a. olivera et al. research article j clin invest s1p and ...a. olivera et al. research article j...
TRANSCRIPT
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
1
Supplemental Figure 1. SphK1-/- and SphK2-/- mice differ in the onset of IgE/Ag-
induced anaphylaxis. A) WT (n=23), SphK1-/- (n=15), Sphk2-/- (n=19) mice were injected
(i.v.) with DNP-specific IgE (3 µg). After 24 hrs, mice were challenged with Ag (250 µg
DNP36-HSA) to induce systemic anaphylaxis. Plasma histamine concentration was
measured from plasma 1.5 min after challenge by ompetitive ELISA. B) Body
temperature changes during the anaphylactic shock induced by Ag 5 and 10 min after
challenge in the WT, SphK1-/- , Sphk2-/- mice (n=12/group), measured as described in
Methods. ***p<0.001 was determined by Student’s t-test.
WT SphK1-/- SphK2-/-
2
6
10
14
18
22
26
***
n=23
n=15
n=19
Pla
sm
a H
ista
min
e (!
M)
WT
SphK
1 -/-
SphK
2 -/-
WT
SphK
1 -/-
SphK
2 -/-
-6
-5
-4
-3
-2
-1
5 min 10 min
***
***
Te
mp
era
ture
Ch
an
ge
(oC
)
A
B
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
2
Supplemental Figure 2. Induction of anaphylaxis by histamine is mast cell-
independent. DNP-specific IgE (3 µg) was injected into C57BL/6 or mast cell-deficient
(Wsh/Wsh) mice. After 24 hrs, mice were challenged with Ag (250 µg DNP36-HSA)
(C57BL/6 and Wsh/Wsh) or saline pseudo challenged (Wsh/Wsh). Anaphylaxis in mast cell-
deficient (Wsh/Wsh) mice was induced by injecting 5 µmol of histamine. Body
temperature during anaphylaxis was monitored by an electronic transponder implanted
under the skin and read at 1 min and subsequently at 5 min intervals for a total of 60 min
using an electronic probe.
5 10 15 20 25 30 35 40 45 50 55 60 65
-8.5
-7.5
-6.5
-5.5
-4.5
-3.5
-2.5
-1.5
-0.5
0.5
WSh/WSh-Histamine
WSh/WSh-IgE/Ag
WSh/WSh-PBS
C57BL/6-IgE/Ag
Time (min)
Te
mp
era
ture
Ch
an
ge
(oC
)
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
3
Supplemental Figure 3. Levels of S1PR1 expression in different organs from
S1PR1loxP/loxP-Mx and S1PR1loxP/loxP mice. Mice were injected with pIpC for 4 weeks and
subsequently used for anaphylaxis studies. Organs were collected 7-14 days after
anaphylaxis studies to confirm excision of the floxed S1PR1 gene in the mice carrying
the Mx-Cre transgene. RNA was extracted from the indicated organs and expression of
S1PR1 mRNA was determined by quantitative real time PCR (TaqMan® Gene
Expression Assays, Applied Biosystems).
