Exogenous Sphingosine 1-Phosphate

Protects Murine Splenocytes Against

Hypoxia-Induced Injury

Sonam Chawla, Chayanika Sahni,

Rajkumar Tulsawani, Mrinalini Singh,
Deepika Saraswat, Anju Bansal & Shweta
Saxena
Lipids

ISSN 0024-4201

Lipids
DOI 10.1007/s11745-013-3860-9

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 Author's personal copy
Lipids
DOI 10.1007/s11745-013-3860-9

ORIGINAL ARTICLE

Exogenous Sphingosine 1-Phosphate Protects Murine Splenocytes

Against Hypoxia-Induced Injury
Sonam Chawla • Chayanika Sahni •
Rajkumar Tulsawani • Mrinalini Singh •
Deepika Saraswat • Anju Bansal • Shweta Saxena

Received: 9 May 2013 / Accepted: 21 October 2013

Ó AOCS 2013

Abstract Sphingosine-1-phosphate (S1P), a biologically
active pleiotropic lipid, is involved in several physiological
processes especially in the area of vascular biology and
immunology encompassing cell survival, angiogenesis, vas-
cular tone, immune response etc. by interacting with specific
cell surface receptors. Hypoxia, a condition common to
innumerable pathologies, is known to lethally affect cell
survival by throwing off balance global gene expression,
redox homeostasis, bioenergetics etc. Several molecular
events of cellular adaptations to hypoxia have been closely
linked to stabilization of hypoxia inducible factor-1a (HIF-
1a). Signalling functions of S1P in physiological events
central to hypoxia-induced pathologies led us to investigate
efficacy of exogenous S1P in preconditioning murine
splenocytes to sustain during cellular stress associated with
sub-optimal oxygen. The present study recapitulated the pro-
survival benefits of exogenous S1P under normobaric
Electronic supplementary material The online version of this
article (doi:10.1007/s11745-013-3860-9) contains supplementary
material, which is available to authorized users.
S. Chawla  C. Sahni  M. Singh  D. Saraswat  A. Bansal 
S. Saxena (&)
Experimental Biology Division, Defence Institute of Physiology
and Allied Sciences (DIPAS), Ministry of Defence,
Defence Research and Development Organisation (DRDO),
Lucknow Road, Timarpur, Delhi 110054, India
e-mail: shweta.dipas@gmail.com
C. Sahni
Amity Institute of Biotechnology, Amity University,
Noida, Uttar Pradesh, India
R. Tulsawani
PACT Division, Defence Institute of Physiology and Allied
Sciences, Defence Research and Development Organisation,
Lucknow Road, Timarpur, Delhi 110054, India
hypoxia. Results indicate a direct effect of S1P supplemen-
tation on boosting cellular adaptive responses via HIF-1a
stabilization and, activation of pro-survival mediators ERK
and Akt. Overwhelming anti-oxidative and anti-inflamma-
tory benefits of S1P preconditioning could also be captured
in the present study, as indicated by improved redox
homeostasis, reduced oxidative damage, balanced anti/pro-
inflammatory cytokine profiles and temporal regulation of
nitric oxide secretion and intra-cellular calcium release.
Hypoxia induced cell death and the associated stress in cel-
lular milieu in terms of oxidative damage and inflammation
could be alleviated with exogenous S1P preconditioning.
Keywords Sphingosine-1-phosphate 
Pro-survival  Hypoxia  Anti-oxidant 
Anti-inflammatory  HIF-1a
Abbreviations
HIF-1a Hypoxia inducible factor-1a
OD Optical density
ROS Reactive oxygen species
RFU Relative fluorescence units
S1P Sphingosine 1-phosphate
SOD Superoxide dismutase
TBARS Thiobarbituric acid reactive substances
Introduction
Hypoxia is a hallmark of diverse pathologies, whereby the
cellular demand of oxygen outstrips the supply. This defi-
ciency of oxygen limits aerobic respiration and energy
generation; disrupts the cellular redox balance propelling the
milieu towards inflammation and apoptosis/necrosis [1]. The
cellular machinery thus resorts to adaptive biochemical and

