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* Lung Inflammatory Group, Center for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; and
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and the substance P analog
[D-Arg6,
D-Trp7,9,NmePhe8]-substance
P(6–11) (SP-G) stimulated a rapid increase in sphingosine kinase
activity in freshly isolated human neutrophils. This activity was
blocked by preincubation with the sphingosine kinase inhibitor
N,N-dimethylsphingosine (DMS). DMS also inhibited the
increase in intracellular calcium concentration stimulated by
platelet-activating factor, fMLP, and SP-G. This suggests that the
increase in intracellular calcium concentration by these agents is
dependent on sphingosine kinase activation and the generation of
sphingosine-1-phosphate. Changes in cell polarization and the
augmentation of the fMLP-induced superoxide anion generation, by all
priming agents were also inhibited by DMS, while only the superoxide
anion release was blocked by the phosphatidylinositol 3-kinase
inhibitor LY294002. Moreover, SP-G and GM-CSF inhibited constitutive
neutrophil apoptosis which was completely blocked by DMS. These results
suggest a novel role for sphingosine kinase in the regulation of
neutrophil priming. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubation of unprimed neutrophils with a secretagogue-agonist (e.g., fMLP) produces only limited cell activation. Exposure to a priming agent, which on its own does not activate neutrophil secretory pathways, causes the initial shape change and polarization necessary for migration to the site of inflammation and greatly amplifies secretagogue-induced respiratory burst activity and cytotoxic degranulation (2). Agents such as GM-CSF, which prime neutrophils to subsequent fMLP-stimulated superoxide anion release, also delay neutrophil apoptosis and prolong neutrophil function at the inflamed site (3, 4). Apoptosis leads to the resolution of inflammation by facilitating the removal of neutrophils from the inflammatory site by phagocytic cells without rupture of the neutrophil membrane or disgorgement of the cytotoxic granule contents (5, 6, 7, 8). Despite the importance of neutrophil function in host defense and inflammatory disease states much remains unknown about the intracellular signaling pathways regulating neutrophil activity. Priming plays a central role in neutrophil activation (2) and apoptosis and investigating the mechanisms regulating neutrophil priming has important consequences for the development of therapeutic strategies for the treatment of inflammatory disease (9, 10).
The wide variety of priming/chemotactic agents identified so far e.g.,
TNF-, GM-CSF, platelet-activating factor
(PAF),3 and substance
P and its analogs has made it difficult to dissect out the common or
shared signal transduction mechanisms underlying priming. At least
three different classes of receptor are involved: 1) G protein-coupled
receptors (GPCR) (e.g., fMLP and PAF); 2) transmembrane domain
receptors such as integrins and FcRs; and 3) single transmembrane
domain receptors for growth-regulating cytokines (e.g., TNF- and
GM-CSF). A clue to the mechanism of priming may be provided by
searching for a common intracellular signal transduction mechanism that
is activated both by low concentrations of the activating agent and by
all receptor classes of dedicated primers. A number of signaling
mechanisms have been implicated in priming neutrophils, such as changes
in receptor number/affinity, changes in intracellular calcium
concentration ([Ca2+]i),
phospholipase D (PLD), and phosphatidylinositol 3-kinase (PI3K)
activity (3, 11, 12), but a direct role for these second
messengers in priming, as opposed to activation, has not been
confirmed. To date, no single mechanism has been described that is
triggered by all classes of neutrophil priming agent. Furthermore,
evidence is emerging that several check points exist during the
transition from the inactive state to the primed, activated state and
eventually to apoptosis and that more than one type of pharmacological
intervention may be possible to modulate neutrophil activity
(13).
