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Regular Articles |
From the Cancer Research Campaign Laboratories,*
the
Beatson Institute for Cancer Research, Bearsden, Glasgow, Scotland; the
Academic Transfusion Medicine Unit,§
Department
of Medicine, University of Glasgow, Glasgow Royal Infirmary, Glasgow,
Scotland; the Department of Pathology,¶
University
of Glasgow, Glasgow, Scotland; the Departments of
Dermatology
and
Pathology,||
University of Vienna, Vienna, Austria;
Novartis Forschunginstitut,**
Vienna, Austria;
and Leukosite Incorporated,
Cambridge, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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isoform
MIP-1P, we have extended these studies to further support
the contention that the in situ chemokine binding to
afferent lymphatics exhibits specificity akin to that observed
in vitro with the promiscuous ß-chemokine receptor D6.
We have generated monoclonal antibodies to human D6 and showed D6
immunoreactivity on the ECs lining afferent lymphatics,
confirmed as such by staining serial skin sections with antibodies
against podoplanin, a known lymphatic EC marker. In
parallel, in situ hybridization on skin with
antisense D6 probes demonstrated the expression of D6 mRNA by lymphatic
ECs. D6-immunoreactive lymphatics were also abundant in mucosa and
submucosa of small and large intestine and appendix, but not
observed in several other organs tested. In lymph nodes, D6
immunoreactivity was present on the afferent lymphatics and also in
subcapsular and medullary sinuses. Tonsilar lymphatic sinuses were also
D6-positive. Peripheral blood cells and the ECs of blood vessels and
high endothelial venules were consistently nonreactive with anti-D6
antibodies. Additionally, we have demonstrated that D6
immunoreactivity is detectable in some malignant vascular tumors
suggesting they may be derived from, or phenotypically similar
to, lymphatic ECs. This is the first demonstration of chemokine
receptor expression by lymphatic ECs, and suggests that D6 may
influence the chemokine-driven recirculation of leukocytes through the
lymphatics and modify the putative chemokine effects on the development
and growth of vascular tumors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Migration of leukocytes and their coordinated encounters are crucial
for efficient inflammatory and immune responses. For example, in lymph
nodes, naïve T lymphocytes attracted from blood via the high
endothelial venules (HEVs) interact with antigen-presenting cells, such
as DCs, that have migrated from the peripheral tissue via the
lymphatics.8,9
Chemokines, members of the family of
structurally related chemotactic cytokines, have been implicated in
driving and directing these tightly choreographed leukocyte
movements.8-12
Chemokines are subclassified according to
the relative position of conserved cysteine
residues:10-12
the members of the most numerous CC or
ß-chemokine subfamily have the first two cysteines adjacent, whereas
in the CXC or -chemokines the first two cysteines are separated by a
single amino acid. CX3C and C chemokine
subfamilies are characterized by the first two cysteine residues being
separated by three intervening amino acids, and the absence of the
first and third cysteine, respectively. Although chemokines are
classically viewed as leukocyte chemoattractants, they have emerging
roles in cell proliferation control, embryogenesis, development, and
neuronal function.13-15
Of particular relevance here, is
their role in EC biology in which chemokine interaction with ECs
contributes to the pathophysiological processes as diverse as
angiogenesis, vascular remodeling, and the transcytosis and surface
presentation of chemokines during leukocyte
transmigration.14,16,17
Classically, chemokine receptors
are members of the G-protein-coupled heptahelical receptor
superfamily.8-12
In addition to these, other molecules
bind chemokines specifically, with high affinity and can either produce
a functional response or modify chemokine action. These molecules
include glycosaminoglycans (most notably heparan sulfate),
2-macroglobulin, virally-encoded secreted proteins, and
virally-encoded heptahelical viroceptors.12,18-21
To examine the location and specificity of chemokine binding sites in
skin, we have previously used an in situ binding analysis of
several chemokines using intact pieces of normal human
skin.22
This revealed that in addition to chemokine
receptors on resident leukocytes, chemokines show specific saturable
binding to the ECs of postcapillary and collective venules, and also
afferent lymphatics. Binding cross-competition studies showed that
venular EC display a chemokine binding fingerprint identical to Duffy
antigen/receptor for chemokines (DARC) that indeed is expressed on
postcapillary and collective venules, and also on
HEVs.23-25
DARC is unusual in that it acts as a
promiscuous receptor for - and ß-chemokines, and additionally, is
currently classified as nonsignaling because of the absence of data
demonstrating signal transduction or any cellular response on ligand
binding.26,27
Surprisingly, the profile of chemokine
binding to the lymphatic endothelium was different from venular
ECs:22
RANTES, monocyte chemotactic proteins (MCPs)-1 and
-3, but not macrophage inflammatory protein-1 (MIP-1) or
interleukin (IL)-8, showed saturable and cross-competable binding.