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molecular re-programming responses which shield the cell
from hypoxia and associated maladies [2, 3].
Sphingosine 1-phosphate (S1P), a bioactive lipid, pres-
ent in the systemic circulation in the nanomolar range is
synthesized and released by platelets and red blood cells
primarily [4]. It exerts secondary messenger effects to
affect cell survival, migration, angiogenesis, intracellular
calcium concentrations and inflammation in endocrine and
paracrine manner, although intracrine actions have also
been suggested recently [5]. S1P turn-over is majorly
controlled by sphingosine kinase (SphK) 1 and 2, and S1P
lyase [6]. Moreover, the relative intracellular concentration
of S1P along with its precursor ceramide, constitute the
‘‘sphingolipid rheostat’’, which is an important regulator of
survival and redox balance in the cell during ischemia-
reperfusion injury [7, 8]. The pro-survival effect of S1P
spans a wide range of cell types exposed to hypoxia—S1P
supplementation to cardiomyocytes mimics ischemic pre-
conditioning and preserves viability over a period of
18–20 h via enhanced activation of survival signals—ERK
and Akt [9, 10], in endothelial and smooth muscle cells the
pro-survival hypoxia inducible factor-1a (HIF-1a) stabil-
ization could be brought about by micromolar quantities of
S1P supplementation [11], in cancerous cells death resis-
tance is associated with up-regulated Sphk1/S1P [12].
Cells having proliferative activity are, in general, the more
hypoxia sensitive therefore the lymphohematopoietic system,
including the spleen and thymus, is known to be adversely
affected by exposure to hypobaric hypoxia [13]. Considering
that results in primary cultured cells can be closely correlated
with hypoxia pathogenesis in vivo, splenocytes such as lym-
phocytes and granulocytes are a good in vitro model to assess
the pre-conditioning potential of S1P to alleviate hypoxia
mediated injuries to lymphohematopoietic system.
In the present study, we hypothesize that exogenous S1P
supplementation can propel the molecular milieu of
splenocytes toward a pro-survival outcome; along with
conferring an anti-oxidative and anti-inflammatory pro-
tection and adaptive cellular physiological state which may
assist splenocytes to successfully cope with hypoxia asso-
ciated cellular injuries. Thus the objective of this study was
to investigate the efficacy of exogenous S1P in precondi-
tioning splenocytes to endure prolonged durations of sub-
optimal oxygen levels, a condition experienced during
exposure to environmental or pathological hypoxia.
Materials and Methods
Materials
S1P, RPMI-1640 and fetal bovine serum were purchased
from Sigma Aldrich., ELISA kits and biochemical
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estimation kits were purchased from BD Biosciences,
Biovision, Inc., eBiosciences, Cayman, Invitrogen and
R&D Systems.
S1P Treatment and Hypoxia Exposure of Splenocytes
The present study was approved by the DIPAS Animal
Care and Use Committee (Project No: DIP 251 A2.3).
Mouse spleens were aseptically removed from 6–8-week-
old Balb/c mice obtained from the DIPAS experimental
animal facility. Single-cell suspensions were obtained by
using a cell strainer and erythrocytes were lysed in
ammonium chloride-potassium (ACK) buffer (150 mM
NH4Cl and 10 mM KHCO3). Following washing with
incomplete RPMI-1640 medium (Sigma Aldrich) these
purified cells were used for experiments and were seeded in
culture flasks at a seeding density of 40 9 106/T-75 flask
(BD Biosciences) and 1 9 106 cells/well in a round-bottom
96 well culture plates (BD Biosciences) in RPMI-1640
media supplemented with 10 % fetal bovine serum. Cells
were treated with different doses of S1P (50, 100, 200, 400
and 800 nM) 30 min prior to exposure to hypoxia or nor-
moxia for 24 and 48 h durations. The hypoxia exposure
conditions were 0.5 % O2/5 % CO2/37 °C. S1P was pre-
pared as 1 mM stock in 10 mM NaOH which was diluted
to required concentrations using 0.1 % BSA in normal
saline (pH 7.8) (vehicle).
MTT Assay and Pro-survival Indicators
Cells seeded in 96-well round-bottom plate were treated
with 0.5 mg/ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyl tetrazolium bromide (MTT) for 3 h at the end of
hypoxia exposure. Formazan crystals formed in the viable
cells were dissolved with DMSO and the optical density
(OD) of each well was measured at 570 nm [14]. Survival
percentages were calculated as: survival percentage = (OD
of drug group/OD of control group) 9 100. Status of
p-ERK1(T202/Y204)/p-ERK2(T185/Y187) (KCB1018,
R&D systems), p-Akt (S473) (KCB887, R&D systems)
was estimated using cell-based ELISA as indicated by the
manufacturer’s protocol to substantiate the pro-survival
benefits observed. ATP levels in the cell lysate were
evaluated to assess the bioenergetics status of surviving
cells, using the kit (A22066, Invitrogen) protocol.
Cell Lysate Preparation
Following exposures, 80 9 106 cells/T-75 cultures were
washed with PBS (pH 7.4) and dispersed in RIPA lysis
buffer (fortified with protease inhibitor cocktail and phos-
phatase inhibitor cocktail, SIGMA) for 30 min at 4 °C.
Cell lysates were centrifuged at 9,000g for 20 min to
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remove the cell debris and the supernatants were stored at
-20 °C for further analysis [15]. Protein estimations were
carried out using Lowry’s method [16].
Western Blot Analysis
Western blot analysis for p-ERK1/2, ERK1/2, p-Akt1/2/3
and Akt1/2/3 was performed to substantiate the ELISA data.
The protein sample for SDS-PAGE was prepared by mixing
cell lysate with 69 Laemelli buffer (0.25 M Tris–HCl pH
6.8, 10 % SDS, 0.5 % bromophenol blue, 0.5 M di-dithio-
threitol and 50 % glycerol) and boiling the samples for
10 min. Then, 50 lg protein was loaded and resolved on a
12 % acrylamide gel. Resolved proteins were blotted on the
nitrocellulose membrane and blocked overnight in 5 %
BSA solution. The blocking solution and antibody dilutions
were prepared in Tris-buffered saline with 0.1 % Tween-20.
Blots were incubated with anti-ERK1/2 (#4695, Cell Sig-
nalling Technologies, Inc.), anti-pERK1/2(Thr202/Tyr204)
(#4377, Cell Signalling Technologies, Inc.), anti-Akt1/2/3
(sc-8312, Santa Cruz) and anti-pAkt1/2/3 (Ser 473) (sc-
7985. Santa Cruz) overnight and then with anti-goat HRP
labelled secondary antibody (1:30,000, Santa Cruz) for 2 h,
at room temperature. An enhanced chemiluminescence
detection kit (SIGMA) was used to develop the blots and
they were captured on X-ray film. Loading control used was
a-tubulin (1:1,000, Santa Cruz). Densitometric analysis was
done using Image J software.
Hypoxia Inducible Factor-1a (HIF-1a) Quantification
The intracellular HIF-1a level in the cell lysates was
quantified using total HIF-1a DuoSet IC kit using the
manufacturer’s protocol and reported as picograms of HIF-
1a per mg protein (DYC1935-5, R&D Systems). Intracel-
lular HIF-1a level are indicative of pro-adaptive hypoxia
responsive transcriptional changes.
Oxidative Stress
Intracellular reactive oxygen species (ROS) formation was
measured using the 20 ,70 -dichloro-fluorescein diacetate
(DCFH-DA) method [17]. Briefly, splenocytes were seeded
in 96-well black plates, treated with increasing S1P doses
and after the completion of hypoxia exposure DCFH-DA
