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ZIB Seminar, original report 12.10.2009 Asolina Braun
Immunoglobulin D enhances immune surveillance
by activating antimicrobial, proinflammatory and
B cell-stimulating programs in basophils
Kang Chen, Weifeng Xu, Melanie Wilson...Andrea Cerutti
Antibody structure
Vortrag.ppt
14.12.2006
Antibody isotypes
Functions of different Ab isotypes
Vortrag.ppt
14.12.2006
Site of IgD production
•
IgD+IgM-
B cells are abundant in tonsil
and nasal mucosa tissues
• 0.5-1% IgD+IgM- of all CD19+ B cells in
the blood
• IgD+IgM- B cells showed a semidifferentiated plasma cell state (Blimp-1+,
BCMA+ but CD138-; still Pax5+ & TACI+)
flow cytometry data
• AID (activation-induced cytidine
deaminase) expression suggests
ongoing class switch recombination
(CSR)
tonsil tissue, 10x
IgD class switch recombination
• IgD switch occurs in respiratory mucosa
Southern blot analysis of σδ-Sμ switch circles amplified by nested PCR from mononuclear cells of various tissues and hybridized with σδ or Sμ probes
IgD class switch recombination
• T-cell dependent follicular CSR: CD40 on B cells and CD40L on
activated CD4+ T cells
• T-cell independent extrafollicular CSR: TACI & BAFF-R on B cells
ligated by APRIL & BAFF (DC, MΦ, mucosal epithelial cells)
• test of combinations: CD40L/BAFF/APRIL alone or with IL-2, IL-4,
IL-10, IL-13, IL-15, IL-21
• isolation of IgD+IgM+ B cells => stimulation with ligand (and cytokine)
for 4d/7d => southern blot to detect switch circles (4d), flow
cytometry (7d), ELISA (7d)
IgD class switch recombination
IgD CSR in immunodefficient patients
•
CD40L deficiency => HIGM1(hyper-IgM type1 syndrome)
CD40 deficiency => HIGM3
AID deficiency
=> HIGM2
=> CD40 indispensable for
systemic but not mucosal IgD
IgD in respiratory mucosa
•
isolation of circulating IgD+IgM+ B cells => stimulation 7d w/wo BAFF, IL-15, IL-21
=> supernatant IgD for ELISA (n=5)
MID = Moraxella catarrhalis IgD-binding protein
LPS = Lipopolysaccharide
CPS = capsular polysaccharide
M. = Moraxella
H. = Haemophilus
Function of circulating IgD
• polyclonal anti-IgD antibody used in flow cytometry
IgD on basophils
• IgD intracellularly in circulating and mucosal basophils
• binding capacity of IgD on basophils is saturated
• binding of IgD to pre-basophilic cell line KU812, mastocytoid HMC-1
& LAD-2, monocytic U937, but not to T cell, NK cell or myeloid cell
lines
• KU812 and LAD-2 bound more IgD after stimulation with IL-3 and/or
IL-4 (and upregulated IL-4 R, IL-3 R, IgE R)
• Mannose and mannan slightly inhibited IgD binding to HMC-1
• IgD binding was abolished by denaturation of IgD or pretreatment of
cells with trypsin or papain
 IgD binding on basophils and mast cells depends on a protease
sensitive receptor that is IL-3 and IL-4 inducible
Implications of IgD binding
•
•
basophils are known to bind IgE, crosslinking of bound IgE leads to degranulation and histamin
release
isolation of basophils => stimulation with beads or Ig-coated beads => 30min/5h => flow cytometry
=> granular CD63 as a marker for activation
Implications of IgD binding
• incubation of basophils with microbeads w/wo IgE/IgD & w/wo Il-3
=> 16h/48h => ELISA
IL-4:
B cell activation, IgE switch
IL-13:
B cell growth & differentiation
BAFF:
B cell proliferation
IL-8:
chemokine for basophils, neutrophils, T cells
CXCL10: chemokine for monocytes, NK cells, naive T cells
Implications of IgD binding
• IgD-stimulated basophils produce antimicrobial, opsonizing and alarmsignaling factors (ß-defensins,cathelicidin, CRP, SPAG11, PTX3)
• supernatant of activated basophils (IgD beads) inhibits bacterial
replication:
Summary
• IgD class switch recombination (CSR) in respiratory mucosa
produces IgD+IgM- B cells
• IgD CSR depends on CD40L, BAFF, APRIL and needs IL-2, IL-15
and IL-21
• mucosal IgD recognizes respiratory bacteria
• secreted IgD binds to basophils and mast cells
• IgD binding to basophils does not elicit degranulation or histamine
release, but
– upregulates IL-4, IL13, BAFF, IL-8, CXL10 (B cell activation)
– produce antimicrobial, opsonizing and alarm-signaling factors
– inhibit bacterial replication