The lipid second messenger sphingosine 1-phosphate (S1P) has been
implicated in the regulation of a variety of cell processes including
cell proliferation, differentiation and apoptosis. More specifically,
increases in S1P levels have been shown to be involved in the
activation of the transcription factor AP-1, the stimulation of the
mitogen-activated protein kinase (MAPK) pathways, activation of PLD,
and the increased expression of adhesion molecules (14, 15). In differentiated HL-60 cells (a neutrophil-like cell
line), fMLPRs can mobilize
[Ca2+]i via the
stimulation of sphingosine kinase and the subsequent production of S1P
(16). This pathway is thought to mobilize
[Ca2+]i directly via a
novel sphingolipid-gated calcium channel present on the endoplasmic
reticulum (17, 18, 19). Sphingosine kinase activity has also
been shown to be stimulated by other GPCRs (20, 21),
growth factor receptors such as platelet-derived growth factor and
nerve growth factor receptors (22, 23), FcRs
(24), and TNF- receptors (25). The object
of this study was to examine the role of sphingosine kinase in human
neutrophil priming and apoptosis using four structurally distinct
priming agents PAF, GM-CSF, TNF-, and the substance P analog
[D-Arg6,
D-Trp7,9,NmePhe8]-substance
P(6, 7, 8, 9, 10, 11) (SP-G).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBS, HBSS, Dextran 500, Percoll, dihydrorhodamine (DHR),
glutaraldehyde, fMLP, PAF, GM-CSF, TNF-,
N,N-dimethylsphingosine (DMS), and S1P were purchased from
Sigma-Aldrich (Poole, U.K.); fura 2 tetraacetoxymethyl ester (fura
2-AM) and LY294002 were obtained from Calbiochem (Nottingham,
U.K.); annexin V was obtained from Bender Medsystems (Vienna, Austria);
[3H]sphingosine (23 Ci/mmol) was obtained from
New England Nuclear (Zaventim, Belgium); SP-G was a kind gift from the
Imperial Cancer Research Fund (London, U.K.). All other reagents
were of the purest grade available.
Neutrophil preparation
Peripheral venous blood was taken from healthy adult volunteers, anticoagulated with 4 ml of 3.8% sodium citrate/40 ml of blood and centrifuged (300 x g) for 20 min. Neutrophils were isolated as previously detailed (8, 9) using dextran sedimentation and discontinuous plasma-Percoll gradients. The purified neutrophils were washed three times in PBS without CaCl2 and MgCl2. Cell purity and viability were routinely >95%.
Measurement of [3H]S1P formation
S1P was measured essentially as described (20). Neutrophils (1 x 107 cells in 250 µl) were suspended in HBSS containing 1 mg/ml fatty acid-free BSA and incubated in the presence or absence of 10 µM DMS for 10 min at 37°C. Cells were sedimented and [3H]sphingosine (15 nM) was added to the cells for 1 min at 37°C before addition of agonist. Following incubation at 37°C for the indicated times, 1 ml of methanol, followed by 0.5 ml of chloroform, was added and the samples were vortexed vigorously. The extracts were cleared by centrifugation and the supernatants were evaporated to dryness in a Speedvac centrifuge. Samples were reconstituted in 25 µl of ethanol and unlabeled S1P was added before spotting onto silica gel 60 TLC plates. The samples were separated in 1-butanol-acetic acid-water (3:1:1). Bands were visualized with iodine and the S1P spots were scraped and radioactivity was measured by scintillation counting. For [3H]S1P release, after stimulation with agonist, cells were rapidly sedimented at 4°C and the supernatant (200 µl) was removed. [3H]S1P in the cellular and extracellular fraction was extracted and quantitated as described above. Alternatively, mass S1P release was measured by alkaline solvent extraction, dephosphorylation to sphingosine and rephosphorylation by recombinant sphingosine kinase in the presence of [32P]ATP (26).
Determination of [Ca2+]i by spectrofluorometry
Freshly isolated neutrophils were incubated at 107 cells/ml in CaCl2 and MgCl2-free HBSS with fura 2-AM (2 µM) for 30 min at 37°C (27). Cells were washed twice and resuspended at 3 x 106 cells/ml in HBSS containing CaCl2 and MgCl2. The cells were transferred to a cuvette and maintained at 37°C. Agonists were added as described in the figure legends. Ratiometric fluorescence was monitored in a PerkinElmer fluorometric spectrophotometer (Wellesley, MA) with dual excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. [Ca2+] was calculated according to the equation [Ca2+]i = K(F - Fmin)/(Fmax - F), where F is the ratio of the unknown sample, Fmax is the ratio after the addition of 0.2% Triton X-100 and Fmin is the ratio after [Ca2+] chelation with 10 mM EGTA. K is the dissociation constant for fura 2-AM which is 224 nM.
