD72 was identified by immunoprecipitation (IP), and pY levels were determined by immunoblotting (IB). B, Tyrosine phosphorylation of Ig_α and Ig_β after stimulation with HEL. Ig_β_ was identified by IP, and pY levels were determined by IB. Bands for Ig_α_ were determined on the basis of reactivity with anti-Ig_α_ antibodies and the molecular weight, which differs from that for Ig_β_. Total Ig_α_ is shown as a control. C, Tyrosine phosphorylation of Syk after stimulation with HEL. Syk was identified by IP, and total and tyrosine phosphorylated Syk were analyzed by IB. Total Syk is shown as a control. D, Tyrosine phosphorylation of Cbl-b after stimulation with HEL. IP and IB results for Cbl-b were equal. The pY levels were normalized to those for total Cbl-b at time 0 for all time points, due to the reduced ability of the anti–Cbl-b antibody to recognize phosphorylated Cbl-b. E, Constitutive interaction of CD72 with Cbl-b. Syk and I_κ_B_α_ are shown as positive (Pos.) and negative (Neg.) controls, respectively, for Cbl-b interaction. IP and IB results for Cbl-b were equal. F, Effects of preligation of CD72 with anti-CD72 on proliferation and self tolerance, with or without stimulation with HEL. Bars show the mean and SD representative results from 1 of at least 2 independent experiments in triplicate cultures. * = P < 0.01. See Figure 1 for other definitions.
Figure 4
Autoantibody production in vivo in CD72-deficient mice. A–C, HEp-2 cells were stained (1:100) for antinuclear antibodies (ANAs) in the sera of 9-month-old wild-type (WT) and CD72−/− mice; MRL/lpr mice were used as a positive control. Results are representative of 1 of 5 samples from each genotype. D and E, Levels of anti–double-stranded DNA (anti-dsDNA) IgG autoantibodies (D) and total IgG (E) were determined in WT and CD72−/− mice (n = 5 per group). Horizontal bar indicates the mean. ** = P < 0.007. **F**–**I**, IgG immune complex deposition was determined in the glomeruli of WT mice (**F** and **G**) and CD72−/− mice (**H** and **I**) by assessing >12 glomeruli on each kidney section. Two representative samples from each genotype are shown.
Figure 5
Development of multiorgan autoimmune disease in vivo in CD72-deficient mice. A–F, Histopathologic analyses of the kidneys of 1-year-old WT and CD72−/− mice revealed features of glomerular enlargement and segmental mesangial thickening (A and B), glomerular basement membrane condensation, thickening and hyperplasia of the Bowman's capsule, proteinaceous material in the dilated capillaries (C and D), and fibrosis of the glomerular tuft and Bowman's capsule (E and F) in CD72−/− mice. Results were determined by staining with hematoxylin and eosin (H&E) (A and B), periodic acid–Schiff (C and D), and trichrome (E and F). Bars = 50 _μ_m. G–L, Staining with H&E revealed moderate, multifocal lymphoplasmacytic interstitial nephritis in the kidneys (G and H), moderate, multifocal lymphoplasmacytic and histiocytic interstitial sialadenitis in the salivary glands (I and J), and moderate, multifocal and coalescing lymphoplasmacytic and histiocytic perivascular infiltrates in the lungs (K and L) of CD72−/− mice. Bars = 200 _μ_m. M–P, Histopathologic scores were used to assess the severity of glomerulitis (n = 9–14) (M), interstitial nephritis (n = 9–15) (N), sialadenitis (n = 7–11) (O), and lung inflammation (n = 7–9) (P) on a scale of 0–3, in which 0 = no change, 1 = minimal to mild change, 2 = moderate change, and 3 = severe change. * = P < 0.01; ** = P < 0.001. See Figure 4 for other definitions. Color figure can be viewed in the online issue, which is available at
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Figure 6
Working model of the role of CD72 in maintaining B cell anergy. CD72 is essential for the maintenance of B cell tolerance through the interaction with Cbl-b and in the negative regulation of B cell receptor (BCR) signaling. During foreign antigen engagement (immunogenic signaling) in the presence of CD72, some Cbl-b is recruited to the immunogenic signalosome. This results in moderate Cbl-b–mediated inhibition of BCR signaling, thereby attenuating cell cycle entry and survival of mature B cells. In the absence of CD72, less or no Cbl-b is recruited to the immunogenic signalosome, resulting in uninhibited BCR signaling and enhanced cell cycle entry and survival. The effects of CD72 deficiency on cell cycle entry and survival are consistent with the enhanced antigen-induced in vitro proliferation of _IgHEL_-transgenic CD72−/− B cells (see Figure 1). During self antigen engagement (tolerogenic signaling), self-reactive B cells transduce low-intensity signals, leading to an anergic phenotype and functional nonresponsiveness. In the presence of CD72, Cbl-b is recruited to the tolerogenic signalosome, which, through induced proximity and direct activation, allows Cbl-b to inhibit BCR signaling (45,46). This attenuated self antigen–directed signaling (tolerogenic signaling) is not sufficient to induce cell cycle entry, promote survival, or result in autoantibody production. Thus, B cell anergy is maintained. In the absence of CD72, Cbl-b is not recruited to the tolerogenic signalosome, resulting in high-intensity (immunogenic) BCR signaling, which leads to cell cycle entry, cell survival, and autoantibody production (see Figure 1). Thus, binding to self antigen is misinterpreted by the anergic B cells in the absence of CD72, which results in the development of autoimmunity. HEL = hen egg lysozyme; dTg = double-transgenic; SHP-1 = SH2-containing protein tyrosine phosphatase 1; Ub = ubiquitination. Color figure can be viewed in the online issue, which is available at
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References
- Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. Predominant autoantibody production by early human B cell precursors. Science. 2003;301:1374–7. -PubMed
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