Immunity Supplemental Information Leukotriene B 4 -Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo Bartomeu Colom, Jennifer V. Bodkin, Martina Beyrau, Abigail Woodfin, Christiane Ody, Claire Rourke, Triantafyllos Chavakis, Karim Brohi, Beat A. Imhof, and Sussan Nourshargh
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Immunity
Supplemental Information
Leukotriene B4-Neutrophil Elastase Axis
Drives Neutrophil Reverse Transendothelial
Cell Migration In Vivo
Bartomeu Colom, Jennifer V. Bodkin, Martina Beyrau, Abigail Woodfin, Christiane Ody,
Claire Rourke, Triantafyllos Chavakis, Karim Brohi, Beat A. Imhof, and Sussan
Nourshargh
SUPPLEMENTAL INFORMATION
SUPPLEMENTAL FIGURES
A
C
JAM-A
VE-Cadherin
PECAM-1
Control LTB4B
Control
IR
1 2
3
4 5 6
1=C5a, 2=IL1α, 3=IL6, 4=KC, 5=MCP-1, 6=MIP-2
1 2
3
4 5 6
Figure S1
Figure S1. Related to Figure 1. Characterization of the inflammatory mediator
generation and expression profile of EC adhesion molecules in response to
cremaster muscle ischemia-reperfusion injury or intradermal injection of LTB4,
respectively. (A) The inflammatory mediator expression profile in sham (control)
and I-R stimulated cremaster muscles was measured in tissue homogenates (pooled
samples from 3 mice per group) using a Mouse Cytokine Array Panel A kit.
Densitometry from the blots was analyzed with ImageJ software. Images are
representative of 2 independent experiments. (B-C) Locally administered LTB4 does
not impact the expression profile of EC JAM-A, VE-cadherin or PECAM-1. Images
(B) and quantification of protein expression levels (C) of the indicated adhesion
molecules at EC junctions of mouse ear dermal post-capillary venules in control (B,
left panels) and in stimulated tissues (4h i.d. LTB4) (B, right panels), as analyzed by
immunofluorescent staining and confocal microscopy (n=4 mice) from 3
independent experiments. Data are percentage change in mean fluorescent intensity
(MFI) of signals acquired from stimulated samples relative to controls and presented
as mean ± SEM. Scale bars, 20µm.
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Figure S2
Figure S2. Related to Figure 2. JAM-C is expressed at different levels in different
types of microvessels. (A) Mouse ears were immunostained for JAM-C and α-SMA. The
images show JAM-C expression in all microvessels but with differing levels: JAM-C
expression was greatest in capillaries (c, α-SMA negative), followed by venules (v) and
was low in arterioles (a). JAM-C was also noted in nerves (n). (B) High magnification
images of mouse ear dermal blood vessels illustrating localization of JAM-C to EC
junctions (as shown by co-localisation with VE-cadherin) and again indicating different
expression levels of JAM-C in capillaries, venules and arterioles. (C-D) Quantification of
JAM-C protein levels at junctions of ECs in different blood vessel types in ear skin (C)
and cremasters (D), as analyzed by confocal microscopy (n=3-7) from 5 independent
experiments. Data indicate mean fluorescent intensity (MFI) ± SEM. ** P<0.01 and ***
P<0.001 as indicated by lines. Scale bars, 100µm (A), 20µm (B).
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Figure S3. Related to Figure 3. Neutrophil elastase mediates LTB4-induced
cleavage of EC JAM-C. LTB4-stimulated cremaster muscles of WT, Elane-/- or
WT mice treated with the NE inhibitor GW311616A were analysed for junctional
expression of EC JAM-C and compared to control unstimulated tissues (n=3-4)
involving 4 independent experiments. Data indicate mean ± SEM. ***P<0.001 as
compared to controls and #P<0.05 and ###P<0.001 as indicated by lines.
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Figure S4
Figure S4. Related to Figure 6. The LTB4-NE axis promotes distant organ damage.