was added to a final concentration of 25 lM per well and
the plates were incubated for 30 min at 37 °C and fluo-
rescence was measured at 485-nm excitation and 530-nm
emission, hydrogen peroxide (75 lM) was used as the
positive control for the assay and wells with no DCFH-DA
were used to measure background fluorescence which was
subtracted from each reading. Controls with no cells were
also analyzed and display no increased fluorescence over
time. ROS-mediated damage to cellular components was
also measured by estimating lipid peroxidation and protein
carbonylation. Lipid peroxidation was determined in the
cell lysate by measurement of thiobarbituric acid reactive
substances (TBARS) using a QuantiChromTM TBARS
assay kit (DTBA-100). Protein carbonyl content was
determined in the cell lysate by 2,4-dinitrophenylhydrazine
(DNPH) method [18]. Cellular anti-oxidant enzyme
defenses—superoxide dismutase (SOD) and catalase, were
estimated by using superoxide dismutase activity kit (Cat
No: K335-100, BioVision) and catalase activity kit (Cat
No: 707002, Cayman Chemical) in the cell lysate.
Cytokine Profile
Secretion of pro-inflammatory and anti-inflammatory cyto-
kines was assessed in media supernatant using commercial
ELISA kits—interleukin-6 (IL-6), interleukin-10 (IL-10),
interleukin-1b (IL-1b), interferon-c (IFN-c), interleukin-4
(IL-4), transforming growth factor-b (TGF-b), INTERLEU-
KIN-2(IL-2) (BD OptEIATM), TNF-a (eBiosciences), vas-
cular endothelial growth factor (SMMV00, R&D Systems).
Nitric Oxide Release and Intracellular Calcium
Mobilization
Nitrite content, an indicator of nitric oxide release, in the
culture media was estimated using the Griess reaction [19].
Arginase activity, which shares L-arginine as a common
substrate with nitric oxide synthase (NOS) thus recipro-
cally regulates NOS activity, was measured in cell lysate
using the standard method. Briefly, samples were incubated
with activation buffer (10 mM MnCl2 in 50 mM Tris-HCl,
pH 7.5) to activate the arginase, and then with 0.5 M
arginine (pH 9.7) at 37 °C for 1 h. Reaction was terminated
by addition of an acid solution and urea formation was
detected using a iso-nitrosopropiophenone solution, the
absorbance was recorded at 540 nm [20]. The intracellular
calcium level was estimated using a fluo-4AM based
detection kit using the manufacturer’s protocol (F36206,
Invitrogen). Briefly, post-exposure cells were washed and
incubated with fluo-4 for 30 min at 37 °C and at room
temperature for another 30 min and fluorescence was
measured at an excitation of 430 nm and an emission of
540 nm. Data represented as Relative fluorescence units
(RFU)/well, being proportional to Ca2? binding to fluo-4.
Statistical Analysis
All experiments were performed at least in triplicate (n = 3)
and all data were expressed as means ± standard devia-
tions. For multiple groups’ comparison, data were analyzed
by one-way ANOVA/Post hoc Bonferroni’s. Significance
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has been denoted as * indicates p B 0.01, # indicates
p B 0.05, $ indicates p B 0.001 vs. normoxia vehicle con-
trol, ** indicates p B 0.01, ## indicates p B 0.05, $$ indi-
cates p B 0.001 vs. hypoxia vehicle control.
Results
S1P Promotes pERK and pAKT Mediated Survival
of Splenocytes
Hypoxia exposure up to 48 h caused a 30–40 % fall in
splenocytes viability in comparison to the normoxia con-
trols. The pro-survival effect of S1P, in the range of
50–800 nM, under hypoxia was used as a screening assay
to determine the effective dose range. S1P supplementation
at 50 and 100 nM doses had minimal or no effect on cell
survival, whereas 200 nM dose conferred maximum pro-
tection to splenocytes against hypoxia-induced cell death
and led to a twofold increase in cell viability at 24 h
(p B 0.001) and a 1.6-fold increase at 48 h (p B 0.001) of
hypoxia exposure (Fig. 1a). Since 200 nM was the lowest
dose with a maximum protective effect observed in the cell
viability assay, further studies were conducted in the range
of 200–800 nM S1P dose. The cyto-protective effect was
supported by a concomitant increase in phosphorylation
and activation of ERK1/2 (Fig. 2a) and Akt1/2/3 (Fig. 2b)
while no significant changes were observed in the basal
levels of ERK1/2 and Akt1/2/3. A significant boost in the
cellular ATP content was also observed, maximally at
200 nM S1P dose (Fig. 1b).
HIF-1a Expression
S1P induced stabilization of HIF-1a under normoxic con-
ditions (p B 0.001) which was further boosted upon
hypoxia exposure for 24 and 48 h (p B 0.001). A dose of
200 nM S1P showed the strongest boost in HIF-1a stabil-
ization under hypoxia (Table 1).
Anti-oxidant Activity of S1P
Exposure of splenocytes to sub-optimal oxygen imposed
severe oxidative stress as indicated by significant increase
in ROS, TBARS and protein carbonyl content. Precondi-
tioning the splenocytes with S1P, prior to hypoxia expo-
sure, could boost redox homeostasis by lowering
intracellular ROS generation, TBARS and protein carbonyl
formation in all the dose groups at the end of 24 h.
Although, S1P could reduce TBARS formation in all the
dose groups following 48 h of hypoxic stress, only 200 nM
dose could prevent protein oxidation effectively (Fig. 3a–
c). Exogenous S1P could attenuate the ROS generation by
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boosting the anti-oxidant enzyme system as evidenced by
an increase in SOD and catalase activities at 24 and 48 h of
hypoxia exposure (Fig. 3d).
Pro-inflammatory and Anti-inflammatory Cytokine
Response
Hypoxia exposure of splenocytes caused up regulation of
pro-inflammatory cytokine levels (TNF-a, IFN-c, IL-b and
IL-6, IL-2 TGF-b and VEGF) both at 24 and 48 h durations
(Fig. 4a–h and Supplementary Fig. 3a–h). Preconditioning
of splenocytes with exogenous S1P, dose dependently,
down regulated pro-inflammatory cytokines, along with
boosting anti-inflammatory cytokine response (IL-10)
(Fig. 4a–h). No change was observed in IL-4 levels in
hypoxia controls as well as following S1P supplementation
in comparison to normoxia control (data not shown).
Secretory Nitric Oxide Response and Intracellular
Calcium Release
S1P mediated regulation of nitric oxide secretion was
confirmed by differential temporal profiles of nitrite level
and arginase activity. Interestingly, exogenous S1P caused
increased nitrite content and decreased arginase activity at
24 h while this profile was reversed at 48 h. The effect was
most significant at 200 nM dose group (Table 2). [Ca2?]i
mobilization, in the presence of exogenous S1P, followed a
similar differential temporal pattern as nitric oxide release.
Both 200 and 400 nM dose groups maintained the [Ca2?]i
at the same levels (p B 0.05) following 24 and 48 h of
hypoxia exposure (Fig. 5).
Discussion
The present study was undertaken to evaluate the protec-
tive efficacy of exogenous S1P in alleviating hypoxia-
induced stress in murine splenocytes. Cellular hypoxia is
known to be intimately associated with ATP depletion,
ROS generation, inflammation, and mitochondrial release
of cytochrome c, leading to apoptotic cell death as a ter-
minal stress response [2, 3]. In the current study, hypoxia-
induced cell death of murine splenocytes could be reduced
in the presence of exogenous S1P, as an outcome of
enhanced activation of AKT1/2/3 and ERK1/2 and
