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J. Exp. Med. Vol. 207 No. 1 17-27
Brief Definitive Report
Lymphocyte egress from LNs into lymph
requires lymphocyte-intrinsic sphingosine-1-
phosphate receptor 1 (S1P1), a G protein–cou-
pled receptor (Matloubian et al., 2004). S1P1
promotes migration into exit structures, the
lymphatic vessel endothelial hyaluronan recep-
tor 1+ (LYVE-1+) cortical sinuses of the LNs,
within which lymphocytes may be captured by
lymphatic flow and transported to the efferent
lymph (Pham et al., 2008; Grigorova et al.,
2009; Sinha et al., 2009). S1P is normally low
in the lymphoid tissue and abundant in blood
and lymph, and disruption of this S1P gradient
results in an egress block (Schwab et al., 2005;
Pappu et al., 2007). However, despite its impor-
tance for lymphocyte egress, the cellular source
of lymph S1P remains unknown (Schwab and
S1P production is dependent on sphingo-
sine kinase (Sphk) 1 and 2, enzymes that are ex-
pressed in most eukaryotic cell types (Kono
et al., 2008). Recent work has demonstrated
that red blood cells are a major source of plasma
S1P, whereas all lymph S1P and 5% of plasma
S1P are supplied by a distinct, radiation-resistant
source (Pappu et al., 2007). In vitro studies have
shown that blood endothelial cells (BECs) can
act as a source of S1P (Venkataraman et al., 2008).
However, it has not been determined whether
endothelial cells are an important source of S1P
Lymphatic endothelial cells (LECs) arise from
the venous endothelium during embryonic de-
velopment at around embryonic day (E) 9–9.5,
when a subpopulation of endothelial cells of
the anterior cardinal vein commit to the lym-
phatic lineage by turning on Prox1 expression
(Karpanen and Alitalo, 2008). LYVE-1 is the
earliest expressing and one of the most specific
and widely used markers for LECs (Karpanen
and Alitalo, 2008; Oliver and Srinivasan, 2008).
Mice lacking Sphk1 and Sphk2 die in utero
between E11.5–13.5 because of blood vascular
Jason G. Cyster:
Abbreviations used: BEC, blood
endothelial cell; FRC, fibroblas-
tic reticular cell; LEC, lymphatic
endothelial cell; LYVE-1,
lymphatic vessel endothelial
hyaluronan receptor 1; OB,
oligomer-B; PTX, pertussis
toxin; S1P, sphingosine-1-
phosphate; Sphk, sphingosine
kinase; VE-cadherin, vascular
Lymphatic endothelial cell sphingosine
kinase activity is required for lymphocyte
egress and lymphatic patterning
Trung H.M. Pham,1 Peter Baluk,2 Ying Xu,1 Irina Grigorova,1
Alex J. Bankovich,1 Rajita Pappu,3 Shaun R. Coughlin,3
Donald M. McDonald,2 Susan R. Schwab,4 and Jason G. Cyster1
1Howard Hughes Medical Institute and Department of Microbiology and Immunology, 2Department of Anatomy, and
3Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143
4Skirball Institute, New York University, New York, NY 10016
Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate
(S1P), but the cellular source of this S1P is not defined. We generated mice that expressed
Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that
showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report
that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking
Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+
Sphk-deficient mice, lymphocyte egress from LNs and Peyer’s patches is blocked. Treatment
with pertussis toxin to overcome Gi-mediated retention signals restores lymphocyte
egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in
nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial
cadherin distribution at cell–cell junctions. Our data provide evidence that lymphatic
endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and
Peyer’s patches, and suggest a role for S1P in lymphatic vessel maturation.
© 2010 Pham et al. This article is distributed under the terms of an Attri-
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The Journal of Experimental Medicine
Lymphatic S1P promotes egress | Pham et al.
YFP expression (Fig. 1 C). YFP was variably observed in non–
LYVE-1+ endothelial cells, as well as some other cells that are
likely radiation-resistant lymphocytes. YFP was also observed
in LYVE-1+ lymphatic vessels in the ear dermis and small
intestine (Fig. S2, A and B). Collectively, these findings indi-
cate the Lyve-1 Cre+ mice exhibit efficient recombination of
a floxed gene element in LYVE-1+ cells within lymphoid and
S1P metabolic enzyme expression by LECs
By quantitative PCR analysis we detected selective expression
of Lyve-1 and Prox1 in the isolated LN LEC population, con-
firming their lymphatic identity (Fig. 1 D). LECs expressed
Sphk1 and Sphk2 abundantly compared with spleen cells (Fig.
1 D). Reciprocally, analysis of enzymes that degrade S1P revealed
that LECs have a lower abundance of transcripts for S1P lyase,
lipid phosphate phosphatase 3, and S1P-phosphatase 1 than
one or more of the other cell types analyzed (Fig. 1 D).
Recently, SPNS2 was implicated as an S1P transporter in ze-
brafish (Osborne et al., 2008; Kawahara et al., 2009). Tran-
scripts for the mouse SPNS2 orthologue were abundantly
expressed in LECs (Fig. 1 D). Thus, LN LECs have a tran-
scriptional profile consistent with their functioning as a source
of extracellular S1P.