Shape change assay
Neutrophils (0.5 x 106/ml) were incubated in PBS with CaCl2 and MgCl2 for 5 min at 37°C. Priming agents were added (10 µl) and the cells were incubated at 37°C with gentle shaking, as described in the figure legends. An equal volume of 2.5% glutaraldehyde was added and the cells were analyzed for changes in cell polarity by flow cytometry (Coulter EPICS Profile II; Coulter Electronics, Luton, U.K.). Shape change was calculated as a percentage from the mean forward light scatter of each sample by gating on the nonshape-changed population (28).
Measurement of reactive oxygen species release
Superoxide anion release was estimated as an increase in the generation of reactive oxygen species as determined by DHR fluorescence. Neutrophils were preincubated with inhibitor (DMS, 10 µM) for 10 min at 37°C before the addition of priming agent (SP-G or PAF). Cells were then stimulated by the addition of fMLP (100 nM) for 5 min in the presence of DHR (1 µM). Samples were placed on ice and fluorescence was measured by flow cytometry (Coulter EPICS Profile II; Coulter Electronics, Luton, U.K.).
PtdIns(3, 4, 5)P3 mass assay determination
PtdIns(3, 4, 5)P3 levels were measured as described (29). In brief, neutrophils (8 x 106) were subjected to standard Folch extraction and lipid extracts containing PtdIns(3, 4, 5)P3 were then subjected to alkaline hydrolysis resulting in the release of the polar head group Ins (1, 3, 4, 5)P4. The mass of Ins(1, 3, 4, 5)P4 was measured by radioligand displacement of [3H]Ins(1, 3, 4, 5)P4 to a recombinant binding protein (GST-GAP1IP4BP expressed in Escherichia coli and purified on glutathione-agarose beads) using unlabeled Ins(1, 3, 4, 5)P4 as standard.
Measurement of apoptosis by morphology
Neutrophils (0.5 x 106 cells/ml) were incubated for 20 h in Iscove’s medium containing 10% autologous serum at 37°C. Cells were cytocentrifuged, fixed in methanol, stained with Diff-Quik, and counted using oil immersion microscopy (x100 objective) to determine the proportion of cells displaying highly distinctive apoptotic morphology (9, 30).
Measurement of apoptosis by annexin V binding
A separate assessment of apoptosis was performed by flow cytometry using FITC-labeled human annexin V which binds to phosphatidylserine exposed on the surface of apoptotic cells. Stock annexin V was diluted 1/200 with binding buffer and then added (25 µl) to 75 µl of the recovered cell samples. Following a 10-min incubation at 4°C, samples were fixed by the addition of 3% paraformaldehyde (100 µl) in PBS before analysis using an EPICS Profile II (Coulter Electronics, Luton, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The secretagogue agonist fMLP has been shown to increase
sphingosine kinase activity in neutrophil-like HL-60 cells
(16). In this study, sphingosine kinase activity was
measured in freshly isolated human neutrophils by the formation of
[3H]S1P from
[3H]sphingosine. Basal activity was routinely
450 ± 52 dpm (mean ± SEM)
[3H]S1P/107 cells. fMLP,
PAF, and SP-G consistently produced a rapid and transient increase in
sphingosine kinase activity in human neutrophils (Fig. 1
). A maximal stimulation of sphingosine
kinase activity was observed at 20–30 s giving a mean increase (±SEM)
relative to control of 81 ± 15% (n = 5),
151 ± 17% (n = 3), and 64 ± 35%
(n = 3), for SP-G, fMLP, and PAF, respectively. TNF-
produced a more sustained activation with maximal activation (245
± 16%, mean ± SEM) reached after 15 min. DMS is a sphingosine
kinase inhibitor which has been widely used to investigate the role of
sphingosine kinase in a number of experimental systems (16, 20, 31). Pretreatment with DMS inhibited sphingosine kinase activity
down to or below basal levels (Fig. 1). The observed increase in
[3H]S1P was derived mainly from the cellular
fraction as the amount of [3H]S1P detected in
the supernatant in response to PAF or fMLP was <5% of the total and
was at the lower limit of detection using this assay. We also measured
total mass S1P release from alkaline extracts of supernatants
which had been dephosphorylated by alkaline phosphatase
(26). In this method, the resultant sphingosine is
rephosphorylated in the presence of [32P]ATP by
recombinant sphingosine kinase and [32P]S1P
(26). Using this method, no S1P (<1 pmol) was detected in
the supernatants from fMLP-stimulated neutrophils (data not shown).
