Download Where is Plexin-A1 located in the cell? - lamsam

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Highly professional antigen presenting cells
DC reside in the peripheral tissues
Upon exposure to antigen they migrate to the
lymphoid organs via the lymphatics
In the lymphoid tissue (e.g. lymph nodes)
they activate naïve T cells
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This is an active process
Involves chemokines
adhesion molecules
Inflammatory molecules
The dendritic cell must undergo
morphological changes in order to pass
through the endothelial junctions
Non-muscle myosin II is known to propel DCs
forward and pass through narrow gaps.
Initially identified as axonal guidance cues that
determine the direction and migration of
neurons.
•Their discovery was in regards to axon guidance
in the limb buds of grasshoppers in 1992
• subsequently found to be involved in many
other systems.
• such as
• heart development
• Vascular growth
•Tumour progression
• Immune responses
• Bone development
•
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In the nervous system semaphorins regulate
cell motility and morphology through
interactions with receptors of the plexin
family
The plexin family includes plexin-A1
This is the main receptor for class III and
class IV semaphorins
In the immune system plexin-A1 is expressed
specifically in dendritic cells
Plexin-A1 deficient mice have severe defects
in antigen-specific T cell responses
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Plexin-A1 has a crucial role in DC trafficking
In particular, entry into the lymphatics
Sem3A produced in the lymphatics is a ligand
for plexin-A1
Sema3A acts on the rear side of DCs where
plexin-A1 is also localised
Sem3A induced phosphorylation of the
myosin light chain resulting in the ability of
DCs to squash through narrow gaps.
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CFSE labelled activated OT-II T cells were introduced
into WT and Plxna1-/- mice
These OT-II T cells specifically recognise and respond
to ova peptide
Then ova peptide administered in freunds adjuvant
subcutaneously into the footpads.
• T cells were isolated from the draining LNs
• They found that antigen specific T cell
proliferative response was much lower in
Plxna1-/- mice then in WT.
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Ova-pulsed DCs from WT and Plxna1-/- mice labelled with
CMTMR (orange) were injected into footpads and analysed for
their ability to move to the popliteal lymph nodes. OT-II CD4
T cells were labelled green and injected IV.
Yes!!
In WT mice DC traffic into the T cell area of LNs within 24hrs
In Plxna1-/- mice only a few DCs made it into the T cell area of
the LN
Thus Plxna1 is required for the migratory ability of DCs.
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CFSE labelled DCs injected into footpads of WT and Plxna1-/mice and their arrival in draining LNs was determined (1C)
Applied FITC epicutaneously to the shoulder skin of WT and
Plxna1-/- mice and looked for FITC+ DCs in draining Brachial
LNs (1D).
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Migrating DCs in 1C calculated
as follows:
% input cells=total cells x
(%CFSE+cells/input cells).
ID shows that actual number
of endogenous DCs isolated
from the brachial LN 24 and
48hrs following epicutaneous
administartion of FITC-isomer
to the shoulder skin
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Cells were cultured with FITC-dextran at 37C for
30 minutes. Cells were analysed for presence of
FITC+ cells
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Uptake of FITC-dextran
at 37C for 30 mins was
the same in WT and KO
mice
Plexin-A1 not required
for antigen uptake by
DCs
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Analysis of chemotaxis of WT and KO DCs towards
a gradient of CCL19,CCL21 or CXCL12 in transwell
system was unaltered in the KO mice
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As was their ability to sense
a directional gradient : see
movie 1
• CFSE labelled wildtype or Plexin-A1-/- DCs
(green) were injected intradermally into the
ears of oxazalone-treated mice.
• lymphatic endothelial cells were stained with
anti-LYVE-1 (red)
• DCs from Plxna-1-/- mice were retained
along the endothelium lining the lymphatics
located in the dermis
• DCs from wildtype mice were not retained
• using time-lapse imaging DCs were investigated for their
ability to move across a lymphatic endothelial cell monolayer
•Yellow dotted lines indicate junctions of endothelial cells.
•White arrows indicate DCs in contact with lymphatic endothelial cells
•Red arrows indicate the transmigration process of wildtype DCs
•Show movie 2
• CFSE labelled wildtype or Plxna-1-/- DCs
(green) were added to monolayers of
endothelial lymphatic cells which had been
stained with F-actin marker phalloidin (red)
•Migration of DCs monitored by z-stack
imaging (confocal microscopy)
• wildtype DCs observed from the top to the bottom
of the stack
•In contrast Plxna1-/- DCs were able to make
contact with the endothelial cells but could not
trnasmigrate across them
•Show movie 3
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Plexin-A1 is a receptor for semaphorin 3A
and semaphorins 6C and 6D.
All are expressed in lymphatic endothelial
cells
Plexin-A1 binds to Sem3A in association with
NRP-1
Sem3A is soluble
Sem6C and Sem6D are both transmembrane
molecules
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Wildtype DCs were transferred into Sem3A
Sem6C -/- and Sem 6D -/- mice.
Presence of DCs in draining lymph nodes
estimated
DCs from Sem3A-/mice only, showed
impaired migration
-/-,
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Sem3A has been identified as a
chemorepellent factor that guides the
direction of neurons.
This data shows that it promotes trafficking.
the authors hypothesise that cell polarity
generated by chemokines during DC
migration may be influenced by Sem3A
They test this using chemotaxis assays
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Sem3A was added to the
upper or lower chamber of
a Transwell system in the
presence or absence of
chemokines
In the absence of chemokines Sem3A
did not show an effect on DC migration
In the presence of chemokines Sem3A
enhanced DC migration when added to
the upper chamber only
In this set-up Sem3A is acting on the
rear of the cells
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Authors used confocal time-lapse video microscopy
Plexin-A1transfected DCs (green) were treated with
LPS and suspended in type 1 collagen.
Gels were then placed in a Zigmond chamber with a
chemokine gradient applied to them
DC locomotion was examined at 1min intervals
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Plexin-A1 found
to be localised in
the trailing edge
of migrating DCs
Play Movie 5
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Myosin II is known to be involved in DC
trafficking
It is phosphorylated by MLC and the Rho
kinase : ROCK
Localisation of these molecules resembles
that of Plexin-A1
Myosin II is believed to be required for
squeezing of the cell body and induction of
actinomycin contraction when cells pass
through narrow gaps or constricted areas.
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Confocal Z stack imaging
was performed on DCs on
fibronectin-coated
coverslips treated with
human IgG or recombinant
Sem3A fusion protein
Stained with antibody to
phosphorylated MLC
(pMLC)
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Phosphorylation of MLC enhanced in the presence of
Sem3A
This affect not observed in Plxn-A1 -/- mice thus
Sem3a promotes MLC phosphorylation via its
interaction Plexin-A1
Phosphorylation of the myosin light chain promotes
actinomycin contraction.
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Semaphorins are immunomodulatory
molecules
Here the authors demonstrate that
Semaphorin mediated signals are crucial for
DC trafficking
In particular in their entrance into the
lymphatics
Sem3A promotes actinomycin contraction at
the trailing edge of migrating DCs so that
they may pass through narrow gaps.