increased ATP levels, most effectively at 200 nM S1P dose
(Fig. 1b). Although, S1P induced a boost in cell survival
under normoxia as well, which could be owing to its known
mitogenic properties, however a further robust cell survival
under severely stressful condition like hypoxia appears to
be cytoprotective rather than mitogenic (Supplementary
Figs. 1, 2a, b).
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Fig. 1 Pro-survival action of S1P: S1P supplementation preserves
viability under hypoxia. a Cell viability was determined in S1P
supplemented splenocytes by the MTT assay 24 and 48 h after
hypoxia exposure and in parallel vehicle controls. Data is presented as
percentage survival relative to the respective normoxia vehicle
control cells for each time point (survival percentage = OD of drug
group/OD of control group). Splenocytes were pre-treated with
50–800 nM S1P and the optimum dose range was determined for
further assays. S1P at the dose of 200 nM shows maximal cyto-
protection. b Intracellular ATP levels were estimated in cell lysates of
S1P supplemented (200, 400, 800 nM) splenocytes and respective
Studies have indicated that exogenous S1P benefits cells
under hypoxic stress via HIF-1a stabilization [11] and this
phenomenon was recapitulated in our experiments where
significant HIF-1a levels were observed following pre-
conditioning with exogenous S1P. Further, Sphk1 is known
to be an upstream activator of HIF-1a and also it has been
proved that exogenous S1P stimulates SphK1 activity, a
robust pro-survival signal [21]. In the present study, since
HIF-1a stabilization and cell survival effects were
observed in the S1P preconditioning group, we hypothesize
that this exogenous S1P might have facilitated viability by
stimulating SphK1 activity, thereby raising intracellular
S1P which when exported out is known to affect NF-kB
vehicle controls under normoxic and hypoxic conditions (NVC
normoxia vehicle control, HVC hypoxia vehicle control, 24 H 24 h
of hypoxia exposure, 48 H 48 h of hypoxia exposure). Values are
given as means ± SD of n = 3 independent experiments and
triplicate measurements and one-way ANOVA/Post hoc Bonferroni’s
analysis was used for calculating statistical significance (# indicates
p B 0.05, * indicates p B 0.01 and $ indicates p B 0.001 vs.
normoxia vehicle control and ** indicates p B 0.01, ## indicates
p B 0.05, $$ indicates p B 0.001 vs. hypoxia vehicle control) (NVC
normoxia vehicle control, HVC hypoxia vehicle control, 24 H 24 h of
hypoxia exposure, 48 H 48 h of hypoxia exposure)
mediated pro-survival signalling via cell surface S1P
receptors [22]. We further propose that since this mecha-
nism may not be noticeable, if cells are stimulated with
overwhelming concentrations of S1P which might preoc-
cupy all the receptors leaving no available binding sites for
the intracellular S1P exported out of the cells, a possible
explanation for the optimum effects of the 200 nM S1P
dose.
HIF-1a facilitates adaptation to hypoxia by metabolic
reprogramming and overcoming the energy deficit due to
sub-optimal oxygen availability [23, 24]. In the present
study, the S1P induced HIF-1a stabilization facilitated
survival of hypoxia exposed splenocytes by regulating bio-
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Fig. 2 S1P mediated activation of pro-survival indicators ERK1/2
and Akt1/2/3 under hypoxia: phosphorylation and activation of
ERK1/2 at Thr202/Tyr204 and Akt at Ser473 residue is a known pro-
survival indicator. The activation states of ERK1/2 and Akt in S1P
supplemented (200, 400, 800 nM) cells and respective vehicle control
splenocytes post-normoxic/hypoxic exposure, was determined using
cell based ELISA, as well as using standard immunoblotting and
densitometry of blots using Image J (see the ‘‘Materials and Methods’’
section). a Activation of ERK1/2 on S1P supplementation. i Cell
based ELISA to detect ERK1/2 phosphorylation and basal levels of
ERK1/2, performed in 2 9 106 cells for each group. Estimation was
reported in relative fluorescence units (RFU)/2 9 106 cells. ii Rep-
resentative immuno-blot of ERK1/2 phosphorylation at Thr202/
Tyr204 residue and basal ERK1/2 levels following S1P precondi-
tioning of splenocytes prior to hypoxia exposures. Densitometric
analysis of blots was normalized against the loading control (a-
tubulin) and is reported as the fold change from the normoxia vehicle
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control (NVC) of the respective time point-24/48 h. b Activation of
Akt1/2/3 on S1P supplementation. i Cell based ELISA to detect Akt1/
2/3 phosphorylation and basal levels of Akt1/2/3, performed in
2 9 106 cells for each group. Estimation was reported in relative
fluorescence units (RFU)/2 9 106 cells. ii Representative immune-
blot of Akt1/2/3 phosphorylation at Ser473 residue and basal Akt1/2/3
levels following S1P preconditioning of splenocytes prior to hypoxia
exposure. Densitometric analysis of blots was normalized against the
loading control (a-tubulin) and is reported as fold change from
normoxia vehicle control (NVC) of the respective time point-24/48 h.
Values are given as means ± SD. One-way ANOVA/Post hoc
Bonferroni’s analysis was used for calculating statistical significance
(# indicates p B 0.05, * indicates p B 0.01, $ indicates p B 0.001 vs.
normoxia vehicle control and ** indicates p B 0.01, ## indicates
p B 0.05, $$ indicates p B 0.001 vs. hypoxia vehicle control) (24 H
24 h of hypoxia exposure, 48 H 48 h of hypoxia exposure, NVC
normoxia vehicle control, HVC hypoxia vehicle control)
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Fig. 2 continued
energetic homeostasis as evidenced by a manifold increase
in ATP levels in the 200 nM S1P supplemented cells even
after 48 h of hypoxic stress. Further, HIF-1a stabilization is
tightly coupled to the cellular redox status. A low level of
ROS is essential as a signalling moiety in the cell, acti-
vating HIF-1a at initial stages of hypoxic stress [25]. But
when the intracellular ROS exceeds the anti-oxidant buffer
in the cellular milieu, the highly reactive ROS can cause
damage to cellular proteins, lipids and DNA. Direct oxi-
dation of lysine, arginine, proline, and threonine residues
may yield carbonyl derivatives and presence of carbonyl
groups in proteins has been used as a marker of ROS-
mediated protein oxidation [26]. Lipids are another cellular
component vulnerable to ROS induced peroxidation
leading to generation of malondialdehyde (MDA) [27]. In
the present study, both 24 and 48 h of hypoxia exposure
caused immense oxidative damage to cellular components
as indicated by the accumulation of protein carbonyls and
MDA in the splenocytes (Fig. 3b, c). But the S1P-induced
boost in anti-oxidant enzyme activities could protect these
cells against protein and lipid oxidation due to the resultant
reigning of intracellular ROS generation and was show-
cased by a significant reduction in protein carbonyls and
TBARS (Fig. 3d). Most importantly, the steeper fall in
intracellular ROS production at 48 h exposure following
S1P treatment could be an outcome of a parallel and
stronger boost in anti-oxidant enzymes SOD and catalase
compared to 24 h hypoxia exposure (Fig. 3a). Finally, it is
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Table 1 S1P induced HIF-1a stabilization under normoxia and hypoxia

