ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

tissues is characteristic for a number of these pathological states. Thus, chemoattractantdirected
migration of mature mast cells or their progenitors might be one of the key
mechanisms responsible for local accumulation of these cells under physiological or
pathological conditions.
Aggregation of high-affinity receptors for IgE (FcεRI), expressed in the plasma membrane
of mast cells, with polyvalent antigen (Ag) leads to cell activation and release of a
broad spectrum of chemical mediators. FcεRI is also involved in mast cell chemotaxis
towards Ag. Mast cells express a variety of chemokine receptors which bind various
chemoattractants, including stem cell factor, sphingosin-1-phosphate, and arachidonic
acid metabolites as leukotriens and prostaglandins (2). Prostaglandin E 2
(PGE 2
) is one
of the major eicosanoids generated during inflammation and potent mediator which
substantially influence mast cell responses, including chemotaxis (3). PGE 2
binds to
E-prostanoid receptors and engage cAMP/PKA pathway (4). However, detail signaling
process leading to mast cell chemotaxis mediated by PGE 2
is incompletely understood.
In this study we analyzed the effect of different drugs affecting cAMP/PKA signaling
in PGE 2
stimulated mouse bone marrow derived-mast cells (BMMCs). We focused on
characterisation of cell spreading on fibronectin as well as changes in cell motility after
stimulation. For analysis of these responses we introduced several quantitative imagebased
cytometry techniques which facilitate large scale image acquisition and data
processing. Using these and other methods we found that potentiation or blocking of
cAMP/PKA signaling has signifcant effect on mast cell signaling pathways induced by
PGE 2
. Consequently, these signaling pathways might contribute to pathological states
occuring in allergy and inflammatory diseases.
Acknowledgements
This work was supported by COST CZ project No. LD12073.
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Legend to figure
Fig. 1. Images and quantification of changes in mast cell morphology after activation
with Ag or PGE 2
. BMMCs were sensitized with IgE and attached to fibronectin. Cells
were activated with Ag (B), PGE 2
(C) or were not activated (A). After fixation and
permeabilization the cells were stained with Alexa Fluor 488-phalloidin for filamentous
actin (F-Actin; green) and with Hoechst stain for nuclei (blue). Images were acquired
on Olympus Scan^R system and analyzed using CellProfiler image analysis software
86 Analytical Cytometry VII