Rosen Daneman News Views 2014

n e w s and vi e w s
High endothelial venules through a transcriptomics lens
Steven D Rosen & Richard Daneman
Transcriptional profiling of endothelial cells from diverse secondary lymphoid organs reveals distinctions that
underlie their functional specification.
npg
© 2014 Nature America, Inc. All rights reserved.
H
igh endothelial venules (HEVs) are the
portals of entry for blood-borne lymphocytes into secondary lymphoid organs such as
lymph nodes and Peyer’s patches (PPs). Lee
et al. now provide a genome-wide transcriptomics analysis of endothelial cells, including those
isolated from HEVs1. Their study contributes
what is expected to be an essential resource for
future work on this critical microvasculature
and provides many new insights into HEV biology and lymphocyte migration, including a previously unknown role for the B cell lectin CD22
(Siglec-2) in the homing of B cells to PPs.
The field of lymphocyte homing and recirculation began with the monumental work of
James Gowans, who first described the extensive migration of small lymphocytes from the
blood into lymph nodes and PPs through specialized high-walled venules, now called ‘high
endothelial venules’2. Noting that HEVs are
infiltrated extensively, yet very selectively, by
lymphocytes, Gowans posited that there must
be a “special affinity” between lymphocytes
and the endothelium of these vessels2.
The past 50 years of research have seen
tremendous progress in understanding the
migration (‘homing’) of lymphocytes across
HEVs and how this “special affinity” is
achieved. Naive B lymphocytes and T lymphocytes and certain memory populations interact
with HEVs through a multistep adhesion
cascade3–6. Homing to different lymphoid
organs involves distinct mechanisms, with
those for peripheral lymph nodes (PLNs) being
the best understood. Homing to PLNs begins
with the tethering and rolling of lymphocytes
on HEVs, mediated by L-selectin’s engaging
the sulfated, sialylated and fucosylated determinant sialyl α2-3-galactose β1-4[fucose
α1-3][SO3-6] N-acetylglucosamine (6-sulfosialyl Lewis X) that is presented on a set of
HEV-expressed mucins (the peripheral lymph
node addressin (PNAd) complex). Rolling lymphocytes encounter immobilized chemo­kines
Steven D. Rosen is in the Department of Anatomy
and Program in Immunology, University of
California, San Francisco, California, USA. Richard
Daneman is in the Department of Pharmacology
and the Department of Neuroscience, University of
California, San Diego, California, USA.
e-mail: [email protected]
906
(such as CCL21) on the luminal aspect of
HEVs and, through signaling by chemokine
receptors (such as CCR7 for CCL21), undergo
activation of the integrin LFA-1. The activated
integrin engages intercellular adhesion molecules expressed on HEVs, which leads to the
arrest of the lymphocytes. The cells migrate
intralumenally on HEVs and subsequently
transmigrate into the node. For homing to
PPs, overlapping but distinct mechanisms are
involved. L-selectin mediates the initial rolling step, but the nature of the carbohydraterecognition determinant on PP HEVs differs
from that on PLN HEVs. In a further distinction, the velocity of rolling is reduced through
the interaction of integrin α4β7 (expressed on
lymphocytes) with its ligand MAdCAM-1
(on HEVs). Activation by chemokines (by an
overlapping set, including CCL21) leads to
integrin-mediated arrest of the lymphocytes
through both interactions between LFA-1 and
intercellular adhesion molecules and those
between the integrin α4β7 and MAdCAM-1.
It is now clear that the “special affinity” of
lymphocytes for HEVs (as well as that of
various leukocyte subpopulations for other
vascular beds) is determined by the combinatorial use of adhesion molecules and chemo­
kines in a multistep process. Despite this new
understanding, many questions remain. For
example, are there additional adhesive and
signaling mechanisms that confer specificity in
homing? How are the intraluminal migration
and transendothelial migration of lymphocytes
regulated? What are the programs for the specification and maintenance of HEVs?