Ablation of lymph S1P by conditional deletion of Sphk1
in Sphk2 null mice
To test the role of LYVE-1+ cells in generating lymph S1P, we
intercrossed Lyve-1 Cre+ mice with animals carrying floxed
and null alleles of Sphk1 and null alleles of Sphk2. Flow cyto-
metric analysis of lymphocytes in the lymph of Lyve-1 Cre+
Sphk1f/ or f/f Sphk2/ mice showed high surface S1P1 as
compared with controls, which had undetectable amounts of
receptor (Fig. 2 A). Lymphocyte S1P1 is very sensitive to S1P-
induced down-modulation, and 1 nM S1P down-modulates
S1P1 on mature thymocytes (Schwab et al., 2005). Thus, the
high surface S1P1 on lymphocytes in the lymph suggested a
marked reduction from the normal 100–300-nM lymph S1P
concentration (Pappu et al., 2007). In contrast, lymphocytes
from the blood of lymphatic Sphk-deficient and control mice
both had undetectable surface S1P1 (Fig. 2 A), consistent
with blood S1P being maintained mostly by red blood cells
(Pappu et al., 2007). Using a sensitive S1P bioassay in which
S1P concentration is measured by the extent of Flag-tagged
S1P1 receptor down-modulation in a cell line (Schwab
et al., 2005; Pappu et al., 2007), the lymphatic Sphk-deficient
mice had undetectable amounts of S1P in lymph, indicat-
ing that levels were at least 50-fold lower than in control
mice (Fig. 2 B). There was no difference in blood S1P
abundance between lymphatic Sphk-deficient and control
mice (Fig. 2 B).
LYVE-1–expressing macrophages are not a significant
source of lymph S1P
Besides LECs, a subset of macrophages has been suggested to
express LYVE-1 (Jackson, 2004). To determine the radiation
defects (Mizugishi et al., 2005). In vitro, stimulation of BECs
with S1P increases localization of vascular endothelial cadherin
(VE-cadherin) at cell–cell junctions and induces tubular mor-
phogenesis (Lee et al., 1999). Recently, S1P was demonstrated
to promote tubular formation of human dermal LECs in vitro
and lymphangiogenesis in Matrigel in vivo (Yoon et al., 2008).
However, whether S1P signaling normally plays a role in the
development of the lymphatic system is not known.
In this report, by examining mice that lack Sphk2 and
have Sphk1 conditionally deleted by a CRE recombinase
expressed from the Lyve-1 locus, we provide evidence that
LECs are the major source of lymph S1P. Lymphatic Sphk-
deficient mice experienced a block of T and B cell egress
from LNs. Additionally, lymphatic Sphk-deficient mice dis-
played altered initial lymphatic vessel morphology and junc-
tional VE-cadherin patterning in the trachea and diaphragm.
RESULTS AND DISCUSSION
Specificity of Lyve-1 CRE-mediated gene deletion
To achieve ablation of loxP-flanked genes in LECs, we gener-
ated a knockin mouse line in which an EGFP-hCre transgene
preceded by an internal ribosomal entry site was inserted into the
3 untranslated region of the Lyve-1 gene (Fig. S1, A and B). Im-
munofluoresence analysis of tissue from Lyve-1 EGFP-hCre+ mice
showed selective GFP staining in the nuclei of LYVE-1+ cells
(Fig. S1 C). In the absence of antibody staining, however, the
eGFP fluorescence was not readily detected, and for simplicity
we refer to the knockin mice as Lyve-1 Cre+ mice. To deter-
mine the efficiency and specificity of Lyve-1 Cre-mediated gene
deletion, Lyve-1 Cre+ mice were intercrossed to mice carrying
YFP preceded by a floxed transcriptional stop in the Rosa26 locus
(Srinivas et al., 2001). Activation of reporter expression was exam-
ined in LNs. By flow cytometric analysis, we identified LN LECs
as CD45 CD31hi gp38 (podoplanin)hi cells (Fig. 1 A; Link et al.,
2007). To confirm the identity of these cells as LECs, we further
demonstrated that they express high amounts of surface LYVE-
1 (Fig. 1 A) as compared with CD45 CD31hi gp38lo BECs,
CD45 CD31lo gp38hi fibroblastic reticular cells (FRCs), and
other CD45 LN stromal cells. When analyzed for reporter ex-
pression, >90% of LECs were YFP+ (Fig. 1 B), indicating efficient
CRE-mediated gene deletion in these cells. A varying fraction of
BECs was also positive for YFP reporter expression, probably be-
cause of the differential expression of LYVE-1 in subsets of BECs
that has been observed during embryonic development (Gordon
et al., 2008). In contrast, few FRCs were YFP+. We also ob-
served that a fraction of CD45+ cells (42.4 ± 6.8%), including
lymphocytes (41.8 ± 5.8%) and myeloid cells (42.9 ± 2.4%), were
YFP+, suggesting that there is some Cre activity in hematopoietic
precursor cells. It will be valuable in future studies to examine
hematopoietic precursors for LYVE-1 expression.
Immunofluorescence analysis of LN tissues obtained from
Lyve-1 Cre+ YFP reporter mice that had been lethally irradi-
ated and reconstituted with wild-type BM showed extensive
overlap between YFP and LYVE-1 immunoreactivity (Fig. 1 C).
In particular, the cells lining cortical sinusoids, identified by
their LYVE-1 staining and location in the T cell zone, showed
JEM VOL. 207, January 18, 2010
Brief Definitive Report
et al., 2008), CD11bhi CD11cmed and CD11bhi CD11clo cell
subsets were found to express LYVE-1 (Fig. 3, A and B).
Importantly, these LYVE-1+ macrophage subsets exhibited
>85% chimerism (Fig. 3 C). There was similarly high re-
placement of all the myeloid cell subsets (Fig. 3 C) as well as
sensitivity of LYVE-1+ LN macrophages and test if they
made a contribution to lymph S1P, we performed BM chimera
studies, reconstituting CD45.2+ lymphatic Sphk-deficient
mice with CD45.1+ wild-type BM. Using a gating scheme
previously used to identify LN macrophage subsets (Jakubzick
Figure 1. Efficiency of Lyve-1 CRE-mediated gene deletion and S1P metabolic enzyme expression in LECs. (A) Isolation and identification of LECs.
