Effect of FTY720P on expression of S1P receptors

and associated signaling in human myeloid cells

Caroline Lambert, Trina A. Johnson, Bryce.A. Durafourt, Veronique E. Miron, Bar-Or, A. , Jack P. Antel
Neuroimmunology Unit, Montreal Neurological Institute,
M Gill University,
McGill U i it Montreal,
M t l Quebec,
Q b Canada
C d
FTY720P does not modify S1P receptor FTY720P treatment of macrophages
Abstract Materials & Methods expression during monocyte results in increased signaling
Background: Fingolimod (FTY720) retains a subset of lymphocytes in lymph nodes. This Myeloid cell isolation and generation
To obtain monocytes, PBMCs collected from venous blood of healthy volunteers were separated on a
differentiation into macrophages through the RhoA/ROCK pathway
redistribution creates an expected reduction in peripheral blood. Yet, fingolimod patients have
normal numbers of circulating monocytes, as this cell type is not sequestered. FTY720P, the active Ficoll density gradient (GE Healthcare). CD14+ monocytes cells were positively selected to >95% purity by
phosphorylated form of the drug, signals through S1P receptors (S1PRs). Studies in glial cells MACS using anti-CD14 beads (Miltenyi), and then seeded at 1 x 106 cells/well in 24-well plates in RPMI
with 10% FBS, penicillin/streptomycin and glutamine (Invitrogen). To generate monocyte-derived DCs,
have indicated that activation of the S1P1R isoform results in the phosphorylation of the MAPK
cells were treated with human recombinant GM-CSF (50 ng/ml, PeproTech) and IL-4 (20 ng/ml,
pathway protein, ERK1/2, whereas activation of S1P3R is associated with the phosphorylation of PeproTech) for 6 days. LPS (serotype 0127:B8, 100 ng/ml, Sigma) or irradiated CD40L-expressing 3T3
myosin light chain II (MLCII).
(MLCII) cells
ll (originally
( i i ll obtained
bt i d form
f P t i k Hwu,
Patrick H MD Anderson
A d C
Cancer C t University
Center U i it off Texas)
T ) was added
dd d
on day 6 for 24 hours to induce DC maturation. Macrophages were derived in a similar fashion using M-
Objective: To characterize the S1PR mRNA expression profiles in monocytes, monocyte- derived CSF (25 ng/ml) as driving factor. FTY720-P (Novartis) was added to a final concentration of 1 uM or 0.1
macrophages and dendritic cells, and to determine which signaling pathways are induced by uM as indicated in figure legends either every 2 days or acutely prior to add the activation stimulus (LPS or
FTY720P. CD40L).
RNA isolation, reverse transcription and real-time PCR
Methods: Peripheral blood mononuclear cells were isolated from healthy human donors; Total RNA was isolated from the myeloid cells by lysing them in TRIzol (Invitrogen Life Technologies)
followed by isolation using the Qiagen RNeasy mini kit according to manufacturer’s instructions.
monocytes were isolated by immunomagnetic bead separation. Monocytes were stimulated with
Expression levels for S1P1 and 3 for all cell types were determined by qPCR using Assays-on-Demand
FTY720P or differentiated into macrophages or dendritic cells using either M-CSF or GM-CSF and primers and probe sets (Applied Biosystems). β-actin was used as an endogenous control. Relative
IL-4 respectively. S1PR expression was assessed by quantitative RT-PCR and signaling was expression was obtained by interpolation of the Ct of each sample to a standard curve generated from 10-
assessed by immunoblotting for phospho-ERK and phospho-MLCII. fold serial dilutions of mRNA derived from peripheral mononuclear cells (PBMCs, S1P1) and Jurkat cells
(S1P3).
Results: Ex vivo, monocytes were found to express S1PRs in relative abundance of S1P3R/4R > Western Blot
S1P1R. Differentiation of monocytes into macrophages or dendritic cells led to an altered Cells were treated for the indicated time with 1 μM FTY720, lysed in RIPA buffer (0.1% SDS, 1%
expression profile of S1P1R > S1P3R/4R. Treatment with FTY720P (1µM) did not alter the pattern deoxycholate, 1% Igepal, 150 mM NaCl, 50 mM Tris) supplemented with BD Baculogold protease inhibitor
of S1PR mRNA expression
p on macrophages
p g derived from monocytes. y Treatment of monocytes
y (BD Biosciences) and 1 mM Na3VO4 (phosphatase inhibitor). Macrophages were subjected to M-CSF and Figure 2. Human myeloid cell expression of the receptors S1P1, S1P3, and S1P4
serum free media for 20-24
serum-free 20 24 hours prior to treatment.
treatment Blots were incubated overnight with antibodies
with FTY720P did not induce phosphorylation of ERK1/2 or MLCII. In contrast, macrophages in monocytes and monocyte-derived macrophages in the presence of 1 µM
against phospho-ERK (1:2000; Cell Signaling Technology) or phospho-MLC II (1:500; Cell Signaling).
