Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 10 (8), 889-98

Immunoglobulin D Enhances Immune Surveillance by Activating Antimicrobial, Proinflammatory and B Cell-Stimulating Programs in Basophils

Affiliations

Immunoglobulin D Enhances Immune Surveillance by Activating Antimicrobial, Proinflammatory and B Cell-Stimulating Programs in Basophils

Kang Chen et al. Nat Immunol.

Abstract

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

Figures

Figure 1
Figure 1. Upper respiratory mucosa B cells generate IgD+IgM plasmablasts by undergoing Cμ-to-Cδ CSR in situ
(a) Proportion of IgD+IgM plasmablasts in various tissues calculated by immunofluorescence as described in Methods. PLN, peripheral lymph nodes; BM, bone marrow. (b) Tonsil tissue stained for IgD (green) and IgM (red). DAPI (blue) counterstains nuclei. Arrowheads point to IgD+IgM plasmablasts. Original magnification ×10. (c) Representative IgD+IgM B cells from tonsils stained for IgD (green), Pax-5, Blimp-1, BCMA, CD138, κ, λ, AID or CD19 (red), and IgM or DAPI (blue). (d) Diagram of CSR from Sμ to σδ. Germline Iμ-Cμ and Iμ-Cδ transcripts, σδ-Sμ switch circles, and post-switched Iμ-Cδ transcripts are shown. Arrows indicate primers. (e) Southern blots of σδ-Sμ switch circles PCR amplified from mononuclear cells of various tissues and hybridized with σδ or Sμ probes. Rightmost lane shows a typical multi-banded σδ-Sμ smear in enriched tonsillar IgD+ B cells. Kb, kilobases. (f) Germline Iμ-Cμ transcripts and germline or post-switch Iμ-Cδ transcripts in Reh IgDIgM+ pre-B-like cells, 2E2 pre-germinal center IgD+IgM+ B cells, Ramos germinal center-like IgDIgM+ B cells, and MM-M1 IgD+IgM plasma cells. Genomic β-actin is a loading control. (g) Representative σδ-Sμ and Iμ-Cδ DNA sequences from tonsillar IgD+IgM and IgD+IgM+ B cells, respectively. Panel a summarizes 1 of 3 experiments (bars indicate s.e.m.), whereas panels b, c, d, f, g and h show 1 of 5 experiments yielding similar results.
Figure 2
Figure 2. Cμ-to-Cδ CSR occurs through both TD and TI pathways, requires AID, and leads to the production of IgD antibodies that bind to respiratory bacteria
(a) σδ-Sμ switch circles from circulating IgD+IgM+ B cells stimulated with or without CD40L, BAFF or APRIL plus IL-15 and IL-21 for 4 d. Genomic β-actin is a loading control. Kb, kilobases. (b, c) Flow cytometric analysis of surface IgD and IgM and ELISA of secreted IgD from circulating IgD+IgM+ B cells stimulated as in a for 7 d. Control indicates medium alone. (d) Percentage of circulating IgD+IgM B cells in a healthy donor and patients with TNFSF5 (HIGM1), AICDA (HIGM2), TNFRSF5 (HIGM3), TNFRSF13b (CVID) or MVK (HIDS) gene defects. (e) Induction of σδ-Sμ switch circles in circulating mononuclear cells from a HIGM2 patient and a control healthy donor incubated with medium alone (control) or BAFF, IL-15 and IL-21 (stimulated) for 4 d. An anti-IgM antibody was added to optimize the activation and expansion of IgD+IgM+ B cells. (f) Binding of secreted IgD to MID, CPS, LPS, M. catarrhalis, and H. influenzae type a or type b as determined by ELISA. IgD secretion was obtained by incubating circulating IgD+IgM+ B cells with or without BAFF, IL-15 and IL-21 for 7 d. Panels a, b, d and e show 1 of 5 experiments yielding similar results, whereas panels c and f summarize 3 experiments (bars indicate s.e.m.; *, p < 0.05).
Figure 3
Figure 3. IgD binds to basophils and mast cells in vivo