LNs were minced and digested as detailed in Materials and methods. CD31 and gp38 expression allows separation of LECs from other CD45 cells: FRCs, BECs,
and double-negative stromal cells (Others). (left plot) LYVE-1 on indicated populations; (right plots) Control stains for each cell type. (B) Flow cytometric analy-
sis of YFP in cells isolated from LNs of Lyve-1 Cre+ Rosa26-YFP reporter mice (percentages are shown). The four cell populations are gated according to the
scheme in A. Peripheral LNs (pLN; axillary, brachial, and inguinal nodes) and mesenteric LNs (mLN) are shown. The shaded histogram represents Cre-negative
cells. (C) Immunofluorescence analysis of YFP in LNs of Lyve-1 Cre+ or Rosa26-YFP mice that had been reconstituted with wild-type BM. Fixed LN sections
were stained as indicated. Data in A–C are representative of at least three experiments with one to two mice of each type per experiment. (D) Quantitative
RT-PCR analysis of S1P metabolic genes in cells sorted from a pool of peripheral and mesenteric LNs using the scheme in A and from spleen tissue or splenic
B cells. Data are representative of three separate sorts with 10–15 mice in each sort, with each gene expression measured at least twice. Bars represent means.
Lymphatic S1P promotes egress | Pham et al.
patches of lymphatic Sphk-deficient mice as compared with
the increase exhibited in the LNs, suggesting a more complete
loss of S1P availability in the Peyer’s patches (Fig. 4 F). The
reduced T cell numbers in LNs cannot be adequately ac-
counted for by the accumulation in Peyer’s patches, and we
did not find an accumulation of T cells in the liver or lung
(Fig. S2 D). The reduction in LN T cells may reflect a require-
ment for T cells to recirculate between lymphoid tissues (Link
et al., 2007), or possibly a more direct effect of S1P on T cells
or the LN microenvironment that promotes T cell survival
(Kennedy et al., 2009).
Consistent with the reduced lymphocyte numbers in the
lymph, LYVE-1+ sinuses in lymphatic Sphk-deficient LNs
contained almost no lymphocytes and appeared collapsed
(Fig. 4 G). In contrast, LYVE-1+ sinuses in control LNs were
extended with cells (Fig. 4 G). A similar emptying of cortical
sinuses was seen 6 h after treatment of wild-type mice with
the S1P1-modulating and egress-inhibiting drug FTY720
(Rosen and Goetzl, 2005; Schwab and Cyster, 2007), indicat-
ing that sinus emptying can occur rapidly and need not reflect
a developmental abnormality (Fig. S2 E). Lymphatic Sphk-
deficient animals also showed a deficiency of lymphocytes in
intestinal lymphatic sinuses adjacent to and likely draining
from (Azzali, 2003) Peyer’s patches (Fig. 4 H). These findings
provide evidence for the involvement of lymphatic-derived
S1P in the earliest step of lymphocyte egress from LNs and
lymphocytes (not depicted). In these BM chimeras, lymph
S1P was reduced to the same extent observed in nonchimeric
Sphk-deficient animals, as indicated by the presence of surface
S1P1 on T cells in the lymph (Fig. 3 D) and by S1P bioassay
(Fig. 3 E). These experiments suggest that hematopoietic
cells within LNs, including the LYVE-1–expressing macro-
phage subpopulations, do not make a significant contribution
to lymph S1P.
Impaired lymphocyte egress in lymphatic Sphk-deficient mice
Flow cytometric analysis of lymph isolated from the cysterna
chyli of lymphatic Sphk-deficient mice revealed that T and B
lymphocyte numbers were markedly reduced, in some cases
up to 50-fold (Fig. 4 A), indicating that egress from LNs was
strongly reduced. We detected a similar extent of egress
impairment in lymphatic Sphk-deficient mice that had been
reconstituted with wild-type BM (Fig. S2 C). There was also
a decrease in lymphocyte numbers in the circulation and the
spleen (Fig. 4, B and C). Because blood S1P levels were
normal in lymphatic Sphk-deficient mice, lymphocytes pre-
sumably could egress from the spleen and traffic to the LNs,
where their egress is impaired. Interestingly, we did not ob-
serve an expansion of the lymphocyte compartment in the
LNs but saw a consistent reduction of T cell numbers (Fig. 4 D).
Concomitantly, there was an accumulation of naive T cells in
the Peyer’s patches (Fig. 4 E). We observed a larger increase
of surface S1P1 on naive T cells recovered from Peyer’s
Figure 2. Ablation of lymph S1P by conditional deletion of Sphk1 in Sphk2-deficient mice. (A) Flow cytometric analysis showing S1P1 on CD4+
CD62Lhi T cells from the indicated circulatory fluids and tissues. indicates Lyve-1 Cre+ Sphk1f/ or f/f Sphk2/ mice; C indicates littermate control. The
shaded histograms show staining with control antibody of cells from LN and blood. Bld, blood; Lym, lymph; Spl, spleen. (B) Measurement of S1P level by
bioassay. Lymph fluid and plasma samples were prepared (see Materials and methods) and titrated onto WEHI231 cells expressing FLAG-S1P1. The x axis
shows dilution of the samples. The y axis indicates mean fluorescence intensity (MFI) of FLAG antibody staining. Data are representative of at least five
experiments with one to two mice each.
JEM VOL. 207, January 18, 2010
Brief Definitive Report
treated T cells in lymph was about one third the frequency of
OB-treated cells in the control hosts, their frequency was up
to eightfold higher than that of OB-treated cells in the lymph
of lymphatic Sphk-deficient hosts (Fig. 5, A and B). These
data suggest that although egress of control (OB)-treated T
cells was blocked in lymphatic Sphk-deficient animals, PTX-
treated cells continued to exit into the lymph. Moreover, the
appearance of cotransferred PTX-treated but not control
cells in the lymph also makes it unlikely that the marked re-
duction in control cells in the lymph is caused by a loss of
lymph flow through the LN. When absolute cell numbers
were plotted, there was no difference in the numbers of
PTX-treated T cells in lymph of control and lymphatic Sphk-
deficient recipients, suggesting that these cells can undergo
egress to a similar extent in both types of hosts (Fig. 5 C).