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PAF, fMLP, and SP-G all increased
[Ca2+]i within 20 s
of addition to intact fura 2-AM-loaded isolated neutrophil suspensions.
S1P (1 nM-1 µM) had no effect on
[Ca2+]i (not shown). The
response to PAF (33.9 ± 22.3 to 315.4 ± 66.1 nM with 3 nM
PAF; n = 3; Fig. 2, top panel) and fMLP (31.4 ± 10.1 to 285 ± 31 nM
with 10 nM fMLP; n = 4; Fig. 2, middle
panel) was primarily from mobilization of intracellular stores as
the peak response to PAF and fMLP were both abolished by the
intracellular Ca2+ antagonist
8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate
hydrochloride (TMB)-8 (Fig. 2) and only marginally
affected by the store-operated channel blocker SKF 96365. However, the
response to SP-G was significantly reduced by SKF 96365 (62 ±
12% inhibition, mean ± SEM, n = 3; Fig. 2). This
suggests that SP-G increases
[Ca2+]i both by
intracellular Ca2+ mobilization and from external
Ca2+ entry. As expected, neither TNF- nor
GM-CSF produced any change in
[Ca2+]i in neutrophils
(data not shown) which is in agreement with previous studies (see Ref.
11 and references therein).
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Preincubation for 2 min with 10 µM DMS significantly reduced the
PAF- and fMLP-induced rise in
[Ca2+]i and completely
abolished the response to SP-G (Fig. 3).
A similar degree of block was observed if total
[Ca2+]i was measured as
area under the curve (data not shown). DMS did not inhibit the mean
[Ca2+]i observed with
ionomycin (Fig. 3) confirming that DMS is not emptying intracellular
Ca2+ stores. The effect of DMS on SP-G-induced
[Ca2+]i was concentration
dependent with an IC50 of 6.47 ± 0.32 µM
(Fig. 3).
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Changes in neutrophil polarization provide a good indication of
neutrophil priming. All priming agents induced morphological changes
characteristic of neutrophil shape change. Preincubation for 5 min with
DMS (10 µM) significantly inhibited PAF-, GM-CSF-, TNF--, and
SP-G-induced shape change (Fig. 4) at all
concentrations of priming agents tested. In contrast, the PI3K
inhibitor LY294002 at 10 µM had no effect on neutrophil shape change
induced by any priming agent (Fig. 4), suggesting that PI3K, although
critical for respiratory burst activation and degranulation responses,
is not essential for neutrophil polarization responses. Addition of S1P
at concentrations up to 10 µM had no effect on neutrophil
polarization (data not shown).
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Previous studies have shown that priming agents strongly
potentiate the fMLP-mediated increase in neutrophil superoxide anion
release. In this study, PAF, GM-CSF, TNF-, and SP-G did not induce
spontaneous neutrophil superoxide anion release but caused a rapid and
concentration-dependent increase in fMLP-stimulated superoxide anion
release as measured by an increase in DHR fluorescence (Fig. 5). Addition of exogenous S1P (1 nM to 10
µM for 10 min) had no effect on fMLP-induced superoxide anion release
(data not shown). The fMLP-stimulated increase in DHR fluorescence in
primed cells was fully inhibited both by DMS (10 µM) and LY294002 (10
µM). Together this suggests a role for sphingosine kinase in
neutrophil priming, and for PI3K in fMLP-induced neutrophil activation.