(a) Flow cytometric analysis of IgD on circulating or tonsillar CD19+CD3 B cells, CD19CD3+ T cells, CD16highCD56low or CD16lowCD56high NK cells, CD19CD14+ monocytes, CD11c+CD83 or CD11c+CD83+ myeloid dendritic cells (mDCs), CD15+CD123 neutrophils, CD15+CD123low eosinophils, CD123+HLA-DR+ or CD123+FcεRIlow plasmacytoid dendritic cells (pDCs), CD123+HLA-DR or CD123+FcεRI+ basophils, and CD117+FcεRI+ mast cells. Gray and black histograms depict control unstained cells and IgD, respectively. A F(ab’)2 pAb was used to detect IgD. Dead cells were excluded using 7-AAD staining. (b) Immunofluoresence analysis of circulating and tonsillar basophils stained for IgD (green) and FcεRI, CD123 or tryptase (red). DAPI (blue) counterstains lobated nuclei of basophils. A F(ab’)2 pAb was used to detect IgD. (c) Flow cytometry of IgD levels on circulating basophils before (black histogram) and after exposure to exogenous monoclonal IgD (10 μg/ml, brown histogram), treatment with acidic buffer (blue histogram), and treatment with acidic buffer followed by addition of exogenous IgD (red histogram). A F(ab’)2 pAb was used to detect IgD. Unstained basophils were used as control (gray histogram) and dead cells were excluded using 7-AAD staining. (d) Binding of labeled monoclonal IgD to circulating basophils stripped of endogenous IgD and incubated with 0 μg/ml (green histogram), 100 μg/ml (orange histogram) or 500 μg/ml (purple histogram) of unlabelled monoclonal IgD. A F(ab’)2 pAb was used to detect IgD. Unstained basophils were used as control (gray histogram) and dead cells were excluded using 7-AAD staining. Panels a-d show 1 of 5 experiments yielding similar results.
Figure 4
Figure 4. IgD binds to basophilic and mast cell lines in vitro
(a) Binding of monoclonal IgD (50 μg/ml) to various lymphoid and myeloid cell lines. A F(ab’)2 pAb was used to detect IgD. Mo, MC and Basø indicate monocytic, mast cell and basophilic cell lines, respectively. (b) Binding of increasing amounts (0.5, 5 and 50 μg/ml) of monoclonal IgD to the mast cell line HMC-1 and binding of monoclonal IgD (50 μg/ml) to the basophilic cell line KU812 before and after treatment with IL-4 for 1 d and with IL-3 for 4 d. Control indicates medium alone. A F(ab’)2 pAb was used to detect IgD. Gray histogram depicts background fluorescence of cells that were not incubated with monoclonal IgD. (c) Binding of fluorochrome-conjugated monoclonal IgD to HMC-1 or KU812 cells in the presence of increasing amounts (0, 5, 50, 250 or 1000 μg/ml) of unlabeled monoclonal IgD. (d) Binding of untreated or denatured monoclonal IgD (50 μg/ml) to HMC-1 cells pre-incubated or not with IgG, IgA, IgE, mannose, mannan, trypsin, pepsin, papain, or papain plus leupeptin. A F(ab’)2 pAb was used to detect IgD. In all the experiments dead cells were excluded using 7-AAD staining. Panel a summarizes 3 experiments (bars indicate s.e.m.), whereas panels b-d show 1 of 3 experiments yielding similar results.
Figure 5
Figure 5. Basophils release immunostimulating and pro-inflammatory factors upon IgD cross-linking
(a) Flow cytometry of CD63 on basophils exposed to microbeads alone (control), microbead-bound monoclonal anti-IgD, or microbead-bound monoclonal anti-IgE for 30 min or 5 h in the presence or absence of IL-3. (b) ELISA of histamine from basophils exposed to microbeads alone (open bar), microbead-bound monoclonal anti-IgD (gray bar), microbead-bound monoclonal anti-IgE (black bar), or microbead-bound isotype-matched control monoclonal antibody (striped bar) for 30 min in the presence or absence of IL-3. (c) ELISA of IL-4, IL-13 or BAFF from basophils exposed to microbeads alone (open bar), microbead-bound monoclonal anti-IgD (gray bar), or microbead-bound