Transcript Group_6_Presentation - Mast Cell

Group 6
In vitro models for rodent mast cells
Trainer: Ronit Sagi-Eisenberg
Marek Grosicki
Carl-Fredrik Johnzon
Ahlam Barhoum
Nicole Meyer
Luca Danelli
In vitro strategies for rodent mast cells culture
Ex vivo isolation from tissues – tissue homogenization and
separation steps affect MC activation – low cell yields
Peritoneal Mast Cells – easily to obtain but further purification can
affect activation response
Bone marrow or blood-derived Mast cells – long culture time,
difference between in vivo and in vitro derived cells
RBL, MCBS1 – cell lines with high homogeneity, marginally
representative of mature, tissue bound mast cells
In vitro strategies for rodent mast cells culture
Ex vivo isolation from tissues – tissue homogenization and
separation steps affect MC activation – low cell yields
Peritoneal Mast Cells – easily to obtain but further purification can
affect activation response
Bone marrow or blood-derived Mast cells – long culture time,
difference between in vivo and in vitro derived cells
RBL, MCBS1 – cell lines with high homogeneity, marginally
representative of mature, tissue bound mast cells
Heterogenous cell cultures of peritoneal cells (3% mast cells, 30%
macrophages, 55% B cells, 7% T cells)
Method:
• Peritoneal lavage (complete media)
• Plated on fibroblasts
• Cultured overnight (with IgE)
Mast cells activation (IgE/Ag)
•
•
•
•
Heterogenous vs homogenous
Time
Concentration
Species differences
• More “realistic” ex vivo microenvironment
But.. Difficulties to dissect MC behavior and single cell population
contribution in the heterogeneous cell cultures
Background
Activating mutations in KIT are associated with specific disease states such as
mastocytosis and gastrointestinal stromal cell tumors.
Aim
Definition of functional MC line which doesn’t express native KIT and which would
facilitate the examination of human KIT
Methods
Detection of a rapidly proliferating MC population from mTOR knock/in mice
(mTOR transcription disruption).
Replicating BMMCs lose kit expression
Reconstitution with huKIT
Chemotactic and degranulation response to huSCF
Advantages
• Screening and identifying potential novel inhibitors of KIT and potentially
mutated KIT signaling capacity
Limitations
• mTOR regulates fundamental physiologic functions, including
nutrient sensing ,metabolism, cell growth, proliferation, and
migration …
relevance in molecular pathways in MC biology?
• Investigating human receptor functionality in a murine model
Munc18 protein in mast cell exocytosis model
Exocytosis: energy-consuming process by which a cell directs the
contents of secretory vesicles out of the cell membrane and into the
extracellular space.
SNARE protein
SNARE protein complex:
-SYNTAXIN
- SNAP-25
- VAMP
complex
Munc18 protein:
Family of proteins that play
multiple functions:
- Chaperoning cognate syntaxin
- Priming/stimulating membrane fusion,
interacting with SNARE complex
•
•
Patients with familial hemophagocytic lymphohistiocytosis (FHL) types 3, 4, and 5
identified mutations in Munc13-4, syntaxin-11, and Munc18Markedly reduced degranulation of lytic granules in cytotoxic T lymphocytes (CTLs),
natural killer (NK) cells, and platelets. Disrupted degranulation of mast cells and
neutrophils?
Stimulation with
DNP-HSA
Model RBL2H3
• Munc 18 is crucial for mast cell degranulation
• RBL-2H3 cells would be ideal model systems to study
immune cell exocytosis WHY?
RBL2H3
BMMC
• RBL cell line is a good model in constitutive IgE
dependent degranulation, as this mechanism is
conserved in these cells.
• This founding has been confirmed on BMMC model
• But we have to take in consideration that RBL shares
some characteristics with basophils rather than of
those of mast cells.
RBL2H3 as a useful model for MC degranulation and exocytosis
but …
Conclusions
• Time, “microenvironment” (population and culture conditions),
different stimuli affect MC responses.
• Keep in mind the background of your MC in vitro model
depending in your experimental setup.
• These different models highlight the importance of selecting an
appropriate mast cell model when studying mast cell
involvement in allergic response and inflammation.