Recently, we have cloned a human chemokine receptor, named D6, that
we demonstrated is able to bind with high affinity to a wide array
of pro-inflammatory ß-chemokines including RANTES, MCP-1, and MCP-3,
but not human MIP-1, despite very high-affinity binding of murine
MIP-1 to murine and human D6.28,29
Thus, D6, but not
any other currently known chemokine receptor, exhibits ligand
specificity similar to the receptor identified on lymphatic
endothelium. Just like DARC, human D6 does not transduce detectable
signals on ligand binding when expressed in heterologous cell
lines.28
Although the function of D6 still remains
uncertain, it is likely to bear more functional similarity to DARC than
to other classical chemokine receptors.
More recently, we have resolved the anomaly of human D6 high-affinity
binding of murine, but not human, MIP-1.30
In all of
the above experiments, one particular isoform of human MIP-1 was
used, namely MIP-1S (or LD78). However, an alternative nonallelic
isoform exists called MIP-1P (or LD78ß). These genes are nearly
identical with the notable exception that MIP-1P encodes a proline
residue at position 2 of the mature protein, whereas in MIP-1S it is
a serine.31,32
This proline at position 2 dramatically
alters the properties of MIP-1P, now allowing its high-affinity
binding to D6.30
MIP-1P is therefore viewed as the
human functional homologue of murine MIP-1.
Here, we have capitalized on these data to further demonstrate that the
chemokine receptor on lymphatic vessels bears ligand specificity
similar to D6. We have used the in situ binding assay to
demonstrate specific binding of radioiodinated MIP-1P to these
vessels. Moreover, we have raised monoclonal antibodies against human
D6 and demonstrate D6 immunoreactivity on dermal lymphatics, confirmed
as such by staining serial sections with antibodies against podoplanin,
a lymphatic EC marker.4
In addition, in situ
hybridization has been used to show that D6 mRNA is expressed by
lymphatic ECs in the dermis. Subsequent analysis of sections taken from
a panel of tissues showed D6 immunoreactivity on lymphatics in the
gastrointestinal tract and lymphatic sinuses within the parenchyma of
secondary lymphoid organs. Furthermore, we have used the anti-D6
antibodies to demonstrate the presence of D6 immunoreactivity in a
subset of vascular tumors. The possible functional roles of D6 in the
biology of lymphatic endothelium and vascular tumorigenesis are
discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The production of mouse monoclonal anti-D6 antibody is detailed below. Anti-podoplanin antibody was prepared as described recently.4 Control murine IgGs and rabbit Ig were purchased from Sigma (Poole, UK) and DAKO (Glostrup, Denmark), respectively. Fluorescein isothiocyanate-coupled goat anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA).
In Situ Binding Assay
MIP-1P was labeled with 125I using
Iodogen (Pierce, Rockford, IL) as previously described.33
In situ ligand-binding analysis with this labeled protein on
skin pieces was performed as detailed before.22
In brief,
intact viable skin was removed in the process of elective reductive
surgery on informed consent. Immediately on removal the skin was cubed
into 1 to 2 mm3
pieces by a sharp scalpel blade.
Five to six cubes were incubated with 
10 pg and 100 pg
125I-MIP-1P (specific radioactivity, 10
µCi/µg) in 0.2 ml of Hanks’ balanced salt solution supplemented
with 0.1% bovine serum albumin and 20 mmol/L HEPES for 1 hour at room
temperature on a platform shaker (200 rpm). The pieces were washed
three times for 10 minutes in 2 ml of the buffer on the platform shaker
and then fixed overnight in 4% buffered paraformaldehyde. Afterward
the pieces were washed, dehydrated in increasing concentrations of
ethanol, embedded in paraplast (Monoject Scientific, Athy, Ireland),
and 5-µm-thin sections cut. These were deparaffinized, rehydrated,
air-dried, and coated with K2 nuclear track emulsion (Ilford,
Mobberley, UK). Slides were exposed at 4°C in the dark for 4 to 12
weeks and then developed with D-19 developer (Kodak-Pathe, Paris,
France), fixed (Unifix, Kodak-Pathe), and counterstained with hemalaun.