S1P (nM) HIF-1 a (pg/mg protein) accumulation
Hypoxia Normoxia
24 h 48 h 24 h 48 h

Vehicle control 137.07 ± 2.48 160.27 ± 3.28 51.67 ± 3.57 71.98 ± 9.31
200 305.64 ± 3.78$ 313.86 ± 7.65$ 205.83 ± 7.21$ 215.21 ± 4.24$
400 240.36 ± 6.25$ 305.67 ± 7.45$ 297.07 ± 7.44$ 218.86 ± 8.42$
800 205.42 ± 1.23$ 307.53 ± 3.42$ 199.01 ± 2.80$ 227.75 ± 2.45$
Cell lysate from 80 9 106 cells supplemented with various doses of S1P and exposed to 24 and 48 h of hypoxia/normoxia was analyzed via
sandwich ELISA (see the ‘‘Materials and Methods’’ section) for HIF-1a accumulation. Data are reported as picograms of HIF-1a per mg protein
and representative of three independent experiments carried out in triplicate. Statistical significance was calculated using one-way ANOVA/Post
hoc Bonferroni’s analysis and is denoted as # indicates p B 0.05, * indicates p B 0.01 and $ indicates p B 0.001 vs. respective vehicle control

Fig. 3 Anti-oxidant action of S1P: a intracellular ROS generation
was measured by incubating S1P supplemented (200–800 nM)
splenocytes in a 96-well plate with a DCFH-DA probe for 1 h after
the hypoxia/normoxia exposure. Data are reported as relative
fluorescence units (RFU)/well proportional to the DCF formed per
well. Each point is the mean ± SD. This is a representative
experiment of at least three experiments conducted in quadruplicate
wells. Intracellular damage and the anti-oxidant enzyme activities
were measured in whole cell lysate prepared from approximately
80 9 106 cells treated with various doses of S1P (200–800 nM) and
control cells. b Protein oxidation was measured by detecting carbonyl
groups using DNPH and indicated by micromolar/mg protein. c Lipid
peroxidation was measured as micromoles of TBARS formed per mg
of protein. d SOD and catalase activities were measured in the whole
cell lysate and reported as the fold change in anti-oxidant enzymes
calculated for each drug group against the normoxia vehicle control
group enzyme activity. Data are reported as means ± SD from three
independent experiments carried out in triplicate. One-way ANOVA/
Post hoc Bonferroni’s analysis was used for calculating statistical
significance (# indicates p B 0.05, * indicates p B 0.01, $ indicates
p B 0.001 vs. normoxia vehicle control and ** indicates p B 0.01,
##
indicates p B 0.05, $$ indicates p B 0.001 vs. hypoxia vehicle
control). (NVC normoxia vehicle control, HVC hypoxia vehicle
control, 24 H 24 h hypoxia, 48 H 48 h hypoxia)