Heart
WT-loxP/loxP S1PR1-loxP/loxP-Mx0.000
0.002
0.004
0.006
0.008
Re
lati
ve
Ex
pre
ss
ion
Aorta
WT-loxP/loxP S1PR1-loxP/loxP-Mx0.000
0.002
0.004
0.006
0.008
Re
lati
ve
E
xp
res
sio
n
Lung
WT-loxP/loxP S1PR1-loxP/loxP-Mx0.00
0.02
0.04
0.06
0.08
0.10
Re
lati
ve
E
xp
res
sio
n
Kidney
WT-loxP/loxP S1PR1-loxP/loxP-Mx0.0000
0.0002
0.0004
0.0006
0.0008
Re
lati
ve
E
xp
res
sio
n
Spleen
WT-loxP/loxP S1PR1-loxP/loxP-Mx0.00
0.02
0.04
0.06
0.08
Re
lati
ve
E
xp
res
sio
n
A B C
D E
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
4
Supplemental Figure 4. Changes in vascular permeability in SphK-/- and S1PR2-/- mice
during systemic anaphylaxis and local inflammation. A) Exudation of vascular fluids into
the peritoneum after induction of systemic anaphylaxis by IgE/Ag. Mice from the
different strains (n=4 to 5) were injected (i.v.) with DNP-specific IgE (3 µg) and after 24
hrs, mice were challenged with Ag (500 µg DNP36-HSA) to induce systemic anaphylaxis
and 20 mg/Kg Evans blue dye to examine plasma leakage. After 30 min, the peritoneal
lavage was collected and mice were perfused with saline though the right ventricle until
blood was visibly eliminated. The peritoneal lavage was centrifuged for 10 min at 3000xg
and the amount of Evans blue in the supernatants was determined by absorbance at 620
nm minus absorbance at 720 nm. Lungs were collected, weighted and homogenized in 1
IgE/Ag-induced anaphylaxis
WT
SphK1-/-
SphK2-/-
WT(S1PR2+/+ )
S1PR2-/-
0
2
4
6
Pe
rme
ab
ilit
y P
eri
ton
eu
m [
pg
/ml]
Histamine-induced anaphylaxis
0 40 0 40 0 40 0 40 0 40 0 4040
45
50
55
60
65
WT
SphK1-/-
SphK1-/- SphK2+/-
SphK2-/-
WT (S1PR2+/+)
S1PR2 -/-
NS (p=0.054) *
**NS
(p=0.098)
NS
*
NS
Time (min)
He
ma
toc
rit
(%)
C48/80-induced vascular permeability
PBS C48/800.0
0.1
0.2
0.3
0.4
0.5
0.6WT
SphK1-/-
SphK2-/-
SphK1-/-SphK2+/-
WT (S1PR2+/+)
S1PR2-/-
NSp=0.08
NSp=0.14
Ev
an
s B
lue
ex
tra
cte
d (
A6
20
)
Histamine
0 5 10 15 20 25 300
5
10
15
20
25
30
WT
SphK1-/-
SphK2-/-
SphK1-/-SphK2+/-
*
Pa
w S
we
llin
g (
% in
cre
as
e)
0 5 10 15 20 25 300
5
10
15
20
25
30
35 WT (S1PR2+/+)
S1PR2 -/-
Time (min)
Pa
w S
we
llin
g (
% in
cre
as
e)
A
B
C
D
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
5
ml formamide. The dye accumulated in the extravascular space was extracted by
incubation for 18 hrs at 55oC and the amount extracted was determined by absorbance.
The amounts of dye extracted by g of wet weight were as follows: WT, 8.5±1.0 pg/g;
SphK1-/-, 9.8±2.1 pg/g; SphK2-/-, 12.8±4.2 pg/g; WT(S1PR2+/+), 9.8±1.9 pg/g, and
S1PR2-/-, 12.78±3.8 pg/g. Direct observation of the ears, limbs and nose, did not reveal
visible differences in the intensity of blue between the different genotypes. B) Changes
in hematocrit during histamine-induced anaphylaxis. A baseline hematocrit was taken by
withdrawing 50 µl of blood via the tail vein into a heparinized microhematocrit tube 2 h
to 24 h before the induction of anaphylaxis. Anaphylaxis was induced by injecting
histamine (5 µmol) into the various mice strains (n=4-5/group). After 40 min, mice were
euthanized and blood was collected by cardiac puncture and 50 µl transferred into
heparinized microhematocrit tubes. Blood was centrifuged and the percentage of packed
blood cell volume (hematocrit) determined using a microhematocrit capillary tube reader.
C) Paw edema induced by injection of histamine into the footpad. Mice (n=5 to 6) were
anesthetized (isoflurane (2%):oxygen (98%) mix for 2-3 min) and injected in the footpad
with 20 µl of histamine (90 µg) or 20 µl PBS into the contralateral hindpaw. Paw
thickness in both hindpaws was determined using a caliper (Mitutoyo) before injection
and at 15 and 30 min post-injection. The fold increase in thickness was calculated for
both paws (histamine- and PBS-injected) and the difference in swelling between the
histamine-injected paw as compared to the PBS-injected paw calculated for each time
point and expressed as percentage. *p<0.05 between WT and SphK1-/-SphK2+/- mice
using a two way ANOVA test. D) Changes in vascular permeability in the skin. Evans
blue dye (200 µl of 0.2%) was injected i.v. into the various mice strains. Compound
48/80 (C48/80) (100 µg in 20 µl), a known secretagogue for mast cells, was then injected
intradermally in one of the ears of the various mice strains, while the contralateral ear
was injected with 20 µl of PBS. C48/80 induces the release of mediators from mast cells
that increase the permeability of the local vascular beds and cause edema. After 30 min,
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
6
mice were euthanized and their ears collected, and plasma exudation measured by the
content of blue dye in the ears. The dye was extracted from the minced ears by
incubation for 1h at 55oC with 700 µl of formamide, and the amount of dye determined
by absorbance at 620 nm. *p<0.05 and **p<0.01 compared to the respective baseline
measurements as determined by Student’s t-test.