Slides were studied and photographs taken under a BX 60 Olympus
microscope equipped with a DP10 digital camera (Olympus Austria,
Vienna, Austria).
Generation of Anti-Human D6 Monoclonal Antibodies
Monoclonal antibodies to human D6 were raised using techniques
described elsewhere.34
Briefly, human D6 was expressed in
the murine cell line L1.2 and this transfectant was used to immunize
C57BL/6 mice. Fusion of spleen and myeloma SP2/0 was performed
following standard procedures and the hybridoma supernatants were
screened by flow cytometry for positively staining the D6 transfectant.
Specificity of the antibodies was tested by staining wild-type L1.2 or
Chinese hamster ovary (CHO) cells and L1.2 or CHO cells transfected
with other human chemokine receptors. CHO cell transfectants were
tested using immunohistochemistry as outlined below. To assess L1.2
transfectants, 106
cells were incubated with
50 µl of hybridoma supernatant for 30 minutes at 4°C, washed, then
similarly incubated with goat anti-mouse IgG (fluorescein
isothiocyanate-coupled), and subsequently assessed by
fluorescence-activated cell sorting (FACS). Supernatants from several
clones that specifically stained the D6 transfectant were selected,
further subcloned by limiting dilution, expanded, and tissue culture
supernatant collected. Specific D6 immunoreactivity was again confirmed
and each antibody titrated to give a maximal differential between D6
transfectants and untransfected controls. One antibody (4A5) was
purified using a Protein-G column. Purified antibody and antibody
supernatants were stored at -20°C until use.
Immunohistochemistry
Normal human skin was removed during elective plastic surgery on informed consent. The skin samples were snap-frozen in liquid nitrogen-chilled isopentane. 5-µm-thin serial frozen sections were cut onto glass slides coated with 3-(triethoxysilyl)-propylamin (TESPA; Sigma Chemical Co., St. Louis, MO). The sections were fixed for 10 minutes in acetone at room temperature and thereafter were kept at -20°C for up to 8 weeks until the staining procedure.
The frozen serial skin sections were rehydrated and incubated with anti-D6 mouse monoclonal or anti-podoplanin rabbit polyclonal antibodies. The bound primary antibodies were detected by sequential incubations with alkaline phosphatase-conjugated goat anti-rabbit and rabbit anti-mouse antibodies (for podoplanin and D6, respectively) and an alkaline phosphatase-anti-alkaline phosphatase staining kit (DAKO), according to the manufacturer’s instructions. To control the specificity of antibody binding, mouse and rabbit IgG and Ig control antibodies (DAKO) were used at equimolar concentrations. Each immunostaining protocol was performed on skin from at least three different donors.
All of the other immunohistochemistry was done using 5-µm-thin paraffin sections cut from blocks obtained from the archives of the Department of Pathology, University of Glasgow. The sections were deparaffinized in Histoclear (Fisher Scientific, Loughborough, UK), rehydrated through decreasing concentrations of ethanol and then pure water, and incubated for 10 minutes in 3% hydrogen peroxide. To unmask the antigens, slides were either microwaved for 10 minutes in 1 mmol/L ethylenediaminetetraacetic acid (pH 8) (for D6) or for 15 minutes in 10 mmol/L citrate buffer (pH 6) (for podoplanin). After blocking with serum, avidin, and biotin, the appropriate primary antibodies were added and the sections left for 45 to 60 minutes at room temperature. The bound antibodies were detected using the Vector avidin-biotin-peroxidase detection system and Nova-Red substrate (Vector Laboratories, Peterborough, UK), according to the manufacturers instructions. Sections were fixed with neutral buffered formalin, counterstained with hematoxylin (Surgipath Europe Ltd., Peterborough, UK), dehydrated through increasing concentrations of ethanol, cleared in Histoclear, and mounted in Histomount (Fisher Scientific). The staining was evaluated by at least two independent examiners and photographed with an Olympus BX60 microscope.