PTX treatment also partially restored B cell egress in host
Restoration of egress in lymphatic S1P-deficient mice
by pertussis toxin (PTX) treatment
When Gi-mediated retention signals are blocked, the lym-
phocyte-intrinsic requirement for S1P1 during LN egress is
partially overcome (Pham et al., 2008). To test whether the
lymphocyte egress defect in Lyve-1 Cre+ Sphk-deficient mice
occurs via effects on the lymphocytes, we sought to deter-
mine if inhibition of Gi-mediated retention signaling would
restore egress. Wild-type lymphocytes were treated ex vivo
either with PTX or the nonenzymatic oligomer-B (OB)
subunit of PTX as a control using a pulse-loading procedure
that allowed treated cells to continue entry into LNs for 2–3
h after being transferred into recipient mice, before complete
inhibition of Gi (Lo et al., 2005; Pham et al., 2008). After 1
d of equilibration, the distribution of transferred cells in host
animals was determined. Although the frequency of PTX-
Figure 3. Lack of contribution of myeloid cells to lymph S1P. (A) Flow cytometric analysis of enzyme-digested LN cells to detect CD11b and CD11c.
(B) LYVE-1 or control antibody staining of the six cell populations shown in A. (C) The proportion of each subpopulation replaced by donor-derived cells in lethally
irradiated CD45.2+ Lyve-1 Cre+ Sphk-deficient mice reconstituted with wild-type CD45.1+ BM. Numbers refer to the percentage of cells in the indicated gates.
(D) S1P1 on CD4+ CD62Lhi T cells from the lymph and LNs of mice that had been reconstituted with wild-type BM as in C. Sphk indicates the Lyve-1 Cre+
Sphk-deficient host; C indicates the control host. The shaded histogram shows staining of LN cells from an FTY720-treated mouse. (E) Bioassay measurement
of S1P in lymph and plasma from the chimeric mice. Data in A–E are representative of three experiments with three mice. MFI, mean fluorescence intensity.
Lymphatic S1P promotes egress | Pham et al.
Altered lymphatic vasculature in lymphatic
Because of the established functions of S1P signaling in the
development of the blood vasculature, we sought to deter-
mine if ablation of lymph S1P had an effect on lymphatic
vasculature. Lymphatic vessel morphology and architecture
in the trachea and diaphragm have been well studied (Baluk
et al., 2007), and we therefore examined these tissues. When
visualized by whole-mount staining for LYVE-1, the lym-
phatic vessels in the trachea of lymphatic Sphk-deficient
animals deficient in lymph S1P (Fig. S3, A and B). By immu-
nohistochemical analysis, OB-treated T cells were found in
the LYVE-1+ cortical sinuses of control but not lymphatic
Sphk-deficient mice (Fig. 5 D and Fig. S3 C). In contrast,
PTX-treated cells could be identified within the cortical si-
nuses of both control and lymphatic Sphk-deficient mice
(Fig. 5 D and Fig. S3 C). Collectively, these findings support
the conclusion that LEC-derived S1P acts on lymphocytes to
promote localization of cells within LYVE-1+ sinuses and
egress from LNs.
Figure 4. Impaired lymphocyte egress in Lyve-1 Cre Sphk-deficient mice. (A–E) Cell numbers in the indicated fluids and tissues in Lyve-1 Cre+
Sphk-deficient and control mice. The LN count was from a pool of two axillary, brachial, and inguinal LNs. Enumerated CD4+, CD8+, and CD19+ cells were
CD62Lhi. Points indicate data from individual mice, and white (control mice) and black (Sphk-deficient mice) bars represent means. (F) S1P1 on CD4+
CD62Lhi T cells from the indicated tissues. Shaded histograms show staining with control antibody. The vertical dashed lines mark the peak S1P1
intensity in the LN sample to allow comparison. Data in A–F are representative of at least three experiments with one to two mice per experiment.
(G and H) Immunohistochemial analysis of LNs (G) and Peyer’s patches (H) stained for LYVE-1 (brown) and CD3 (blue) or B220 (blue).
JEM VOL. 207, January 18, 2010
Brief Definitive Report
that lymphatic S1P may help regulate this process under
Early in vitro studies with BECs showed that S1P stimu-
lation increased VE-cadherin localization at cell–cell junctions
and enhanced adherens-junction assembly (Lee et al., 1999).
S1P signaling has also been implicated in promoting tight-
junction formation between BECs (Sanchez et al., 2003; Lee
et al., 2006; Camerer et al., 2009), and N-cadherin–mediated
adhesive interactions between BECs and mural cells (Paik et al.,
2004). Although in control mice the characteristic VE-cadherin+
mice appeared tortuous and ragged, with occasional sprouts,
as compared with the smooth tubular structure of lym-
phatic vessels in the control mice (Fig. 6 A and Fig. S4 A).