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Fig. 4 Anti-inflammatory action of S1P: anti-inflammatory shift in
the secreted cytokines profile of S1P supplemented (200–800 nM)
splenocytes exposed to 24 and 48 h of hypoxia. Media supernatant
from supplemented and hypoxia/normoxia exposed 10 9 106 cells
was snap-frozen and analyzed in sandwich ELISA format according
to the kit manufacturer’s protocol (see the ‘‘Materials and Methods’’
section). The pro-inflammatory cytokines’ secretion—TNF-a, IFN-c,
IL-6, IL-1b, IL-2, TGF-b and VEGF, is attenuated on S1P supple-
mentation and the anti-inflammatory cytokine IL-10 shows a
proposed that the S1P mediated boost in the anti-oxidant
capacity of the cell, propels the milieu towards less oxi-
dizing/more reducing—contributing to HIF-1a stabiliza-
tion [28].
The last decade has encompassed several debates on
whether S1P plays a pro- or anti-inflammatory role, the
present study is one of the pioneer studies implicating S1P
in the control of inflammation under hypoxic stress. An
interesting observation made in this study was a differential
temporal boost of nitric oxide synthesis indicated by a
higher nitrite content and lower arginase activity at 24 h
whereas a reversal of nitric oxide levels at 48 h (Table 2).
Hypoxia induced [Ca2?]i release in the presence of exog-
enous S1P also followed a similar trend as nitric oxide
(Fig. 5). Limited exposure of cells to nitric oxide under
hypoxia is known to allow for full cell recovery and via-
bility, with mitochondrial membrane homeostasis being
significant boost. Values are reported as mean ± SD from three
independent experiments carried out in triplicate. One-way ANOVA/
Post hoc Bonferroni’s analysis was used for calculating statistical
significance (# indicates p B 0.05, * indicates p B 0.01, $ indicates
p B 0.001 vs. normoxia vehicle control and ** indicates p B 0.01,
##
indicates p B 0.05, $$ indicates p B 0.001 vs. hypoxia vehicle
control) (solid line 24 h of hypoxia exposure, dashed line 48 h of
hypoxia exposure, NVC normoxia vehicle control, HVC hypoxia
vehicle control
maintained through utilization of glycolytic ATP. By
contrast, extended continuous exposure to nitric oxide
activates mitochondrial-dependent apoptosis [29, 30].
Similarly, a moderate increase in [Ca2?]i is seen to be pro-
survival via biphasic activation of ERK-1/2 and a disrup-
tion of apoptotic signalling, as against the higher increases
which lead to sustained ERK1/2 activation and progression
to cell death [31]. Furthermore, nitric oxide is known to
influence the mitochondrial membrane permeability lead-
ing to a release of sequestered calcium to the cytosol and
further stimulating the pro-death signals [32, 33]. The S1P
induced restoration of cell viability during prolonged
hypoxia exposure of spleen cells observed in the current
study appears to be an outcome of this intelligent interplay
of differential nitric oxide synthesis and [Ca2?]i release.
Here we also argue for a pivotal role for S1P, as a key
mediator of the cytokine network. The concept that
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Table 2 Nitric oxide secretion regulation