(A-B) Time course of remote organ damage following LTB4 stimulation of cremaster
muscle. Quantification of lung (A) and heart (B) albumin content (plasma
extravasation), as an indicator of tissue damage, following local (cremaster,
intrascrotal) injection of LTB4 at the indicated time points as compared to control (Ctrl)
unstimulated tissues (n=3-20 from 15 independent experiments). (C) Intradermal
administration of LTB4 into the mouse ear skin promotes lung inflammation. LTB4 was
injected into mouse ears intradermally and 4h later, the lungs were excised and
analysed for neutrophil infiltration as quantified by measurement of tissue MPO
enzymatic activity. Unstimulated ears acted as controls (n=4-6 from 2 independent
experiments). (D) Intradermal injection of LTB4 in the mouse ear promotes multi-organ
distant damage in an NE-dependent manner. Quantification of remote tissue albumin
content following local (ear, intradermal) injection of LTB4 (4h) as compared to control
unstimulated tissues in WT and Elane-/- mice (n=4-24 from 11 independent
experiments). (E) LTB4-induced neutrophil recruitment to lungs is NE-independent.
WT, Elane-/- mice or WT mice pretreated (orally, 24h) with the NE inhibitor
GW311616A were stimulated with intranasal LTB4 and 24h later neutrophil infiltration
into the airways was quantified in bronchoalveolar lavage (BAL). Control mice
received LTB4 vehicle (n=3-6 from 3 independent experiments). (F) Cremaster I-R
injury induces multi-organ distant damage. Quantification of remote tissue albumin
content in WT and Elane-/- mice following I-R injury of the cremaster muscle as
compared to sham operated mice (n=3-7 from 5 independent experiments). Data
indicate mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 as compared to controls
and #P<0.05, ##P<0.01 and ###P<0.001 as indicated by lines.
SUPPLEMENTAL MOVIE LEGENDS
Movie S1. Related to Figure 1. Neutrophil reverse TEM as induced by I-R injury. The
movie captures an inflammatory response in a cremasteric venule of a Lyz2-EGFP-ki mouse
(exhibiting GFP myeloid cells), immunostained in vivo for EC junctions with Alexa Fluor-555-
labeled anti-PECAM-1 mAb 390 (red) and stimulated with I-R. The clip shows high optical
zoom of a neutrophil migrating through a multi-cellular junction viewed from the luminal side.
The neutrophil (green) is initially on the abluminal (sub-EC) side of the endothelial junction
and subsequently migrates through the junction in an abluminal to luminal or 'reverse' direction.
Breaching the EC barrier results in the transient formation of an exit pore, as indicated in the
movie. On the luminal side the leukocyte disengages from the junction and crawls across the
luminal surface. Still images of this sequence are shown in Fig. 1D. Of note, the movie has
been created using a software (IMARIS™, Bitplane) that recreates the structures being imaged
from individual voxels in 3D via a blend projection algorithm whilst maintaining a
transparency function. As a result, when observing neutrophil migration from the luminal side,
neutrophils in the vascular lumen are seen as being fully green (closest to the viewing direction)
whilst cells in the sub-EC space and/or within EC junctions are visualized as green cells with
an overlay of red fluorescence (further away from the viewing direction). Similarly when
viewing events from the abluminal side, the sub-EC neutrophil is seen as being fully green due
to it being close to the viewing direction.
Movie S2. Related to Figure 1. Neutrophil reverse TEM as induced by topical LTB4. The
movie captures an inflammatory response in a cremasteric venule of a Lyz2-EGFP-ki mouse
(exhibiting GFP myeloid cells, green), immunostained in vivo for EC junctions with Alexa
Fluor-555-labeled anti-PECAM-1 mAb 390 (red), as induced by locally administered LTB4.
The clip shows high optical zoom of a neutrophil migrating through a multi-cellular junction
viewed from the luminal side. The neutrophil (green) is initially on the luminal side of the
endothelial cell, transmigrates into the sub-endothelial cell space and subsequently migrates
through the junction back into the lumen in a 'reverse' direction. On the luminal side the
leukocyte disengages from the junction and crawls across the luminal surface. The movie has
been created using a software (IMARIS™, Bitplane) that recreates the structures being imaged
from individual voxels in 3D via a blend projection algorithm whilst maintaining a
transparency function. As a result, since the neutrophil rTEM event is being observed from the
luminal side, neutrophils in the vascular lumen are seen as being fully green (closest to the
viewing direction) whilst cells in the sub-EC space and/or within EC junctions are visualized
as green cells with an overlay of red fluorescence (further away from the viewing direction).
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Reagents/antibodies. Recombinant murine C5a, CXCL2 (MIP-2) and IL1β were purchased
from R&D Systems (Abingdon, Oxford, UK). CXCL1 (KC) was from AbD Serotec (Oxford,
UK). LTB4 and PVDF membranes were purchased from Calbiochem (Merck Millipore,