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
7
Supplemental Figure 5. Glomerular filtration rates in anesthetized mice during
histamine-induced anaphylaxis. Bar graph of GFR values measured between 5 to 60 min
after histamine injection (5 µmol) in anesthetized WT (n=6), SphK1-/- (n=6) and SphK2-/-
mice (n=6). Also shown is the GFR of SphK1-/- mice that were treated with S1P post-
histamine (10 min) administration (n=3). It is important to note that 3 out of the 6 SphK1-/-
mice showed no measurable GFR while all of the WT had values between 131 and 273
µl/min.
WT SphK1-/- SphK2-/- SphK1-/- (+ S1P)0
50
100
150
200
250
300p=0.088
GF
R [µ
l/m
in]
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
8
Supplemental Figure 6. Blood pressure of anesthetized WT and S1PR2-/- mice. Mean
arterial pressure (MAP) before (10 min) and after (30 min) histamine injection (5 µmol)
did not reveal any differences in anesthetized S1PR2-/- (n=5) versus WT mice (n=5).
Graph lines connect mean values of MAP measured at 2 min intervals. *p<0.05
compared to baseline measurements.
-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 300
10
20
30
40
50
60
70
80
90
WT (n = 5)
S1PR2 -/- (n = 5)
injection
Time (min)
MA
P [
mm
Hg
]
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
9
Supplemental Figure 7. Changes in the heart and breathing rates of SphK-/- and S1PR2-/-
mice during histamine-induced-anaphylaxis. Heart (A and B) and breathing (C and D)
rates were recorded using a pulse oximeter with a collar clip sensor both at baseline and
after injection of histamine (5 µmol) at 30, 60 and 120 min in conscious SphK1-/-, /- (A and
C, n=6) and S1PR2-/- mice (B and D, n=5) and compared to their appropriate WT
counterparts (n=4 or 5). *p<0.05 compared to baseline measurements.
baseline post-30 min post-60 min post-120 min500
550
600
650
700
750
800
850WT (n = 5)
SphK1 -/- (n = 6)
SphK2 -/- (n = 6)
****
***
* **
He
art
Ra
te [
bp
m]
baseline post-30 min post-60 min post-120 min100
150
200
250
300WT (n = 5)
SphK1 -/- (n = 6)
SphK2 -/- (n = 6)
*** **
p < 0.05
**
Bre
ath
ing
Ra
te [
bre
ath
s/m
in]
baseline post-30 min post-60 min post-120 min500
550
600
650
700
750
800
850WT (n = 4)
S1PR2 -/- (n = 5)
***
****
****
p < 0.05 *
He
art
Ra
te [
bp
m]
baseline post-30 min post-60 min post-120 min100
150
200
250
300WT (n = 4)
S1PR2 -/- (n = 5)
**
* *
Bre
ath
ing
Ra
te [
bre
ath
s/m
in]
A B
C D
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
10
Supplemental Figure 8- Changes in vascular permeability in S1PR1loxP/loxP-Mx mice
during systemic anaphylaxis and local inflammation and effects of S1PR1 inhibition in
SphK-/- and S1PR2-/- mice during histamine-induced anaphylaxis. A) Exudation of
vascular fluids into the lungs and peritoneum after induction of systemic anaphylaxis by
IgE/Ag. Mice (n=4 ) were injected (i.v.) with DNP-specific IgE (3 µg) and after 24 hrs,
mice were challenged with Ag (500 µg DNP36-HSA) to induce systemic anaphylaxis and
20 mg/Kg Evans blue dye to examine plasma leakage. After 30 min, the peritoneal lavage
was collected and mice were perfused with saline though the right ventricle until blood
was visibly eliminated. The amount of Evans blue in the peritoneal lavage and lungs was