The objective of the study by Lee et al. is
to extend the knowledge of HEVs through
a gene-expression analysis of purified high
endothelial cells (HECs)1. It has long been
established that there is important structural and functional heterogeneity in the
vasculature, both in different segments of
the vascular tree (arteries, capillaries and
veins) and in the same segments in different
organs throughout the body. This heterogeneity is important in allowing region-specific
regulation of blood flow, permeability,
clotting, leukocyte trafficking and other vascular functions. Several groups have used the
purification of endothelial cells and micro­
array analysis to identify regional vascular
gene expression, including the identification
of transcriptomic signatures of endothelial
cells in the central nervous system that form
the blood-brain barrier and liver endothelial
cells that form discontinuous sinusoidal liver
vessels7,8. Here, Lee et al. aim to identify the
molecular signatures of HECs and capillary
endothelial cells (CAP ECs) in different lymphoid organs. In particular, they address two
questions: first, what makes HECs different
from CAP ECs, and second, what are the differences between HECs in these organs?
To accomplish this, Lee et al. isolate HECs
and CAP ECs by flow cytometry from dissociated PLNs, mesenteric lymph nodes (MLNs)
and PPs1. They first enrich the samples for
blood vascular endothelial cells by positive selection with monoclonal antibody to
the adhesion molecule CD31 (PECAM-1)
and by negative selection to remove cells
of hematolymphoid lineages, lymphatic
ECs and stromal cells. They then sort capillary endothelial cells and HECs with the
monoclonal antibody MECA-99 to recognize CAP ECs, with the monoclonal antibody MECA-79 to recognize the sulfated
L-selectin ligands of PLN HEVs and with
monoclonal antibody to MAdCAM-1 to
stain PP HEVs and MLN HEVs. Since Lee
et al. adhere to standards of the Immunological
Genome Project, it is anticipated that their
new data sets can be incorporated into that
resource and thus be generally available to the
community.
Principal-component analysis of genes expre­
ssed differently by the various EC populations
demonstrates that the largest variability is
between CAP ECs and HECs. There are also
tissue-specific differences among HECs (PLN versus
MLN versus PP) (Fig. 1), consistent with known
distinctions in homing mechanisms, as well as
surprising differences among CAP ECs. The ana­
lysis provides lists of signature gene sets for HECs
and CAP ECs (in which differences in expression
hold for all three organs), as well as genes with
different expression between HEC populations.
Lee et al. highlight several examples of
genes with higher expression in HECs than
in CAP ECs1. Consistent with their trafficking functions, HECs have higher expression
of genes encoding certain chemokines (Ccl21,
Cxcl9 and Cxcl10) and molecules involved in
volume 15 number 10 october 2014 nature immunology
n e w s and vi e w s
Lymphatic system
PLN
Principal-component analysis
HEV
Afferent
lymphatic vessel
Capillary
Artery
PLNs
Vein
Efferent
lymphatic vessel
MLN
PP HEC
HEV
Afferent
lymphatic vessel
PC2
PP CAP
Capillary
Kim Caesar/Nature Publishing Group
Artery
© 2014 Nature America, Inc. All rights reserved.
PLN HEC
PLN CAP
MLNs
npg
MLN HEC
MLN CAP
PPs
Vein
Efferent
lymphatic vessel
PC1
PP
Follicle-associated
epithelium
HEV
Capillary
Artery
Vein
Figure 1 The transcriptomic resource. The secondary lymphoid organs (left) include PLNs, MLNs
and PPs (details, middle). Lee et al. isolate HECs and CAP ECs from pooled inguinal, axillary and
brachial lymph nodes for PLN samples, and isolate HECs and CAP ECs from MLNs and PPs. Capillaries
deliver nutrients to the lymph tissues, whereas HEVs regulate the trafficking of lymphocytes from the
blood into the lymphoid organs. Principal-component analysis (right) of gene expression in HECs and
CAP ECs isolated from PLNs, MLNs and PPs shows that the biggest difference in gene-expression profiles
(PC1) is between HECs and CAP ECs, whereas the next biggest difference (PC2) distinguishes the
anatomical source (PLN, MLN or PP) of each subset of endothelial cells.
chemokine transcellular transport (Darc) and
chemokine scavenging (Ackr2). CXCL9 and
CXCL10 have previously been linked to the
recruitment of activated T cells and monocytes across HEVs during inflammation4,5.