A similarly altered morphology was noted in lymphatics
within the diaphragm (Fig. S4 B), and in some cases LYVE-1
staining intensity was reduced (Fig. S4, A and B). The
increased number of “spikes” in the initial lymphatics of
Lyve-1 Cre+ Sphk-deficient mice bears some resemblance
to the lymphatic sprouting observed under conditions fa-
voring lymphangiogenesis (Baluk et al., 2005), suggesting
Figure 5. PTX treatment facilitates lymphocyte egress and localization in cortical sinuses in Lyve-1 Cre+ Sphk-deficient mice. (A–C) Spleno-
cytes were treated with either PTX or OB and cotransferred into recipient hosts. 22 h later, transferred cell numbers were determined in the lymph and
LNs of Lyve-1 Cre+ Sphk-deficient (Sphk ) and control hosts. (A) Flow cytometric analysis of transferred T cells present in the lymph. PTX-treated cells
were CFSE labeled. Numbers refer to the percentage of cells in the indicated gates. (B) Frequency of transferred OB (O)- and PTX (P)-treated cells in pe-
ripheral LNs (pLN), mesenteric LNs (mLN), and the lymph (Lym). (C) Total numbers of transferred CD4 and CD8 T cells in the lymph of control and Sphk-
deficient recipients from the same experiments as in B. In B and C, points indicate data from individual mice, and bars indicate means. (D) Distribution of
transferred OB- and PTX-treated cells with respect to LN cortical sinuses. Purified T cells were treated and cotransferred into control or Sphk hosts as in
A–C. Sections were stained for LYVE-1 (brown) and transferred T cells (blue). Several transferred cells located within sinuses of the Sphk-deficient recipi-
ent are marked by arrows. Data in A–D are representative of at least three experiments with one to two mice per experiment. C, control; , Sphk deficient.
Lymphatic S1P promotes egress | Pham et al.
(Baluk et al., 2005) of nonlymphoid tissue lymphatics (Fig.
S4 E). In the lymphatic Sphk-deficient mice, although the
LN LYVE-1+ sinuses often appeared collapsed, the VE-cad-
herin distribution appeared similar (Fig. S4 E). However, higher
resolution approaches will be required to determine pre-
cisely how VE-cadherin is organized at LN cortical sinus cell
junctions of wild-type and mutant mice.
To determine if the altered morphology of initial lym-
phatics in Lyve-1 Cre+ Sphk-deficient mice led to major defects
in cell trafficking, we examined the numbers of tissue-derived
DCs in skin-draining LNs. By gating on a population that
contains the majority of the skin-derived DCs in the LNs
(Jakubzick et al., 2008), we observed no apparent difference in
the numbers of these cells in lymphatic Sphk-deficient and
control mice under steady state (Fig. S4 F). In a second experi-
ment, BM-derived DCs were transferred subcutaneously to
discontinuous junctions at the initial lymphatics were apparent
and often consisted of a series of “buttons” (Baluk et al., 2005),
the junctions in the affected animals were less defined, usually
being made up by fewer or more diffuse buttons (Fig. 6, B
and C; and Fig. S4 C). Consistent with previous studies (Yoon
et al., 2008; Sinha et al., 2009), we found that LECs expressed
S1p1, and to a lesser extent S1p3 (Fig. S4 D), suggesting that
LECs are capable of transducing signals from extracellular S1P
that mediate organization of lymphatic cell–cell junctions. Tak-
ing these findings together with earlier work, S1P may have
similar actions on blood vessel and lymphatic endothelium,
regulating the localization of cadherins at cell–cell junctions.
LN LECs also express VE-cadherin (Pfeiffer et al., 2008), but its
junctional distribution has not yet been well studied. In wild-
type LNs, we observed a VE-cadherin distribution in LYVE-1+
sinuses that was less well defined than the buttons and zippers
Figure 6. Lymphatic Sphk expression is required for normal lymphatic vessel maturation. (A) Confocal images showing lymphatic vessels stained
with antibody against LYVE-1 in whole mount of mouse trachea. Arrowheads point to the jagged appearance of the lymphatic vessels in Lyve-1 Cre+
Sphk-deficient mice. (B) Confocal images showing the button-like pattern of VE-cadherin at endothelial cell–cell junctions of the diaphragm initial lym-
phatics. Arrowheads mark VE-cadherin+ buttons in the control and a corresponding junctional region in the Lyve-1 Cre+ Sphk-deficient mice. White
squares (top) indicate enlarged regions (bottom). Data are representative of six experiments (n = 6 mice). (C) VE-cadherin buttons per 1,000-µm2 projected
area of lymphatic for littermate control and lymphatic Sphk-deficient trachea (n = 3 mice per group). Bars show means ± SE. *, P < 0.05 (Student’s t test).
JEM VOL. 207, January 18, 2010
Brief Definitive Report
two doses separated by 3 h and injected with 5 × 106 wild-type BM cells pre-
pared from a CD45.1+ donor. In some experiments, 2 × 107 cells/ml were
labeled with 3.3 µM CFSE (Invitrogen) or 10 µM 5-(and-6)-(((4-chlorometh
yl)benzoyl)amino)tetramethylrhodamine (CMTMR; Invitrogen) in RPMI
1640 containing 2% FCS for 20 min at 37°C, and were then washed by spin-
ning through a layer of FCS. Labeled cells were resuspended at 2 × 107
cells/ml, and were treated with 10 ng/ml OB or PTX at 37°C for 10 min,
washed twice in warm RPMI 1640 with 2% FCS and 10 mM Hepes, and
transferred to recipient mice. Lymph collection was performed as previously
described (Matloubian et al., 2004). In brief, under a stereomicroscope, lymph
was drawn from the cysterna chyli using a fine borosilicate glass microcapillary
pipette (Sutter Instrument Co.). Cell numbers determined by flow cytometry
were divided by the volume of collected lymph to determine the concentra-
tion. Protocols were approved by the Institutional Animal Care and Use
Committee of the University of California, San Francisco.
Isolation of LECs. LNs removed from mice were minced into small pieces
and added into RPMI 1640 medium containing 2% FCS, 10 mM Hepes,
100 µg/ml DNase, and 0.2 mg/ml Blendzyme 2 (Roche). The samples were
incubated at 37°C for 25 min, rotating. Midway through the digestion, sam-
ples were passed through glass Pasteur pipettes multiple times to help break
up the pieces of LNs. The digestion was stopped by addition of EDTA and
FCS to a final concentration of 10 mM and 10%, respectively. The samples
were filtered through 100-µM cell strainers and centrifuged at 450 g for 7 min.