In Situ Hybridization
Adjacent 7-µm frozen sections of formaldehyde-fixed human skin were either stained with anti-podoplanin antibodies as above, or used for in situ hybridization as follows. Sections were rehydrated, immersed in 100 mmol/L glycine-phosphate-buffered saline, permeabilized in 0.3% Triton X-100, and treated with Proteinase K (20 µg/ml) for 15 minutes at 37°C in 10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.4 buffer. After fixing in 4% paraformaldehyde at 4°C, sections were acetylated in TE buffer containing 0.25% acetic anhydride. Prehybridization and hybridization were done in buffers supplied by Novagen (Madison, WI). Sense and antisense D6 probes were generated from linearized human D6 cDNA plasmid using T7 or SP6 polymerases in combination with digoxigenin RNA-labeling mix (Roche Molecular Biochemicals, Mannheim, Germany). Transcripts were subjected to alkaline hydrolysis and 100- to 200-bp fragments purified. Probes were added to the hybridization buffer to 100 ng/ml, denatured, hybridized for 16 hours at 42°C, and the sections then washed in several changes of 1x standard saline citrate at 37°C, treated for 30 minutes with RNase A (10 µg/ml), and washed extensively at 42°C with 0.1x standard saline citrate. Pre-incubation for 1 hour in blocking solution (150 mmol/L NaCl, 100 mmol/L Tris, pH 7.5, 1% rabbit serum, 1% goat serum, 1% sheep IgG, 0.15 Triton X-100), was followed by 2 hours in blocking solution containing alkaline phosphatase-conjugated sheep anti-DIG. After washing extensively, sections were equilibrated in AP buffer (100 mmol/L Tris, pH 9.5, 100 mmol/L NaCl, 50 mmol/L MgCl2, 1 mmol/L levamisole) and BCIP/NBT solution applied in AP buffer. Color development was done for 16 hours at room temperature in the dark.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P in
Viable Human Skin
Our previous in situ binding experiments22
had demonstrated the presence on lymphatic endothelium of a chemokine
receptor to which RANTES, MCP-1, and MCP-3 (but not MIP-1S and IL-8)
bind and fully cross-compete for each others binding. To our knowledge
D6 is the only chemokine receptor that can bind RANTES and MCP-1 but
not IL-8 or MIP-1S. This prompted us to investigate the possible D6
expression on lymphatic ECs.
To further assess the specificity of lymphatic chemokine receptor, we
performed in situ ligand-binding experiments on human skin
using 125I-MIP-1P, a MIP-1 isoform, which
unlike the previously studied MIP-1S,22
binds to D6
with high affinity.30
As shown in Figure 1
, MIP-1P bound selectively to the EC
lining of lymphatics and some resident dermal cells but weakly, if at
all, to vascular ECs. Thus, our current results, together with our
previous findings,22,30
suggest the presence of chemokine
receptors with ligand specificity identical to D6 on the surface of
dermal lymphatic ECs. The almost complete cross-competition of RANTES
binding by MCP-1 and vice versa22
excludes the
significant involvement of specific chemokine receptors, eg, CCR-2 and
CCR-5 in chemokine in situ binding to lymphatic ECs.
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To further examine tissue distribution and confirm lymphatic EC
expression of D6, a panel of monoclonal antibodies was generated
against cells transfected with the human D6 gene (see Materials and
Methods). Supernatants from hybridomas were screened by
immunohistochemistry and FACS analysis for binding to D6 expressed on
heterologous cell lines. Several supernatants contained D6 binding
antibodies (Figure 2)
. One clone (4A5)
appeared to have the highest titer and was further purified on a
protein G column. None of the antibody supernatants tested was able to
block 125I-MIP-1 binding to D6 transfectants,
or detect human D6 in D6 transfectant cell extracts using Western
blotting (not shown), suggesting that they recognize conformational
epitopes outside the ligand-binding domain. Clones were tested for
their ability to bind to a panel of cell lines stably transfected with
individual human chemokine receptors [CCR1–5 (Figure 2)
, CXCR1–3
(not shown)] and no cross-reaction was observed. Furthermore, using
FACS analysis, the 4A5 monoclonal did not bind to any cell types from
peripheral blood, including permeabilized cells or cells fixed before
antibody staining. Human D6 transfectants and the Jurkat T cell line
that expresses the D6 gene, both bound the 4A5 antibody in FACS
analysis (data not shown). Moreover, we have not observed staining by
4A5, or other clones, of cells within blood vessels in any of the
tissue sections that we have screened (see below). These results
effectively exclude cross-reaction with a wide array of chemokine
receptors known to be expressed on peripheral blood cells. Also,
importantly, these data show that D6 expression on, or within,
peripheral blood cells is below the level of detection with our
monoclonal antibodies. These results are in accordance with gene
expression analysis that has shown that D6 mRNA is of low abundance in
peripheral blood samples.28
However, it remains a
possibility that hemopoietic cells express this protein if they are
subjected to the appropriate stimulation or activation.