S1P (nM) Nitrite (lM/mL)
[arginase (U/mg protein)]
Hypoxia Normoxia
24 h 48 h 24 h 48 h

Vehicle control 1.92 ± 0.17 1.54 ± 0.24 0.98 ± 0.12 1.76 ± 0.24
[26.23 ± 0.31] [35.75 ± 0.10] [30.13 ± 0.29$] [34.59 ± 1.54]
200 2.76 ± 0.12* 0.84 ± 0.07* 1.75 ± 0.17* 1.90 ± 0.24
[24.83 ± 0.02] [96.13 ± 0.69$] [8.81 ± 0.03$] [15.54 ± 0.03$]
400 2.29 ± 0.21# 0.96 ± 0.12* 1.57 ± 0.19* 2.04 ± 0.32
$ $ $
[18.60 ± 0.19 ] [68.62 ± 0.23 ] [7.76 ± 0.61 ] [11.41 ± 0.15$]
# *
800 2.04 ± 0.28 1.16 ± 0.09 1.57 ± 0.12 2.14 ± 0.30
[14.67 ± 1.32$] [64.58 ± 1.69$] [4.78 ± 0.12$] [40.2 ± 1.32$]
Cultures containing 80 9 106 cells, supplemented with various doses of S1P and exposed to 24 and 48 h of hypoxia/normoxia, were analyzed for
nitrite levels using the Griess method and represented as lM/mL media supernatant. Corresponding arginase activity, a reciprocal regulator of
NOS activity, was estimated and represented as the specific activity/mg protein in cell lysate. Data shown are from three independent experi-
ments carried out at least in triplicate. Statistical significance was calculated using one-way ANOVA/Post hoc Bonferroni’s analysis and is
denoted as # indicates p B 0.05, * indicates p B 0.01 and $ indicates p B 0.001 vs. respective vehicle control

