A. Olivera et al. Research Article J Clin Invest S1P and ...A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis 5 ml formamide. The dye accumulated in

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


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

)


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


0.002

0.004

0.006

0.008

Re

lati

ve

E

xp

res

sio

n

Lung


0.02

0.04

0.06

0.08

0.10

Re

lati

ve

E

xp

res

sio

n

Kidney


0.0002

0.0004

0.0006

0.0008

Re

lati

ve

E

xp

res

sio

n

Spleen


0.02

0.04

0.06

0.08

Re

lati

ve

E

xp

res

sio

n

A B C

D E


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


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,


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.


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]


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

]


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]


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]


550

600

650

700

750

800

850WT (n = 4)

S1PR2 -/- (n = 5)

***

****

****

p < 0.05 *

He

art

Ra

te [

bp

m]


150

200

250

300WT (n = 4)

S1PR2 -/- (n = 5)

**

* *

Bre

ath

ing

Ra

te [

bre

ath

s/m

in]

A B

C D


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


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).


12

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A. Olivera et al. Research Article J Clin Invest S1P and ...A. Olivera et al. Research Article J Clin Invest S1P and Recovery from Anaphylaxis 5 ml formamide. The dye accumulated in