Cell pellets were resuspended in RPMI 1640 medium containing 2% FCS,
10 mM Hepes, and 5 mM EDTA for analysis.
S1P bioassay. The assay was performed as described by Pappu et. al. (2007).
In brief, platelet-poor plasma or cell-depleted lymph was titrated into RPMI
1640 containing 10 mM Hepes and 0.5% fatty-acid free BSA (EMD) in a
96-well U-bottom plate. 4 × 104 WEHI231 cells stably expressing FLAG-
tagged S1P1 (Lo et al., 2005) were added to each well, and the plate was
incubated for 40 min at 37°C. Cells were analyzed by flow cytometry to
measure the surface FLAG-S1P1 level using the M2-FLAG antibody (Sigma-
Aldrich). Lymph was drawn as described in Mice and adoptive cell transfer
into Alsever solution, and cells were removed by centrifugation. Platelet-
poor plasma was prepared by centrifuging whole blood for 10 min at 630 g
at room temperature.
Immunohistochemical and flow cytometric analysis. 7-µm cryostat
sections were fixed and stained as previously described (Reif et al., 2002).
CFSE-labeled cells were visualized in sections with alkaline phosphatase–
conjugated antifluorescein antibodies (Roche). Congenic transferred lympho-
cytes were visualized by staining with biotinylated antibodies to CD45.1
(clone A20) or CD45.2 (clone 104). The LYVE-1–specific antibody Mab22
was generated as previously described (Pham et al., 2008) and was from
R&D Systems. The anti–mouse gp38 (podoplanin) hybridoma was from
American Type Culture Collection. For visualization of LYVE-1+ structures
in sections, either unconjugated or biotinylated Mab22 was used. Lympho-
cyte preparations were stained with various fluorochrome-conjugated anti-
bodies purchased from BD or anti-S1P1 as previously described (Lo et al.,
2005), and data were acquired on a FACS LSRII (BD) and analyzed with
FlowJo software (Tree Star, Inc.).
Immunofluorescence analysis. 7-µm sections were prepared from para-
formaldehyde-fixed tissues, dried, and blocked with immunomix (1× PBS,
5% normal serum, 0.3% Triton X-100, 0.2% BSA, and 0.1% sodium azide)
for at least 1 h. Sections were stained with primary antibodies in immuno-
mix: biotinylated anti–LYVE-1 (R&D Systems) and rabbit anti-GFP (Invit-
rogen or Millipore) for 3 h to overnight. After two washes with 1× PBS,
sections were stained with Cy3- and Cy5-conjugated antibodies (Jackson
ImmunoResearch Laboratories, Inc.) and DAPI for 3 h. Sections were ana-
lyzed on a microscope (Axiovert Z1; Carl Zeiss, Inc.). For whole-mount
staining, mice were perfused for 2 min with fixative (1% paraformaldehyde
in PBS, pH 7.4) from a cannula inserted through the left ventricle into the
aorta. The tracheas and diaphragms were removed and immersed in fixative
Lyve-1 Cre+ Sphk-deficient and control mice. 2 d later, similar
numbers of transferred DCs were recovered from the popliteal
LNs of both types of mice (Fig. S4 G), suggesting that the
afferent lymphatics of Lyve-1 Cre+ Sphk-deficient mice can
support DC migration.
Using a genetic approach, we provide evidence in this report
that LECs are the main source of S1P needed for lymphocyte
egress from LNs and Peyer’s patches into lymph. The partial
Lyve-1 Cre activity in BECs is considered unlikely to ac-
count for the reduction in lymph S1P because the amounts of
S1P in blood were unaffected by Lyve-1 Cre-mediated Sphk1
ablation. We also consider it unlikely that Lyve-1 Cre activity
in hematopoietic cells is responsible, because the major he-
matopoietic cell populations were well replaced by wild-type
cells in BM chimera experiments that showed no restoration
in lymph S1P; involvement of some other cell type that was
not tracked in our analysis and that had high Lyve-1 Cre
activity and was not well replaced by irradiation and recon-
stitution with wild-type BM cannot be ruled out, but is con-
sidered the less likely explanation of our observations. The
ability to restore measurable lymphocyte egress in lymphatic
Sphk-deficient mice by antagonizing Gi-mediated retention
provided evidence that the egress defect was caused by a re-
quirement for S1P to act on the lymphocytes rather than on
endothelial egress barriers. These studies support a model in
which S1P produced locally by LYVE-1+ cortical sinus lining
cells is essential in promoting lymphocyte egress from LNs.
In addition, lymphatic Sphk-deficient mice exhibit altered mor-
phology and junctional patterning in initial lymphatic vessels
in nonlymphoid tissues, establishing a role for Sphk activity,
and likely S1P, in maturation of these vessels. The similar lack of
cortical sinus lymphocytes in LNs from Sphk-deficient mice
and 6-h FTY720-treated mice, together with the PTX treat-
ment and DC migration data, suggests that the emptying of
these sinuses is secondary to the block in lymphocyte egress
rather than being a consequence of developmental abnormal-
ities. However, we do not rule out the possibility that cor-
tical sinus endothelial cell junctions are affected by Sphk
deficiency. Finally, our findings suggest that perturbations
altering S1P availability or S1P receptor function may lead to
alterations in lymphatic vessels, and further work will be
needed to define the extent to which such perturbations affect
MATERIALS AND METHODS
Mice and adoptive cell transfer. CD45.2 C57BL/6 (B6) and CD45.1 B6
mice were from the National Cancer Institute or a colony maintained at the
University of California, San Francisco. Mice lacking Sphk2 and carrying
LoxP-flanked Sphk1 were on a B6/129 mixed background (Pappu et al.,
2007). Lyve-1 Cre knockin mice on a B6/129 mixed background were gener-
ated as described in Fig. S1. Lyve-1 Cre+ Sphk1f/ or f/f Sphk2/ mice were
generated by intercrossing. Control mice were usually littermates and were al-
ways from the same intercross and carried at least one wild-type Sphk allele.