Notably, HECs have higher expression of
genes encoding molecules involved in the synthesis of sphingosine 1-phosphate (Sphk1 and
Asah2). Sphingosine 1-phosphate from HEVs
may be relevant to integrin-mediated arrest
of lymphocytes or may exert autocrine activities on the endothelial cells. HECs have higher
expression of Ch25h and Cyp27a1, which
encode enzymes involved in the synthesis of
25-hydroxycholesterol and 27-hydrocholesterol, precursors of B cell chemoattractants.
Lee et al. note that the gene encoding the
7-hydroxylase CYP7B1, which is responsible
for generating active ligands, is expressed in
stromal cells, whereas the gene encoding the
degradative hydroxy­steroid dehydrogenase
HSD3B7 is expressed in HECs1; this suggests
the existence of a HEV–stromal cell gradient of the ligands. Such a gradient could be
pertinent to the transient retention of B cells
around HEVs preceding their entry into follicles6. Genes involved in innate defense also
have higher expression in HECs, including those encoding complement components, a pattern receptor for Gram-negative
bacteria, an antimicrobial protein and inhibitors of neutrophil proteases. These factors
may offer protection to HEVs against various
insults that might otherwise compromise this
critical portal.
The genes with higher expression in CAP
ECs also suggest some interesting physiological possibilities. CAP ECs have higher
expression of genes encoding molecules
involved in angiogenesis, including components of the VEGF, PDGF, Notch, TGF-β and
Wnt signaling pathways. The 'enrichment'
for these pathways in capillaries may reflect
the need to expand the microvasculature
when the lymphoid organ is activated. In
line with more general capillary endothelial
function is the ‘enrichment’ for genes encoding molecules involved in water transport
(Aqp7 and Aqp11), pH control (Car4 and
Car7) and lipid transport (Cd36). There are
also differences in CAP ECs from different lymphoid organs, indicative of distinct,
as-yet-undefined functions of the capillary
bed in each organ.
nature immunology volume 15 number 10 october 2014
A particular strength of this study is its
sophisticated analysis of the glycobiological implications of the HEC transcriptomes.
Applying several gene-ontology approaches,
Lee et al. document that the gene signature
of HECs shows considerable enrichment for
glycosylation-related transcripts relative to
their abundance in CAP ECs, which is not surprising given the critical function of L-selectin
in homing1. N-acetyllactosamine galactosyl
β1-4 N-acetylglucosamine (LacNAc;) serves
as framework for the elaboration of 6-sulfosialyl Lewis X. Notably, genes encoding many
of the enzymes involved in the synthesis of
LacNAc show higher expression in the transcriptomes of HECs than in those of CAP ECs.
Chst4 and Fut7, which encode the key enzymes
that provide the 6-sulfation and α1-3 fucos­
ylation, respectively, of N-acetylglucosamine
(GlcNAc) in LacNAc, are among the top 100
HEC signature genes with the greatest difference in expression in HECs versus CAP ECs.
Although it is expressed in PP HECs, Chst4
has much higher expression (~17-fold) in PLN
HECs, a finding consistent with the much
smaller amount of 6-sulfo-sialyl Lewis X on
PP HEVs and the dispensability of this enzyme
for the homing of lymphocytes to PPs9,10. The
lower avidity of L-selectin ligands in PP HEVs,
which underlies the higher rolling velocity
of lymphocytes in this vascular bed, may be
due to the reduced abundance of 6-sulfation
on sialyl Lewis X. Searching for additional
mucin-like ligands for L-selectin, the authors
spot upregulation of Parm1 in PLN HECs relative to its expression in CAP ECs. They verify
expression of the encoded mucin in PLN
HEVs and document that it reacts with the
monoclonal antibody MECA-79, which supports the proposal that it is another member
of the PNAd complex. As for adhesive ligands
on PP HEVs, it is very satisfying to see that
Madcam1 ranks as the gene with the greatest
difference in expression by PP HECs relative
to its expression in PLN HECs.