determined as described in Supplemental figure 4. B) Changes in hematocrit during
WT-loxP/loxP
S1PR1-loxP/loxP-Mx
0
5
10
15
Pe
rme
ab
ility
Lu
ng
[p
g/g
we
t tis
su
e]
WT-loxP/loxP S1PR1-loxP/loxP-Mx0
2
4
6
8
10
12
Ch
an
ge
in H
em
ato
cri
t
WT-loxP/loxP S1PR1-loxP/loxP-Mx0
1
2
3
Pe
rme
bili
ty P
eri
ton
eu
m [p
g/m
l]
0 5 10 15 20 25 30 350
5
10
15
20
25
WT-loxP/loxP
S1PR1-loxP/loxP-Mx
*
Time (min)
Pa
w S
we
llin
g (%
inc
rea
se
)
PBS C48/800.00
0.04
0.08
0.12
0.16
0.20
0.24
0.28
WT-loxP/loxP
S1PR1-loxP/loxP-Mx
Ev
an
s B
lue
ex
tra
cte
d (
A6
20
)
0 5 10 15 20 25 30 35 40 45 50 55 60 65
-8
-7
-6
-5
-4
-3
-2
-1
0
SphK2-/-
SphK2-/- (VPC23019)
***
Time (min)
Te
mp
era
ture
Ch
an
ge
s (
oC
)
0 5 10 15 20 25 30 35 40 45 50 55 60 65
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
SphK1-/-SphK2+/-
SphK1-/-SphK2+/- (VPC23019)
Time (min)
A B C
D
E
F G0 5 10 15 20 25 30 35 40 45 50 55 60 65
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
S1PR2-/-
S1PR2-/- (VPC23019)
Time (min)
0 5 10 15 20 25 30 35 40 45 50 55 60 65
-14
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
S1PR2-/- S1PR1 -loxP/loxP-Mx
S1PR2-/- S1PR1 -loxP/loxP
Time (min)H I
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
11
histamine-induced anaphylaxis. Hematocrits before and after 30 min of histamine-
induced anaphylaxis were measured as described in Supplemental figure 6. The results
are expressed as ∆increasein hematocrit (n= 4 to 5). C) Paw edema induced by injection
of histamine into the footpad. Mice (n=7 to 9) were anesthetized (isoflurane (2%):oxygen
(98%) mix for 2-3 min) and injected in the footpad with 20 µl of histamine (90 µg) or 20
µl PBS into the contralateral hindpaw. Paw thickness were measured as described in
Supplemental figure 6. *p<0.05 between WTloxP/loxP-Mx and S1PR1loxP/loxP-Mx mice using a
two way ANOVA test. D) Changes in vascular permeability in the skin. Vascular leakage
was determined by quantifying the Evans blue dye extravasated into the ear 30 min after
injection of the secretagogue C48/80 as described in Supplemental figure 4. E)
Immunostaining showing reduced staining of S1PR1 in the endothelium of the aorta.
Cross sections of aorta from WTloxP/loxP-Mx and S1PR1loxP/loxP-Mx were immunostained for
isolectin B4 (green), a marker for endothelial cells, and S1PR1 as described previously
(Allende, M.L, Yamashita, T., and Proia, R.L. Blood 15:3665-3667, 2003). F-H) Effects
of the inhibition of S1PR1 by VPC23019 on histamine-induced anaphylaxis in SphK2-/-
(F)(n=5-7), SphK1-/- SphK2+/- (G)(n=3) and S1PR2-/- mice (H) (n=4-6). VPC23019, an
S1PR1/S1PR3 inhibitor, was injected into the mice i.v. (0.4 mM in 200µl) 10 min prior to
the histamine injection. Body temperature was monitored as described in Methods.
VPC23019 at 0.8 mM did not induce any further effects on body temperature on SphK2-/-
mice as compared to 0.4 mM VPC23019. I) Effect of histamine on body temperature in
S1PR2-/- /S1PR1loxP/loxP-Mx double knockout mice. Anaphylaxis by histamine in these
mice was determined as described in Methods. (*p<0.05 and **p<0.01 was determined
using a two way ANOVA).
A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis
12