Rosa26-YFP reporter mice (Srinivas et al., 2001) were provided by N. Killeen
(University of California, San Francisco, San Francisco, CA). To generate BM
chimeras, recipient CD45.2+ mice were lethally irradiated with 1,300 rads in
Lymphatic S1P promotes egress | Pham et al.
Kono, M., M.L. Allende, and R.L. Proia. 2008. Sphingosine-1-phosphate
regulation of mammalian development. Biochim. Biophys. Acta. 1781:
Lee, M.J., S. Thangada, K.P. Claffey, N. Ancellin, C.H. Liu, M. Kluk, M. Volpi,
R.I. Sha’afi, and T. Hla. 1999. Vascular endothelial cell adherens junction
assembly and morphogenesis induced by sphingosine-1-phosphate. Cell.
Lee, J.F., Q. Zeng, H. Ozaki, L. Wang, A.R. Hand, T. Hla, E. Wang, and
M.J. Lee. 2006. Dual roles of tight junction-associated protein, zonula
occludens-1, in sphingosine 1-phosphate-mediated endothelial chemo-
taxis and barrier integrity. J. Biol. Chem. 281:29190–29200. doi:10.1074/
Link, A., T.K. Vogt, S. Favre, M.R. Britschgi, H. Acha-Orbea, B. Hinz,
J.G. Cyster, and S.A. Luther. 2007. Fibroblastic reticular cells in lymph
nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8:1255–
Lo, C.G., Y. Xu, R.L. Proia, and J.G. Cyster. 2005. Cyclical modulation
of sphingosine-1-phosphate receptor 1 surface expression during lym-
phocyte recirculation and relationship to lymphoid organ transit. J. Exp.
Med. 201:291–301. doi:10.1084/jem.20041509
Matloubian, M., C.G. Lo, G. Cinamon, M.J. Lesneski, Y. Xu, V. Brinkmann,
M.L. Allende, R.L. Proia, and J.G. Cyster. 2004. Lymphocyte egress
from thymus and peripheral lymphoid organs is dependent on S1P re-
ceptor 1. Nature. 427:355–360. doi:10.1038/nature02284
Mizugishi, K., T. Yamashita, A. Olivera, G.F. Miller, S. Spiegel, and R.L.
Proia. 2005. Essential role for sphingosine kinases in neural and
vascular development. Mol. Cell. Biol. 25:11113–11121. doi:10.1128/
Oliver, G., and R.S. Srinivasan. 2008. Lymphatic vasculature develop-
ment: current concepts. Ann. NY Acad. Sci. 1131:75–81. doi:10.1196/
Osborne, N., K. Brand-Arzamendi, E.A. Ober, S.W. Jin, H. Verkade,
N.G. Holtzman, D. Yelon, and D.Y. Stainier. 2008. The spinster
homolog, two of hearts, is required for sphingosine 1-phosphate
signaling in zebrafish. Curr. Biol. 18:1882–1888. doi:10.1016/
Paik, J.H., A. Skoura, S.S. Chae, A.E. Cowan, D.K. Han, R.L. Proia,
and T. Hla. 2004. Sphingosine 1-phosphate receptor regulation of
N-cadherin mediates vascular stabilization. Genes Dev. 18:2392–2403.
Pappu, R., S.R. Schwab, I. Cornelissen, J.P. Pereira, J.B. Regard, Y. Xu, E.
Camerer, Y.W. Zheng, Y. Huang, J.G. Cyster, and S.R. Coughlin. 2007.
Promotion of lymphocyte egress into blood and lymph by distinct
sources of sphingosine-1-phosphate. Science. 316:295–298. doi:10.1126/
Pfeiffer, F., V. Kumar, S. Butz, D. Vestweber, B.A. Imhof, J.V. Stein, and
B. Engelhardt. 2008. Distinct molecular composition of blood and lym-
phatic vascular endothelial cell junctions establishes specific functional
barriers within the peripheral lymph node. Eur. J. Immunol. 38:2142–
Pham, T.H., T. Okada, M. Matloubian, C.G. Lo, and J.G. Cyster. 2008.
S1P1 receptor signaling overrides retention mediated by G alpha i-
coupled receptors to promote T cell egress. Immunity. 28:122–133.
Reif, K., E.H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Förster, and J.G.
Cyster. 2002. Balanced responsiveness to chemoattractants from adja-
cent zones determines B-cell position. Nature. 416:94–99. doi:10.1038/
Rosen, H., and E.J. Goetzl. 2005. Sphingosine 1-phosphate and its recep-
tors: an autocrine and paracrine network. Nat. Rev. Immunol. 5:560–
Sanchez, T., T. Estrada-Hernandez, J.H. Paik, M.T. Wu, K. Venkataraman,
V. Brinkmann, K. Claffey, and T. Hla. 2003. Phosphorylation and ac-
tion of the immunomodulator FTY720 inhibits vascular endothelial cell
growth factor-induced vascular permeability. J. Biol. Chem. 278:47281–
Schwab, S.R., and J.G. Cyster. 2007. Finding a way out: lymphocyte egress
from lymphoid organs. Nat. Immunol. 8:1295–1301. doi:10.1038/
for 1 h at 4°C. Tissues were washed, stained with the antibodies described or
anti–VE-cadherin (clone BV13), and viewed with a confocal microscope
(LSM-510; Carl Zeiss, Inc.) as described previously (Baluk et al., 2007).