The most exciting biological finding of the
study is triggered by the observation that St6gal1
has higher expression in PP HECs than in PLN
HECs or CAP ECs. This gene encodes an α2-6
sialyltransferase that acts on LacNAc to produce
sialyl α2-6-galactosyl β1-4 N-acetylglucosamine.
This structure is a known recognition determinant
for CD22, a B cell Siglec previously linked to the
accumulation of B cells in bone marrow11. Indeed,
PP HECs are stained by a chimera of CD22 and
immunoglobulin Fc, whereas PLN HECs stain
minimally with this chimera. Short-term migration studies of wild-type mice given Cd22–/–
donor lymphocytes or St6gal1–/– recipients given
wild-type lymphocytes point to the involvement
of CD22 in the homing of B cells to PPs. Such a
907
n e w s and vi e w s
Of additional interest will be the investigation
of HEVs in tertiary lymphoid organs (TLOs).
These organized collections of lymphoid cells,
which have HEV-like vessels, arise in settings of
chronic inflammation, infection, autoimmunity
and cancer6,14,15. Increasing evidence indicates
that TLOs initiate adaptive immune responses to
local antigens. The presence of TLOs can result
in either deleterious consequences (chronic
inflammation or graft rejection) or beneficial
consequences (cancer or infection)6,14,15. As
HEV-like vessels are critical to the genesis and
maintenance of TLOs14, fuller understanding of
these vessels gained from the Lee et al. resource1
and follow-up studies may open up new opportunities for therapeutic intervention in a wide
range of diseases.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
1. Lee, et al. Nat. Immunol. 15, 982–995 (2014).
2. Gowans, J.L. & Knight, E.J. Proc. R. Soc. Lond. B Biol.
Sci. 159, 257–282 (1964).
3. Butcher, E.C. & Picker, L.J. Science 272, 60–66
(1996).
4. von Andrian, U.H. & Mempel, T.R. Nat. Rev. Immunol.
3, 867–878 (2003).
5. Miyasaka, M. & Tanaka, T. Nat. Rev. Immunol. 4,
360–370 (2004).
6. Girard, J.P. et al. Nat. Rev. Immunol. 12, 762–773
(2012).
7. Seaman, S. et al. Cancer Cell 11, 539–554
(2007).
8. Daneman, R. et al. PLoS ONE 5, e13741 (2010).
9. Kawashima, H. et al. Nat. Immunol. 6, 1096–1104
(2005).
10.Uchimura, K. et al. Nat. Immunol. 6, 1105–1113 (2005).
11.Nitschke, L. et al. J. Exp. Med. 189, 1513–1518
(1999).
12.Stevens, S.K. et al. J. Immunol. 128, 844–851
(1982).
13.Kimura, N. et al. J. Biol. Chem. 282, 32200–32207
(2007).
14.Ruddle, N.H. & Akirav, E.M. J. Immunol. 183,
2205–2212 (2009).
15.Pitzalis, C. et al. Nat. Rev. Immunol. 14, 447–462
(2014).
npg
© 2014 Nature America, Inc. All rights reserved.
mechanism may help explain why B cells have a
homing ‘preference’ for PPs rather than PLNs, relative to that of T cells12. The more complete recognition determinant for CD22 on HEVs may be a
GlcNAc-6-sulfated version of sialyl α2-6-galactosyl
β1-4 N-acetylglucosamine13, a critical point that
remains to be explored with sulfotransferase-null
mice (in particular Chst2–/– mice)9,10.
Of course, this transcriptomic data set is just
the start. It undoubtedly provides a fantastic
resource, but much hard work still remains to
be done to achieve full understanding of how
the molecules encoded by HEC-expressed genes
of the HEC signature regulate the development,
structure and function of HEVs in the various
lymphoid organs. In the future, combining cellpurification techniques with RNA sequencing, proteomics, glycomics, metabolomics and
other ‘-omics’ approaches will provide even
broader understanding of this microvasculature.
908
volume 15 number 10 october 2014 nature immunology