There is some evidence that sphingosine analogs such as DMS and
dihydrosphingosine also inhibit protein kinase C (PKC) which
could explain the effect of DMS on neutrophil priming. However, Fig. 5
shows that the selective PKC inhibitor GF109203X, although inhibiting
PMA-induced superoxide release (IC50 = 60 nM) had
no effect on primed fMLP-induced PKC activation, suggesting that
inhibition of PKC could not explain the inhibition of primed superoxide
release observed with DMS. We also looked further at the involvement of
PI3K in neutrophil activation by measuring phosphatidylinositol
(3, 4, 5)-triphosphate (PIP3) levels
following TNF--induced priming of the fMLP response, which has been
shown to be increased during neutrophil activation (32, 33). Fig. 6 shows that TNF-
primed the fMLP-induced increase in PIP3 levels
in human neutrophils. This was effectively inhibited by pretreatment
with LY294002 (10 µM) but not DMS (10 µM).
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Neutrophils aged in culture for 20 h undergo constitutive
apoptosis, which can be measured morphologically and by annexin
VFITC binding and flow cytometry. GM-CSF and SP-G
protected neutrophils from undergoing constitutive apoptosis (Fig. 7). SP-G inhibited apoptosis from
60.5 ± 4.1% in control cultures to 30.8 ± 4.1% in treated
cells. The EC50 for SP-G-induced inhibition of
apoptosis was 10.1 ± 0.6 µM. GM-CSF- and SP-G-induced
protection from apoptosis was completely abolished by DMS (10 µM) and
LY294002 (10 µM; Fig. 7) supporting a role for sphingosine kinase and
PI3K in neutrophil apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and SP-G)
all activate sphingosine kinase; 2) inhibition of sphingosine kinase by
DMS abolishes two cardinal markers of neutrophil priming, namely shape
change and augmentation of the fMLP-stimulated superoxide anion
release; 3) GPCR-induced Ca2+ release induced by
PAF, fMLP, and SP-G in neutrophils is partially mediated by
agonist-stimulated increases in sphingosine kinase activity; and 4)
inhibition of sphingosine kinase abolishes the survival effect of SP-G
and GM-CSF. The activation of sphingosine kinase is the first
demonstration of a common signal activated by structurally diverse
priming agents. The stimulation of intracellular sphingosine kinase to
generate S1P may therefore be a proximal and central mechanism
responsible for the priming of human neutrophils.
S1P is produced from sphingosine by sphingosine kinase. It has become
clear that sphingolipids, in addition to being structural components of
cell membranes, are sources of important signaling molecules. S1P, as
well as acting as an intracellular second messenger, is also a high
affinity agonist for the endothelial differentiation gene (EDG) family
of GPCRs, EDG1, EDG3, EDG5, EDG6, and EDG8 which couple via different
heterotrimeric G proteins to activate multiple effectors (15, 34). This makes S1P unique in eliciting both intra- and
extracellular actions. Therefore, it is possible that stimulation of
intracellular sphingosine kinase activity could lead to an increase in
intracellular S1P which is released from cells to activate cell surface
EDG receptors. In this study, we show that S1P released from
neutrophils after stimulation with fMLP or PAF was well below the
Kd concentration for S1P at any of the EDG
receptor subtypes (Kd = 8 nM for EDG1
and 
20–30 nM for EDG3 and EDG5; Refs. 15 ,
31 , and 35). Mass S1P detected in the
supernatant following fMLP stimulation was <1 pmol (<2 nM in the
500-µl supernatant). In addition, exogenous S1P at concentrations
from 1 nM-10 µM had no effect on
[Ca2+]i, cell
polarization, or superoxide release. Furthermore, it has been reported
that neutrophils lack cell surface receptors for S1P (35).
Therefore, even if low concentrations of S1P are released following
stimulation with priming agents, the results from this study suggest
that it is unlikely to be responsible (via EDG receptor stimulation)
for the effects described in this study. Previous work has shown that
TNF--induced priming of fMLP-induced superoxide production involves
sphingomyelin metabolites (36). Moreover, recent work has
shown that Fc
R1 aggregation activates NADPH oxidase in primed U937
cells via a pathway involving PLD1 and sphingosine kinase
(37). The mechanism whereby an increase in S1P in response
to priming agents leads to an increase in superoxide release remains to
be elucidated.
We have shown that PAF, fMLP, and SP-G induce a rapid and transient Ca2+ signal in isolated human neutrophils that is at least partially dependent on sphingosine kinase activation and the generation of the putative second messenger S1P. This suggests that receptors for PAF, fMLP, and SP-G can use dual pathways leading to calcium release: the conventional phospholipase C-inositol 1,4,5-triphosphate pathway and the sphingosine kinase pathway. The results are similar to those described for lysophosphatidic acid and muscarinic acetyl choline receptors in SH-SY5Y cells (21). The effect of DMS is not due to an emptying of the intracellular Ca2+ store, as there was no significant reduction in the ionomycin-sensitive pool. We also show that SP-G, fMLP, and PAF could stimulate sphingosine kinase activity in human neutrophils with similar kinetics to that observed for Ca2+ release.
The role of PI3K in neutrophil activation/apoptosis is unclear. Studies
have shown that IL-8- and GM-CSF-mediated apoptosis delay is inhibited
by LY294002 and the mitogen-activated protein kinase kinase
inhibitor PD98059, suggesting a role for PI3K and extracellular
signal-regulated kinase (ERK) in neutrophil apoptosis
(4). PI3K, but not the MAPKs (ERK, c-Jun-N-terminal
kinase, p38) has been shown to be involved in IL-8-induced
neutrophil migration (38). Other studies have shown that
different chemoattractants (e.g., fMLP and PAF) use different signal
transduction pathways to stimulate neutrophils, where PAF and fMLP
strongly activate p38 MAPK but only fMLP activates ERK
(13). The ability of fMLP to activate more than one member
of the MAPK family as opposed to PAF is reflected by the wider range of
functional responses evoked by fMLP. Recently, fMLP and IL-8 have been
shown to activate the G protein-dependent PI3K, p110, and it has
been suggested that this isoform is the major PI3K activated following
neutrophil activation (12). Previous studies have
established that neutrophil polarization (shape change) and an increase
in fMLP-stimulated superoxide anion release serve as robust and
independent indicators of neutrophil priming and this finding was
confirmed for PAF, GM-CSF, TNF-, and SP-G. Both of these responses
could be inhibited by DMS, while only the superoxide anion release was
blocked by LY294002. Although LY294002 inhibited PAF-, GM-CSF-,
TNF--, and SP-G-induced superoxide release, it did not inhibit
changes in neutrophil polarity suggesting that PI3K is not involved in
the initial reorganization of cytoskeletal actin. This suggests a role
for sphingosine kinase in neutrophil priming and a role for PI3K
primarily in neutrophil activation. This finding is in agreement with
recent in vitro studies indicating the pivotal role of activated
rac and PtdIns(3)P in the assembly and activation of the
NADPH oxidase (39).
The production of superoxide anions by the NADPH oxidase involves
several membrane and cytosolic proteins and can be effected by a
variety of Ca2+ and PKC-dependent and
-independent mechanisms (11). This is illustrated by the
finding that although TNF- and GM-CSF prime the fMLP-induced
superoxide anion release, they have no effect on calcium release (data
not shown and Ref. 40). However, TNF-- and
GM-CSF-induced priming was blocked by DMS, suggesting that sphingosine
kinase may play an important regulatory role in neutrophil priming
irrespective of [Ca2+]i.
This concurs with previous data indicating that a rise in
[Ca2+]i, while necessary
for full neutrophil activation, is not obligatory for priming
(11). The extended time course of TNF--induced
sphingosine kinase activation (Fig. 1
) may reflect a different
mechanism whereby TNF- receptors signal to sphingosine kinase which
would reflect the long time period required for optimal priming of
neutrophils by TNF-.
DMS and its analog DL-threo dihydrosphingosine, in addition to inhibiting sphingosine kinase, have also been shown to inhibit PKC, although work by Edsall et al. (41) showed that 10 µM DMS showed no PKC inhibition in whole cells. It could possibly be construed that inhibition of PKC activity could explain the results observed with DMS in this study. We think this is unlikely for several reasons. Firstly, although the selective PKC inhibitor GF109203X blocked superoxide release by direct PKC stimulation with PMA, it had little effect on the primed response to fMLP. This agrees with previous work suggesting that fMLP may use PKC independent pathways to phosphorylate p47phox (42) and that PKC is not primarily involved in priming (43). Moreover, GF109203X did not inhibit neutrophil shape change or calcium transients induced by fMLP (data not shown). This suggests that although direct stimulation of PKC with PMA activates p47phox, this is not the primary route used by more physiological activating agents. Therefore, inhibition of PKC cannot explain the effects observed with DMS in this study.
The balance between ceramide and S1P can determine the physiological
fate of the cell (14, 15). In human endothelial cells,
TNF- activates sphingosine kinase which was shown to be important
for protection against TNF--mediated apoptosis (25).
These data showed that as well as inducing ceramide accumulation by
sphingomyelin hydrolysis (a pathway normally associated with apoptosis
(44, 45, 46), TNF- also activated sphingosine kinase to
generate S1P that could serve as an important antiapoptotic molecule
for cell survival. The protective role of S1P may also be important in
tumor cells as the relative levels of sphingolipid metabolites (S1P
over sphingosine) has been shown to play a key role in determining the
radiosensitivity of prostate cancer cells (47). In this
study SP-G, which primes neutrophils and stimulates sphingosine kinase
activity also protects against constitutive apoptosis, which again
suggests a protective role for sphingosine kinase. However, SP-G has
been shown to induce apoptosis in some tumor cell types
(48). It will be interesting to assess the role of
sphingosine kinase in SP-G-induced apoptosis in tumor cells.
The present study has highlighted a novel role for sphingosine kinase as a regulator of neutrophil priming by a variety of structurally distinct neutrophil priming agents. Although neutrophils are not amenable to genetic manipulation, we are at present characterizing responses in neutrophilic differentiated HL-60 cells with the view to using antisense or a dominant-negative approach to studying further the role of sphingosine kinase in priming. Future work will be important for the design of new therapeutic strategies to modulate the sphingosine kinase/S1P pathway, with the prediction that such agents could have a profound influence on the adverse effects of neutrophil priming. This would be of value in the regulation of chronic inflammatory conditions where the neutrophilic granulocyte plays such a prominent role.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alison C. MacKinnon, Lung Inflammatory Group, Center for Inflammation Research, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9AG, U.K. E-mail address: a.mackinnon{at}ed.ac.uk
3 Abbreviations used in this paper: PAF, platelet-activating factor; GPCR, G protein-coupled receptor; [Ca2+]i, intracellular calcium concentration; PI3K, phosphatidylinositol 3-kinase; S1P, sphingosine 1-phosphate; MAPK, mitogen-activated protein kinase; PLD, phospholipase D; SP-G, [D-Arg6, D-Trp7,9,NmePhe8] substance P(6–11); DMS, N,N-dimethylsphingosine; DHR, dihydrorhodamine; fura 2-AM, fura 2 tetraacetoxymethyl ester; TMB, tetramethylbenzidine; PKC, protein kinase C; PIP3, phosphatidylinositol (3,4,5)-triphosphate; EDG, endothelial differentiation gene; ERK, extracellular signal-regulated kinase.
Received for publication May 2, 2002. Accepted for publication September 20, 2002.
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  O. Florey and D. O. Haskard Sphingosine 1-Phosphate Enhances Fc{gamma} Receptor-Mediated Neutrophil Activation and Recruitment under Flow Conditions J. Immunol., August 15, 2009; 183(4): 2330 - 2336. [Abstract] [Full Text] [PDF]
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 S. M. Uriarte, K. R. McLeish, and R. A. Ward Anti-proteinase 3 antibodies both stimulate and prime human neutrophils Nephrol. Dial. Transplant., April 1, 2009; 24(4): 1150 - 1157. [Abstract] [Full Text] [PDF]
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 S. Brechard and E. J. Tschirhart Regulation of superoxide production in neutrophils: role of calcium influx J. Leukoc. Biol., November 1, 2008; 84(5): 1223 - 1237. [Abstract] [Full Text] [PDF]
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 D. A. Lebman and S. Spiegel Thematic Review Series: Sphingolipids. Cross-talk at the crossroads of sphingosine-1-phosphate, growth factors, and cytokine signaling J. Lipid Res., July 1, 2008; 49(7): 1388 - 1394. [Abstract] [Full Text] [PDF]
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J. Stie and A. J. Jesaitis Reorganization of the human neutrophil plasma membrane is associated with functional priming: implications for neutrophil preparations J. Leukoc. Biol., March 1, 2007; 81(3): 672 - 685. [Abstract] [Full Text] [PDF]
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 Y. H. Zeidan, B. J. Pettus, S. Elojeimy, T. Taha, L. M. Obeid, T. Kawamori, J. S. Norris, and Y. A. Hannun Acid Ceramidase but Not Acid Sphingomyelinase Is Required for Tumor Necrosis Factor-{alpha}-induced PGE2 Production J. Biol. Chem., August 25, 2006; 281(34): 24695 - 24703. [Abstract] [Full Text] [PDF]
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B. W. Wattenberg, S. M. Pitson, and D. M. Raben The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function J. Lipid Res., June 1, 2006; 47(6): 1128 - 1139. [Abstract] [Full Text] [PDF]
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 M. Rahaman, R. W. Costello, K. E. Belmonte, S. S. Gendy, and M.-T. Walsh Neutrophil Sphingosine 1-Phosphate and Lysophosphatidic Acid Receptors in Pneumonia Am. J. Respir. Cell Mol. Biol., February 1, 2006; 34(2): 233 - 241. [Abstract] [Full Text] [PDF]
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M. Karima, A. Kantarci, T. Ohira, H. Hasturk, V. L. Jones, B-H. Nam, A. Malabanan, P. C. Trackman, J. A. Badwey, and T. E. Van Dyke Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis J. Leukoc. Biol., October 1, 2005; 78(4): 862 - 870. [Abstract] [Full Text] [PDF]
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A. Olivera and J. Rivera Sphingolipids and the Balancing of Immune Cell Function: Lessons from the Mast Cell J. Immunol., February 1, 2005; 174(3): 1153 - 1158. [Abstract] [Full Text] [PDF]
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A. J. Melendez and F. B. M. Ibrahim Antisense Knockdown of Sphingosine Kinase 1 in Human Macrophages Inhibits C5a Receptor-Dependent Signal Transduction, Ca2+ Signals, Enzyme Release, Cytokine Production, and Chemotaxis J. Immunol., August 1, 2004; 173(3): 1596 - 1603. [Abstract] [Full Text] [PDF]
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 P. S. Jolly, M. Bektas, A. Olivera, C. Gonzalez-Espinosa, R. L. Proia, J. Rivera, S. Milstien, and S. Spiegel Transactivation of Sphingosine-1-Phosphate Receptors by Fc{varepsilon}RI Triggering Is Required for Normal Mast Cell Degranulation and Chemotaxis J. Exp. Med., April 5, 2004; 199(7): 959 - 970. [Abstract] [Full Text] [PDF]
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K. Itagaki and C. J. Hauser Sphingosine 1-Phosphate, a Diffusible Calcium Influx Factor Mediating Store-operated Calcium Entry J. Biol. Chem., July 18, 2003; 278(30): 27540 - 27547. [Abstract] [Full Text] [PDF]
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) or control
diluent (•). Neutrophils were loaded with
[3H]sphingosine for 1 min and stimulated with 100 nM
fMLP, 10 nM PAF, 10 ng/ml TNF-
) fractions. The results are expressed as
cpm [3H]S1P and represent the mean ± SD of two
experiments.
)
for 2 min as indicated. Results are representative of at least four
independent experiments.
), LY294004 (10
µM; 