stimulated with FTY720P demonstrated a rapid decrease in phospho-ERK levels and a were incubated for 2 h with goat anti-rabbit (phospho-MLC II blots) or rabbit anti-mouse (phospho-ERK FTY720P. Individuals cDNAs were generated from RNA extracts of ex vivo monocytes
corresponding increase in phospho-MLCII. blots) horseradish peroxidase-conjugated secondary antibodies (1:2000; Calbiochem). Following and cultured M-CSF-generated macrophages. Data indicate differences between
detection, blots were stripped using Reblot Plus Super (Millipore) and reblotted to normalize for loading
Conclusions: Monocytes and macrophages or dendritic cells express distinctive profiles of S1PR. with antibodies against total ERK (1:1000; Stressgen) or total MLCII (1:500; Cell Signaling).
monocytes and macrophages with regard to relative levels of expression of S1P1 and
FTY720P transduces a differential signaling response in macrophages resulting in MLCII S1P3 and S1P4.
phosphorylation. The increase in phospho-MLCII levels suggests that FTY720P mediates its There was no detectable alteration in expression when macrophages cells were
effects downstream of S1P3R and S1P4R and downregulates signaling events through S1P1R. generated in the presence of FTY720P.
The phosphorylation of MLCII is an important step in the regulation of the cytoskeleton. Given the Figure 4. Western immunoblot for phospho-MLCII (p-MLCII) and total MLCII protein
finding that macrophages express lower levels of the S1P3R/4R transcripts compared to S1P1R, Myeloid cells display differential in macrophages and monocytes after FTY720P exposure. (A) M-CSF generated
this signaling may be the result of preferential cell-type specific G-protein coupling or differential macrophages were exposed to either 1uM FTY720P or vehicle for the indicated time
affinities of FTY720P for receptor subtypes. expression of S1P receptors FTY720P treatment of macrophages
before lysis. Densitometry of the protein bands for both the phosphorylated and total
MLCII protein revealed an increase in phospho-MLCII at 10 minutes that decreased
by 30 minutes of exposure. (B) Monocytes were exposed to either vehicle, 10nM or
results in decreased MAPK/ERK signaling
g g 100nM FTY720P, PMA or serum for the indicated time period. Monocytes did not
display phosphorylation of MLCII with any of the indicated stimuli.
stimuli
Background
• Fingolimod (FTY720) is a sphingosine 1-phosphate receptor (S1PR) modulator Discussion
with demonstrated efficacy in reducing inflammation in the CNS in multiple
sclerosis (MS).
•In contrast to lymphocytes, circulating monocytes are not retained by FTY720
macrophages monocytes
therapy. Monocytes have the potential to differentiate into dendritic cells (DCs) •S1PR1 expression is relatively
and macrophages. greater than S1PR3 for dendritic cells
and macrophages while S1PR3
•In active lesions of MS brains blood-derived macrophages and DCs are found in
expression is relatively greater than
the perivascular areas and activated macrophages surround the lesion area in
S1PR1 for monocytes.
the parenchyma.
•The phosphorylated form of FTY720 (FTY720-P) is a sphingosine-1-phosphate •Expression of S1P receptors in
(S1P) analog which binds with high affinity S1P receptors 1,3,4 and 5. Studies in macrophages is not effected by
glial cells have indicated that activation of the S1P1R isoform results in the differentiation in the presence of
phosphorylation of the MAPK pathway protein, ERK1/2, whereas activation of Survival FTY720P.
S1P3R is associated with the phosphorylation of myosin light chain II (MLCII).
•In M-CSF generated macrophages,
Survival
FTY720P promotes Rho/Rock
Figure 3. Western immunoblot for phospho-ERK1/2 (p-ERK1/2) and total Differentiation signaling and inhibits MAPK/ERK
Objective Figure 1. Human myeloid cell expression of S1P1 and S1P3 receptors. Individuals cDNAs were
ERK1/2 protein in macrophages and monocytes after FTY720P exposure.
(A) M-CSF generated macrophages were exposed to either 1uM FTY720P or
Survival
Process extension
Process 
retraction
signaling.
generated from RNA extracts of ex vivo monocytes and cultured cytokine-generated dendritic
cells (DCs) and macrophages.
macrophages Arbitrary units represent expression in relation to expression in vehicle for the indicated time before lysis. Densitometry of the protein bands for •This data supports
pp the p
postulate that
total PBMCs or extracts derived from the Jurkat cell line. S1PR1 expression was relatively both the phosphorylated and total ERK1/2 protein revealed a decrease in distinct populations of myeloid cells
greater than S1PR3 for dendritic cells and macrophages while S1P3 expression was relatively phospho-ERK at 10 minutes. Each line represents the ratio of p-ERK to ERK will show differential responses to
To characterize the S1PR mRNA expression profiles in greater than S1PR1 for monocytes.
densitometry in three individual donors. (B) Monocytes were exposed to either
Funding provided by: S1P receptor-directed therapies.
monocytes, monocyte- derived macrophages and dendritic Addition of lipopolysaccharide (LPS) did not alter the expression pattern of receptors for either vehicle, 10nM or 100nM FTY720P, PMA or serum for the indicated time period.
Monocytes did not display phosphorylation of ERK1/2 with any of the indicated
cells, and to determine which signaling pathways are induced macrophages or dendritic cells suggesting that activation/maturation does not further modify
FTY720P provided by:
S1P receptor expression. stimuli even though total ERK1/2 protein levels were detected. (C) Primary
by FTY720P. human fetal astrocytes were used as a positive control for ERK1/2
phosphorylation by stimulating with either FTY720P or vehicle for the indicated
time period.