Group_1_Presentation - Mast Cell

Models for in vivo
and in vitro studies
on basophils
Trainer: Franco Falcone
• Basophils were discovered more than century ago (1879)
• Only recently they have been identify to play a role in
allergy (1972)
• Compared to human, mice have less basophils which were
initially more difficult to identify. First discovery of mouse
basophils by EM in 1981
How to deplete basophils in mice
• No naturaly occuring basophil defficiency in mice
• Antibody induces defficiency
Anti-FcseRI mAb
anti-CD200R3 mAb
• Not selective only for basophils!
• Posible activation and/or depletion of mast cells
• Depletion of DC subpopulations (antigen presentation properties?)
• Not optimal depletion efficiency (80 – 90% for Ba103)
• Problematic timing in long term experiments
Other models are needed!
Transgenic models
DTR transgene
under CD203c
Diphteria toxin
Cells express DTR
allowing inducible
Depletion of
basophils 84%
• To reveal contribution of basophils and eosinophils in IgE-CAI in vivo.
• To generate a new mouse models lacking basophils and eosinophils.
BasoDTR mice - hDTR espression driven by CD203c promotor
(basophil marker)
EoDTR mice - hDTR espression driven by EPO promotor (eosinophil
peroxidase, granule cationic protein)
Basophil-speciphic ablation after DT administration in BasoDTR
• Decrease in basophils in
peripheral blood (84%) and
• No decrease in basophil
population in bone-marrow!
• qPCR and WB analysis failed to
detect hDTR in bone-marrow
• No effect on peritoneal mast
IgE-mediated chronic allergic (CAI)
reaction in BasoDTR mice
TNP-spec. IgE i.v. 24 hrs
TNP-OVA i.d.
DT treatment in BasoDTR mice almost completely abolished developement of IgE-CAI
DT added -2days before Ag
DT treatment abolished the IgE-CAI
in EoDTR mice
Eosinophils are involved in ear
swelling exacerbation rather than
induction of inflammation
Pros and cons
+ efficient systemic depletion
+ first inducible depletion of eosinophils
- No convincing evidence about CD203c expression in mice basophils and not in other
cell types
- What happens to other cells is not discused (Table 1)
- Only one time poind measured
- Significance for chronic models (IgE-CAI?)
- High concentrations of DT affects also other cells
• Generate mast cell and basophil deficient mouse model
– Based on finding that Myeloid cell leukemia sequence-1 (Mcl-1) is
shown to be an intracellular anti-apoptotic factor
Increased apoptosis of cells that do express Cpa3
• Cpa3 is highly expressed in mast
• Efficient mast cell depletion in all
tested tissues, except for spleen
• Cpa3 is expressed in
subpopulation of
• Efficient basophil
depletion in tested
tissues (56-78%)
• Cpa3 is expressed in
neutrophils, eosinophils,
T cells,b cells
• Other cells type
numbers not affected,
except for RBC
• PCA: Passive cutaneous anaphylaxis,
Mast cell-dependent reaction
PCA (mast cells)
• CAI: Chronic allergic inflammation,
basophil-dependent reaction
(contribution of mast cells ignored
CAI (basophils)
Pros and cons
+ efficient depletion of mast cells
+ constitutive depletion (e.g. chronic models)
- Quite inefficient depletion of basophils; 22-42% of basophils are still present;
what happens in a more subtle model in which basophils are involved?
- Not a good model for investigating basophils; mast cells are depleted
- No depletion of mast cells in spleen
• To asses the role of basophils in acquired tick resistance
– Basophil depleting antibodies used (Ba103, anti-CD200R3)
– Selective inducible ablation of basophils – Mctp8DTR mice
– hDTR expressed under the control of basophil speciphic promoter of
Acquired resistance to ticks
• Resistance measured
as the time of feeding
• Resistance is mast celldependent
• FceRI and antibody
Recruitment of basophils to tickfeeding sites during the second
DT-mediated basophil ablation in
Mctp8DTR mice
• A: DT abolished acquired tick resistance in Mctp8DTR mice
• B: Basophils and mast cell numbers in skin 4 days after the initiation of second
Pros and cons
+ efficient basophil depletion
+ no other cell types appear to be affected
- High concentrations of DT affects also other cells
- Significance for chronic models (IgE-CAI?)
• Selective ablation of basophils showed to be powerful tool in studying their
role in physiological conditions
• Limitations in basophil specific markers
• Limitations in tools for inducing depletion (DT)
• Constitutive depletion might affect also mast cells numbers (Cpa3-Cre)
• Mcpt8-Cre might be a good alternative
• Search for more specific basophil markers?
• Mctp8 is currently the best option
• GFP expression only in basophils is important control (mctp8 promotor
control, different cells from several tissues were checked)
• Best model (Mctp8) is good only for mice, not for human
• Toxic effects of DT, dose-dependent
• Fast recovery of basophils – should be DT administration repeated?
• Hello kitty – only part of the promotor, mediates moderate Cre expression
• Cpa3 KO has different phenotype than Cpa3-Cre
• Side effects of Cre expression in other tissues?
• Overexpression of basophil population is missing in the literature

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