monoclonal anti-IgE (black bar) for 16 h in the presence or absence of IL-3. IL-8 and CXCL10 were measured after 48 h. (d) Intracellular Ca2+ levels of basophils treated as in c. The arrow indicates addition of the cross-linking reagent and 0 sec indicates the start of the kinetic measurement. (e) IgM, IgA and IgG production by peripheral blood IgD+IgM+ B cells exposed for 7 d to basophils treated as in c. Panels a and d show 1 of 3 experiments yielding similar results, whereas panels b, c and e summarize 3 experiments (bars indicate s.e.m.; *, p < 0.01; **, p < 0.001).
Figure 6
Figure 6. Basophils release antimicrobial factors upon IgD cross-linking
(a) QRT-PCR of DEFB103A, CAMP, SPAG11A/F, and SPAG11D/G transcripts from basophils exposed to microbeads alone (control, open bar), microbead-bound monoclonal anti-IgD (gray bar), or microbead-bound monoclonal anti-IgE (black bar) for 6 h. PTX3 and CRP transcripts were measured after 16 h. mRNAs were normalized to ACTB mRNA. (b) ELISA of LL-37 from basophils stimulated as in a in the presence or absence of IL-3 for 8 h. (c) Growth of Moraxella catarrhalis and Haemophilus influenzae type-a and type-b upon 2-h exposure to culture supernatants from basophils stimulated as in a for 8 h or 16 h. CFU, colony forming unit. Panels a-c summarize 3 experiments (bars indicate s.e.m.; *, p < 0.03; **, p < 0.02; ***, p < 0.01).
Figure 7
Figure 7. Increased IgD class-switched plasmablasts and IgD-armed basophils in inflamed tissues from patients with periodic fever syndromes
(a, b) Flow cytometric analysis of circulating IgD+IgM B cells and CD123+HLA-DR basophils in a healthy subject and hyper-IgD patients with MVK (HIDS), unknown (PFAPA), NALP3 (MWS) and TNFRSF1A (TRAPS) gene defects. Numbers indicate percentage of IgD+IgM B cells in CD19+ B cells and of CD123+HLA-DR basophils in mononuclear cells. (c) Immunofluorescence analysis of a tonsil tissue specimen from healthy individual or a PFAPA syndrome patient stained for IgD (green), tryptase (red) and DAPI (blue). Dashed lines demarcate lymphoid follicles. (d) BAFF (green) expression by basophils co-stained for IgD (green), CD123 (red) and FcεRI (blue). Arrowheads show both IgD+CD123 plasmablasts (B) and IgD+CD123+ basophils (Basø). (e) ELISA of TNF and IL-1β from basophils cultured with IL-3 and microbeads alone (control, open bar), microbead-bound monoclonal anti-IgD (gray bar) or microbead-bound monoclonal anti-IgE (black bar) for 48 h in the presence or absence of monocytes. Panels a-d show 1 of 3 experiments yielding similar results, whereas panel e summarizes 3 experiments (bars indicate s.e.m.).

Similar articles

See all similar articles

Cited by 115 PubMed Central articles

See all "Cited by" articles

References

    1. Rowe DS, Fahey JL. A new class of human immunoglobulins. J Exp Med. 1965;121:171–199. - PMC - PubMed
    1. Butler JE, Sun J, Navarro P. The swine Ig heavy chain locus has a single JH and no identifiable IgD. Int Immunol. 1996;8:1897–1904. - PubMed
    1. Ohta Y, Flajnik M. IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates. Proc Natl Acad Sci U S A. 2006;103:10723–10728. - PMC - PubMed
    1. Preud’homme JL, et al. Structural and functional properties of membrane and secreted IgD. Mol Immunol. 2000;37:871–887. - PubMed
    1. Bengten E, et al. The IgH locus of the channel catfish, Ictalurus punctatus, contains multiple constant region gene sequences: different genes encode heavy chains of membrane and secreted IgD. J Immunol. 2002;169:2488–2497. - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback