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Introduction
Dendritic cells (DC) play a key role in the regulation of immune response. DC uptake pathogens or cancer cells, process
them and then present processed peptides to the surface-bound major histocompatibility complex molecule, which is
recognized by T cells[1,2]. DC migration is an important factor for the regulation of proper immune response against invading
antigens. In terms of immature DC (iDC) functioning, trafficking is important for the recognition of antigens and their efficient
uptake. After antigen uptake and processing in the peripheral tissues, DC undergo a maturation process and migrate to
secondary lymphoid organs via the activation of some chemotactic
receptors[3,4]. It has been reported that some extracellular
stimuli, including a series of chemokines, stimulate DC
chemotaxis[3_5]. Although some chemokines have been reported to
regulate DC chemo-
taxis[3_5], further factors including bioactive lipid mediators involved in DC chemotaxis should be considered.
Sphingosylphosphorylcholine (SPC) is a bioactive lipid mediator which is a component of membrane lipids. Previous
studies have demonstrated that SPC induces human neutrophils to generate cellular superoxide and calcium
mobilization[6,7]. Two cell surface G protein-coupled SPC receptors have been suggested, namely, ovarian cancer G
protein-coupled receptor 1 (OGR1) and G protein-coupled
receptor 4 (GPR4)[8,9]. Moreover, as members of the lysopho-spholipid family play an important role in innate immune
response, SPC has proven useful in the study of phagocyte activation mechanisms. However, the effect of SPC on immune
response by DC has not been elucidated. In the present study, we investigated the effects of SPC on DC trafficking in iDC
and mDC, and further investigated the target receptors and signaling pathways involved in the regulation of SPC-induced DC
chemotaxis.
Materials and methods
Reagents RPMI-1640 medium was bought from Invitrogen Corp (Carlsbad, CA, USA). Dialyzed fetal bovine serum was
purchased from Hyclone Lab Inc (Logan, UT, USA). SPC and VPC23019 were purchased from Avanti Polar Lipids Inc
(Alabaster, AL, USA). Lipopolysaccharide (LPS, derived from
Escherichia coli strain 055:B5) was obtained from Sigma (St
Louis, MO, USA). 2'-Amino-3'-methoxyflavone (PD98059), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1
H-imidazole (SB203580) and 1,2-bis (amino-phenoxy)
ethane-N,N,N',N'-tetraacetoxymethyl ester (BAPTA/AM) were from
Calbiochem (San Diego, CA, USA).
2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) was from BIOMOL
Research Laboratories. (Plymouth Meeting, PA, USA). Rabbit anti-human antibodies to extracellular signal regulated protein
kinase (ERK), phospho-ERK (pERK), phospho-p38 (pp38), Akt, and phospho-Akt (pAkt) were purchased from Cell Signaling
Technology, Inc (Beverly, MA, USA), and horseradish peroxidase-conjugated antibodies to rabbit IgG were purchased from
Kirkegaard & Perry, Inc (Gaithersburg, MD, USA).
Generation of human DC Peripheral blood was collected from healthy donors; peripheral blood mononuclear cells were
isolated by separation on a Histopaque-1077 gradient, as previously
described[10]. After washing twice with Hanks' buffered
saline solution (without Ca2+ and
Mg2+), the peripheral blood mononuclear cells were suspended in RPMI-1640 medium
containing 10% FBS and incubated for 60 min at
37 oC to allow monocytes to attach to the culture dish.
Attached monocytes were then collected as described
previously[10]. Peripheral blood monocytes were differentiated to DC
by culture in 6-well plates in 2 mL of complete medium (RPMI-1640 medium supplemented with 10% fetal bovine serum),
supplemented with recombinant human granulocyte macrophage-colony stimulating factor (10 ng/mL; Pierce Endogen,
Rockford, IL, USA) and recombinant human IL-4 (10 ng/mL; Pierce Endogen, Rockford, IL,
USA)[11]. All cultures were incubated at 37
oC in 5% humidified CO2. After 7 d of culture, the DC were matured by stimulation with LPS (100 ng/mL) for
48 h[11]. The generation of iDC and mDC from peripheral blood monocytes was confirmed by FACS analysis using antibodies
against several CD markers, as earlier
described[11,12]. The addition of granulcotye-colony stimulating factor (10 ng/mL) and
IL-4 (10 ng/mL) induced monocyte differentiation of human monocytes into DC. CD14 was down-regulated in the DC, but
CD1a, CD40 and HLA-DR were significantly upregulated in the DC compared with the
monocytes[12]. LPS (100 ng/mL) treatment dramatically
enhanced CD86 and HLA-DR expression, as reported
earlier[11].
Chemotaxis assay Chemotaxis assays were performed using multi well chambers (Neuroprobe Inc, Gaithersburg, MD,
USA)[13]. Briefly, prepared DC were suspended in RPMI-1640 medium at
1×106 cells/mL, and 25 mL of this suspension was
placed into the upper well of a chamber separated by an 8
mm polyhydrocarbon filter from the lipid-containing lower well.
After incubation for 90 min at 37 oC, non-migrated cells were removed by scraping, and cells that had migrated across the filter
were dehydrated, fixed and stained with hematoxylin (Sigma; St Louis, MO, USA). Stained cells from a particular well were
then counted in 3 randomly chosen high power fields
(×400)[14].
Stimulation of DC with SPC for Western blot
analysis
DC (2×106) were stimulated with SPC at the indicated concentrations for predetermined times. After stimulation, the cells
were washed with serum-free RPMI-1640 medium and lysed in lysis buffer (20 mmol/L Hepes, pH 7.2, 10% glycerol,
150 mmol/L NaCl, 1% Triton X-100, 50 mmol/L NaF, 1
mmol/L Na3VO4, 10 µg/mL leupeptin, 10 µg/mL aprotinin and 1
mmol/L phenylmethylsulfonyl fluoride). Detergent insoluble materials were pelleted by centrifugation (12
000×g, 15 min at 4 oC), and
the soluble supernatant fraction was removed and stored at either -80
oC or used immediately. Protein concentrations in the
lysates were determined using Bradford protein assay reagent.
Electrophoresis and immunoblot analysis
Protein samples were prepared for electrophoresis and then separated using
a 10% SDS-polyacrylamide gel and the buffer system described
previously[15]. Following electrophoresis, they were blotted
onto nitrocellulose membranes, which were then blocked by incubation in Tris-buffered saline containing Tween-20 (TBST)
(25 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% TBST) and 5% dried non-fat milk. The membranes were then incubated with
anti-phospho-ERK antibody, anti-phospho-Akt kinase antibody, anti-ERK antibody or anti-Akt antibody and washed with
TBST. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection system by incubating
membranes with 1:5000 diluted goat anti-rabbit IgG or goat anti-mouse IgG antibody, coupled with a horseradish peroxidase.
Reverse transcription polymerase chain reaction (RT-PCR)
analysis mRNA was isolated by using a QIAshredder and
an RNeasy kit (Qiagen, Hilden, Germany). mRNA,
M-MLV reverse transcriptase, and pd (N) 6 primers (Imvitrogen corp; Carlsbad, CA, USA) were used to obtain cDNA. The
primers used for the RT-PCR analysis were reported
previously[16]. The sequences of the primers used were as follows.
Human OGR1: forward, 5'-TTCCTGCCCTA-CCACGTGTTGC-3'; reverse, 5'-CTTCCAGACCCCTAACT-CGCCA-3'; human
GPR4: forward, 5'-ACCTCTATCGGGTG-TTCGTG-3'; reverse, 5'-CCACTCACCTCCAAGAGGAA-3'; and human GAPDH:
forward, 5'-GATGACATCAAGAAGG-TGGTGAA-3', reverse, 5'-GTCTTACTCCTTGGAGGCCA-TGT-3'. Amplification was
performed over 30 cycles (94 oC/1 min [denaturation], 62
oC/1 min [annealing], and 72
oC/1 min [extension]). PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining.
Statistics The results are expressed as Mean±SD of the number of determinations indicated. Statistical significance of
differences was determined by ANOVA. Significance was accepted when
P<0.05.
Results
SPC induces chemotaxis of iDC and mDC
In the present study, we examined the effect of SPC on the migration of iDC.
Various concentrations of SPC caused iDC migration showing a concentration dependency. SPC 100 nmol/L elicited maximal
migration in iDC (Figure 1A). To investigate whether maturation of DC affects responsiveness to SPC, the DC were matured
in the presence of LPS and chemotactic migration by SPC was examined. mDC also migrated to SPC (Figure 1B). In mDC,
SPC-induced chemotactic migration was maximal at 1
mmol/L (Figure 1B). To distinguish between SPC-induced chemotaxis and
chemokinesis, we performed migration assays in the absence or presence of SPC in the upper wells of the chambers as
described previously[17]. As shown in Figure
1, the addition of SPC (10 mmol/L) in the upper chamber reduced the
SPC-induced migrations of iDC and mDC to the lower well, which demonstrates that SPC induces DC chemotaxis.
SPC induces DC chemotaxis via pertussis toxin-sensitive G proteins
Many previous reports have demonstrated that pertussis toxin (PTX)-sensitive G protein-mediated signaling is critically involved in DC
chemotaxis[18,19]. We also examined the effect of PTX on SPC-induced DC chemotaxis. When iDC were preincubated with 100 ng/mL of PTX prior to the
chemotaxis assay, the number of cells migrating
toward SPC was reduced by >95% versus cells not treated with PTX (Figure 2A). Preincubation of mDC with 100
ng/mL of PTX prior to the chemotaxis assay also almost completely inhibited SPC-induced chemotactic migration in mDC
(Figure 2B). These results strongly suggest the involvement of PTX-sensitive G proteins in SPC-induced chemotaxis in iDC
and mDC.
SPC stimulates mitogen-activated protein kinases (MAPK) and Akt activity in DC
MAPK has been reported to mediate extracellular signals to the nucleus in a variety of cell
types[20]. In this study, we attempted to determine whether SPC
stimulated MAPK activity by Western blotting with anti-phospho-specific antibodies for the enzyme. When iDC were
stimulated with SPC (1 mmol/L) for several lengths of time, ERK phosphorylation levels increased transiently and
exhibited maximal activity after 5 min of stimulation (Figure 3A), which returned to baseline after 30 min of stimulation (Figure
3A). SPC also stimulated p38 kinase phosphorylation in a time-dependent manner, showing maximal activity at 2_5 min after
stimulation (Figure 3A). The effect of SPC on Akt phosphorylation in iDC was also checked. SPC caused Akt
phosphorylation at 2_30 min of stimulation with SPC in iDC (Figure 3B).
We then examined the effect of SPC on MAPK phosphorylation in mDC. Treating mDC with 1
mmol/L of SPC caused both ERK and p38 kinase phosphorylation at 2 min of stimulation, which returned to baseline after 30 min of stimulation (Figure
3C). SPC also caused Akt phosphorylation at 2_30 min of stimulation with SPC in mDC (Figure 3D).
Regulation of SPC-induced DC chemotaxis
We observed that SPC dramatically induced DC chemotaxis (Figure 1). In
order to elucidate the intracellular signaling pathways involved in the induction of DC chemotaxis by SPC, we performed
chemotaxis assay using cells treated with an ERK pathway inhibitor (PD98059), which prevents the activation of MAPK
kinase 1/2, an upstream activator of ERK 1/2. PD98059 treatment inhibited the SPC-induced iDC chemotaxis (Figure 4A). This
finding suggests that the PD98059-inhibitable MAPK pathway may be involved in SPC-induced iDC chemotaxis. We also
examined the effects of the p38 kinase, PI3K and
Ca2+ pathways on the SPC-induced iDC chemotaxis. As shown in Figure 4A,
pretreatment of DC with SB203580 (20 µmol/L) or LY294002 (50 µmol/L) for 15 min, before treatment with SPC, blocked the
chemotactic migration by SPC. However, BAPTA/AM, a
Ca2+ chelator did not affect the SPC-induced chemotactic migration
in iDC (Figure 4A), ruling out the role of
Ca2+ in the process. Taken together, these
results suggest that the ERK, p38 kinase and PI3K pathways exert a positive effect on SPC-induced iDC migration.
We also investigated the signaling pathways involved in SPC-induced chemotactic migration in mDC. SPC-induced mDC
chemotaxis was also strongly inhibited by PD98059,
SB203580 and LY294002, but not by BAPTA/AM (Figure
4B). The results also indicate that SPC induces mDC chemotaxis via ERK, p38 kinase and PI3K-mediated pathways.
DC express receptors for SPC
Previous studies have demonstrated that SPC acted on at least 2 different cell surface
receptors, ie, OGR1 and GPR4[8,9]. Since we observed that SPC induced chemotactic migration in iDC and mDC, we examined
the expression pattern of SPC receptors in DC using RT-PCR analysis. As shown in Figure 5, human monocytes express
OGR1, but not GPR4. iDC were not found to express both OGR1 and GPR4 (Figure 5). Like monocytes, mDC express OGR1,
but not GPR4 (Figure 5). The results indicate that the expression patterns of SPC receptors change during the differentiation
and maturation process of human monocyte-derived DC.
Role of VPC23019 on SPC-induced DC chemotaxis
In order to determine whether SPC shows DC chemotaxis via S1P
receptors, we utilized the S1P receptor-selective antagonist, VPC23019. As shown in Figure 6, S1P-induced DC chemotaxis
was completely inhibited by preincubating DC with 10 µmol/L of VPC23019. However, SPC-induced DC
chemotaxis was unaffected by VPC23019 (10 µmol/L; Figure
6). These results strongly indicate that SPC acts as a unique cell surface receptor,
which is different from S1P receptors, resulting in DC chemotaxis.
Discussion
In this study, we investigated the effects of SPC on DC chemotaxis. SPC caused chemotactic migration of iDC and mDC,
suggesting that SPC act as a chemoattractant for DC throughout maturation (Figure 1). We also found the expression of SPC
receptors in iDC and mDC, and the signaling pathways led to chemotactic migration.
Since DC are key players for the induction of immune responses, their migration is a very important factor for the
regulation of immune responses against invading pathogens. Several previously published reports have demonstrated that
some lipid mediators, such as lysophosphatidic acid and S1P, could induce DC migration into distinct anatomical
regions[21,22]. In this study, we showed that another important lipid mediator, SPC, regulated DC chemotaxis in mDC as well as in iDC.
Since S1P, a similar sphingolipid, also acts as a DC chemoattractant, we examined the utilization of S1P receptors by SPC. As
there was no effect of the S1P receptor antagonist, VPC23019, on SPC-induced DC chemotaxis, we can rule out the
involvement of S1P receptors on SPC-induced DC chemotaxis.
SPC is a component of membrane lipids and occurs naturally in blood plasma and in high density lipoprotein
particles[23]. It has been reported that high amounts of SPC are found in the brain of patients with type A Niemann-Pick disease, resulting
from a deficiency of sphingomyelinase
activity[24]. SPC is a potent mitogen that increases intracellular free calcium and free
arachidonate, resulting in the activation of AP-1, which may contribute to the pathophysiology of Niemann-Pick
disease[24]. High levels of SPC have also been reported in the epidermis of atopic dermatitis patients who express abnormally high levels
of sphingomyelin deacylase[25]. It has been suggested that SPC has a pathological effect in skin disease by modulating
inflammatory processes of the epidermis via upregulation of intercellular adhesion molecule-1 and tumor necrosis
factor-a[25,26]. The levels of SPC have been reported to be higher in malignant ascites of patient with ovarian cancer, suggesting that SPC
may be involved in ovarian cancer
development[27]. SPC has been reported to induce chemotactic migration of IL-2 activated
natural killer cells[28] and vascular endothelial
cells[29]. From these reports, SPC has been proposed to be involved in
inflammation, angiogenesis and cancer. In this study, we demonstrated that SPC induces DC chemotaxis. Taken together,
SPC may play important roles in the modulation of several pathological responses, including inflammation and cancer via
inducing DC chemotaxis.
Because some reports have demonstrated that SPC binds to its specific G protein-coupled receptors, including OGR1 and
GPR4[8,9], we also checked the expression pattern of SPC receptors in monocytes, iDC, and mDC. During the differentiation
of human monocytes into iDC, OGR1 was found to be down-regulated, and iDC were found not to express OGR1 (Figure 5).
The functional roles of OGR1 on DC chemotaxis had not been previously determined. Here, we confirmed that mDC, but not
iDC, express OGR1 (Figure 5), suggesting a potential role of OGR1 in mDC chemotaxis. Taken together, we suggest that SPC
induces iDC chemotaxis via a unique receptor which is different from OGR1 or GPR4. Since SPC-induced iDC chemotaxis were
PTX-sensitive, unknown SPC receptors were coupled with the PTX-sensitive G protein. According to our result, even
though OGR1 is not expressed in iDC, it is expressed in mDC
(Figure 5). Furthermore, SPC induced chemotactic migration in
mDC (Figure 1), although it is unclear whether this was done via OGR1.
With the downstream signaling of the SPC receptor in iDC and mDC, the results were perplexing. Although SPC
stimulated chemotactic migration of DC in a PTX-sensitive manner, SPC did not induce calcium release. Until now, SPC receptors,
OGR1 and GPR4 have been reported to stimulate calcium release and calcium release via PTX-sensitive G proteins. Keeping
in mind previous reports and the results of the present study, it is reasonable to assume that SPC also acts on another
receptor which is different from OGR1 and GPR4.
A variety of chemoattractants induce chemotaxis of leukocytes via their own receptors. Chemoattractant-induced
signaling and chemotaxis in DC are PTX-sensitive, indicating the involvement of PTX-sensitive G protein-coupled
receptors[19,30]. In the present study, SPC-induced DC chemotaxis was also found to be PTX-sensitive (Figure 2). Other important
components of the chemoattractant-mediated signaling for DC chemotaxis are PI3K and
MAPK[31,32]. SPC also activates multiple
types of protein kinases, including ERK, p38 kinase, and PI3K activities downstream of the receptor activation. DC
chemotaxis by SPC shows a LY294002, PD98059 and SB203580-sensitivity ,indicating a PI3K, ERK, and p38 kinase-dependency
(Figure 4). Several signaling molecules involved in the regulation of DC chemotaxis have been
reported[32,33]. They include phospholipase C,
Ca2+, PI3K, MAPK, Rho, and
pyk2[33,34]. SPC-induced mDC chemotaxis was more largely inhibited by
PD98059, SB203580 and LY294002 than that of iDC chemotaxis. It suggests that other signaling molecules (except PI3K, ERK
and p38 kinase) will play a role in SPC-induced iDC chemotaxis.
In conclusion, our findings indicate that SPC modulates chemotactic migration in iDC and mDC. Our findings also
suggest a new perspective on the roles of OGR1 or SPC receptors in the regulation of immune responses via their chemotactic
activity for DC. Furthermore, these findings suggest that SPC receptors should be regarded as important chemotherapeutic
targets with respect to the modulation of DC migration.
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