exogenous S1P led to a shift in the cytokine profile from

Fig. 5 Regulation of intracellular calcium by S1P: regulation of
intracellular calcium by S1P supplementation (200–800 nM) in
splenocytes exposed to hypoxia for 24 and 48 h. 0.2 9 106 spleno-
cytes/well were seeded in black 96-well plates and treated with S1P at
various concentrations along with parallel normoxia controls. Post-
exposure, cells were washed and incubated with fluo-4 for 30 min at
37 °C and at room temperature for another 30 min and fluorescence
was measured at excitation 430 nm and emission 540 nm. Data are
reported as relative fluorescence units (RFU)/well, being proportional
to Ca2? binding to fluo-4. The values are reported as means ± SD,
from three independent experiments carried out in quadruplet. One-
way ANOVA/Post hoc Bonferroni’s analysis was used for calculating
statistical significance (# indicates p B 0.05, * indicates p B 0.01,
$
indicates p B 0.001 vs. normoxia vehicle control and ** indicates
p B 0.01, ## indicates p B 0.05, $$ indicates p B 0.001 vs. hypoxia
vehicle control). 24 H 24 h hypoxia, 48 H 48 h hypoxia, NVC
normoxia vehicle control, HVC hypoxia vehicle control
hypoxia can induce inflammation has gained general
acceptance and was recapitulated in our study [34].
Splenocytes, when exposed to hypoxia, secreted high levels
of pro-inflammatory cytokines viz. TNF-a, IL-6, IL-2,
IFN-c, TGF-b, IL-1b and VEGF (Fig. 4a–h, Supplemen-
tary Fig. 3a–h). Interestingly, preconditioning with
pro-inflammatory to anti-inflammatory. There are very few
studies reporting S1P’s potential to favor a shift towards
anti-inflammatory responses by inhibiting TNF-a, IL-12
and increasing IL-10 production in lymphocytes [35, 36],
with our present finding this proposal has been further
strengthened. Recently HIF-1a stabilization has been
attributed with propagation of anti-inflammatory responses
while down regulating pro-inflammatory responses. In the
light of existing evidences, it may be inferred that S1P
mediated HIF-1a stabilization could have modulated
cytokine expression which resulted in a pronounced anti-
inflammatory outcome [37, 38]. In another interesting
observation, the robust increase in VEGF levels following
hypoxia exposure of splenocytes was observed to be dose-
dependently reduced in the presence of S1P (Fig. 4g).
T-lymphocytes are pivotal in the initiation and progression
of inflammation and their homing to inflammatory sites in
intimate contact with endothelial cells could influence the
angiogenesis associated with inflammation [39]. Aberrant
angiogenesis and vascular leakage is a characteristic of
abnormal inflammation in hypoxia-induced pathologies. It
is conceivable that the infiltrating T cells contribute to
pathogenic angiogenesis by secreting VEGF in response to
IL-2 produced by other activated T cells [38]. VEGF
secretion further pushes T cells visiting the site into a pro-
inflammatory Th1 mode and amplifying inflammation, thus
VEGF secreted from immune effector cells is an important
signal in the dialog between the tissues and the immune
system [40, 41]. S1P induced reduction in VEGF secretion
by splenocytes, thus can very well be explained in the light
of established role of S1P in barrier integrity and

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prevention of vascular leakage [42]. Based on these results,
we are performing animal experiments to provide sup-
porting in vivo results to confirm the preconditioning
potential of S1P to alleviate hypobaric hypoxia mediated
injuries.
Conclusion
Through this study, exogenous S1P supplementation has
emerged as an effective strategy for boosting innate
physiologic adaptive responses to hypoxia exposure under
in vitro culture conditions. S1P displayed immense
potential in preconditioning the splenocytes, rendering
them more resistant to hypoxia (via enhanced accumulation
of pro-survival signals—p-ERK1/2 and p-Akt1/2/3), by
boosting HIF-1a stabilization, re-establishing redox
homeostasis and enhanced ATP accumulation, differential
regulation of nitric oxide and [Ca2?]i release, and balanc-
ing pro/anti-inflammatory cytokine pools. Building upon
this study, hypoxia pre-conditioning property of S1P can be
investigated in higher diseased models of pathologies
involving sub-optimal oxygen levels in the tissues.
Acknowledgments The authors acknowledge the Defence Research
and Development Organization (DRDO), Director, Defence Institute
of Physiology and Allied Sciences (DIPAS) and Council of Scientific
and Industrial Research, India for providing necessary facilities and
funding for this study.
Conflict of interest The author(s) declare that they have no com-
peting interests.
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