Projection images were generated using AIM confocal software (version
3.2.2; Carl Zeiss, Inc.) from 10 consecutive frames, each 0.75 µm thick.
Online supplemental material. Fig. S1 shows a map of the Lyve-1 EGFP-
hCRE construct and targeted locus. Fig. S2 shows the efficiency of Lyve-1
Cre-mediated gene deletion, effect on egress, and cortical sinus emptying af-
ter FTY720 treatment. Fig. S3 shows the recovery of B cell egress in Lyve-1
Cre Sphk-deficient mice by PTX treatment and cell distribution in LN cor-
tical sinusoids. Fig. S4 shows the effect of Sphk deficiency on lymphatic
vasculature. Online supplemental material is available at http://www.jem
We thank J. An for excellent care of mouse colonies, X. Wang for performing the
lung and liver lymphocyte stain, and T. Arnon for discussion.
T.H.M. Pham is supported by the Boyer Program in the Biochemical Sciences
and the University of California, San Francisco Medical Scientist Training Program.
J.G. Cyster is an Investigator of the Howard Hughes Medical Institute. This work was
supported in part by grants from the National Institutes of Health.
The authors declare that they have no financial conflicts of interest.
Submitted: 24 July 2009
Accepted: 23 November 2009
Azzali, G. 2003. Structure, lymphatic vascularization and lymphocyte migra-
tion in mucosa-associated lymphoid tissue. Immunol. Rev. 195:178–189.
Baluk, P., T. Tammela, E. Ator, N. Lyubynska, M.G. Achen, D.J. Hicklin, M.
Jeltsch, T.V. Petrova, B. Pytowski, S.A. Stacker, et al. 2005. Pathogenesis of
persistent lymphatic vessel hyperplasia in chronic airway inflammation. J.
Clin. Invest. 115:247–257.
Baluk, P., J. Fuxe, H. Hashizume, T. Romano, E. Lashnits, S. Butz, D.
Vestweber, M. Corada, C. Molendini, E. Dejana, and D.M. McDonald.
2007. Functionally specialized junctions between endothelial cells of lym-
phatic vessels. J. Exp. Med. 204:2349–2362. doi:10.1084/jem.20062596
Camerer, E., J.B. Regard, I. Cornilissen, Y. Srinivasan, D.N. Duong, D.
Palmer, T.H. Pham, J.S. Wong, R. Pappu, and S.R. Coughlin. 2009.
Sphingosine-1-phosphate in the plasma compartment regulates ba-
sal and inflammation-induced vascular leak in mice. J. Clin. Invest.
Gordon, E.J., N.W. Gale, and N.L. Harvey. 2008. Expression of the hy-
aluronan receptor LYVE-1 is not restricted to the lymphatic vascula-
ture; LYVE-1 is also expressed on embryonic blood vessels. Dev. Dyn.
Grigorova, I.L., S.R. Schwab, T.G. Phan, T.H. Pham, T. Okada, and
J.G. Cyster. 2009. Cortical sinus probing, S1P1-dependent entry
and flow-based capture of egressing T cells. Nat. Immunol. 10:58–65.
Jackson, D.G. 2004. Biology of the lymphatic marker LYVE-1 and applica-
tions in research into lymphatic trafficking and lymphangiogenesis. APMIS.
Jakubzick, C., M. Bogunovic, A.J. Bonito, E.L. Kuan, M. Merad, and
G.J. Randolph. 2008. Lymph-migrating, tissue-derived dendritic cells
are minor constituents within steady-state lymph nodes. J. Exp. Med.
Karpanen, T., and K. Alitalo. 2008. Molecular biology and pathology of lym-
phangiogenesis. Annu. Rev. Pathol. 3:367–397. doi:10.1146/annurev.
Kawahara, A., T. Nishi, Y. Hisano, H. Fukui, A. Yamaguchi, and N.
Mochizuki. 2009. The sphingolipid transporter spns2 functions in
migration of zebrafish myocardial precursors. Science. 323:524–527.
Kennedy, S., K.A. Kane, N.J. Pyne, and S. Pyne. 2009. Targeting sphingo-
sine-1-phosphate signalling for cardioprotection. Curr. Opin. Pharmacol.
JEM VOL. 207, January 18, 2010 Download full-text
Brief Definitive Report
Schwab, S.R., J.P. Pereira, M. Matloubian, Y. Xu, Y. Huang, and
J.G. Cyster. 2005. Lymphocyte sequestration through S1P lyase in-
hibition and disruption of S1P gradients. Science. 309:1735–1739.
Sinha, R.K., C. Park, I.Y. Hwang, M.D. Davis, and J.H. Kehrl. 2009. B
lymphocytes exit lymph nodes through cortical lymphatic sinusoids by a
mechanism independent of sphingosine-1-phosphate-mediated chemo-
taxis. Immunity. 30:434–446. doi:10.1016/j.immuni.2008.12.018
Srinivas, S., T. Watanabe, C.S. Lin, C.M. William, Y. Tanabe, T.M. Jessell,
and F. Costantini. 2001. Cre reporter strains produced by targeted inser-
tion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1:4.
Venkataraman, K., Y.M. Lee, J. Michaud, S. Thangada, Y. Ai, H.L.
Bonkovsky, N.S. Parikh, C. Habrukowich, and T. Hla. 2008. Vascular
endothelium as a contributor of plasma sphingosine 1-phosphate. Circ.
Res. 102:669–676. doi:10.1161/CIRCRESAHA.107.165845
Yoon, C.M., B.S. Hong, H.G. Moon, S. Lim, P.G. Suh, Y.K. Kim, C.B.
Chae, and Y.S. Gho. 2008. Sphingosine-1-phosphate promotes lym-
phangiogenesis by stimulating S1P1/Gi/PLC/Ca2+ signaling pathways.
Blood. 112:1129–1138. doi:10.1182/blood-2007-11-125203