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Next, we used the mouse monoclonal anti-D6 antibodies in
immunohistochemical analysis of frozen and paraffin-embedded sections
of human skin. All of the anti-D6 antibody clones tested stained ECs
lining thin-walled channels within the dermis that had the
morphological appearance of afferent lymphatics (Figure 3; A, B, and D
). Blood vessels
corresponding to different segments of the circulatory tree were
immunonegative in all sections tested (Figure 3B
and not shown). In
some skin sections, both frozen and paraffin-embedded, a few individual
cells scattered within the lower and more rarely upper dermis showed D6
immunoreactivity (Figure 3B
, insets). These cells were not associated
with either blood or lymphatic vessels and their identity is currently
under investigation. In these experiments as in all of the subsequent
analyses, immunohistochemistry using anti-D6 antibodies was performed
alongside controls on adjacent sections using no primary antibody and
equimolar isotype-matched irrelevant control antibody that showed no
specific staining (not shown). The stainings presented are
representative of that seen in several sections from skin from at least
five different individuals.
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To confirm the identity of the D6-immunoreactive ECs as lymphatic,
we stained the adjacent serial sections of human skin with antibodies
against podoplanin, a known lymphatic EC marker.4
As shown
in Figure 3, C and D
, all of the vessels in skin that expressed D6
contained podoplanin immunoreactivity. However, podoplanin expression
was considerably broader than D6 with many podoplanin immunoreactive
vessels being D6-negative (not shown). This suggests that only a subset
of lymphatics in skin expresses D6. The exact fingerprint of D6
chemokine specificity observed in the in situ binding to
lymphatics, and the D6 immunoreactivity of lymphatic ECs, together
strongly imply that D6 is specifically expressed on lymphatic
endothelium.
Lymphatic ECs Express D6 mRNA
Next, sections of skin were examined for expression of D6 mRNA
using in situ hybridization. Although sense D6 probes
produced no specific hybridization (Figure 4A)
, antisense D6 probes hybridized to
ECs lining vessels in the dermis, both in regions near the epidermis
(Figure 4B)
and also deeper within the dermis (Figure 4B
, inset). These
vessels had the morphological appearance of lymphatics. To confirm
this, serial sections were either hybridized with antisense D6 probes
or subjected to immunohistochemistry using anti-podoplanin antibodies
(Figure 4
; C, D, E, and F). ECs lining the superficial (Figure 4C)
and
the deep dermal (Figure 4E)
vessels that hybridized effectively to the
antisense D6 probe also showed podoplanin immunoreactivity (Figure 4, D and F)
. ECs lining blood vessels, such as the one seen in Figure 4, E and F
, did not hybridize to the antisense D6 probe and showed no
podoplanin immunoreactivity. These observations provide further
evidence that lymphatic ECs express D6, and strongly suggest that the
anti-D6 antibodies are not cross-reacting with other antigens on these
cells.
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Next we embarked on a search for D6-immunoreactive lymphatics in a
panel of normal human tissues. As shown in Figure 5
, anti-D6 antibodies stained abundant
lymphatics in the wall of large and small intestine and appendix. Small
lymphatics located in the villi of small and large intestine and in the
lamina propria mucosae of colon (Figure 5; A, B, and C
) and large
collective lymphatics located in the muscular layer (Figure 5D)
displayed D6 immunoreactivity. However, not all of the lymphatics
identified as such by their podoplanin immunoreactivity were
D6-positive (not shown). In appendix, a network of D6-positive
lymphatics was observed in the lymphoid tissue of the lamina propria
(Figure 5E)
and in the lamina muscularis externa (Figure 5F)
. As
observed in skin sections (Figure 3)
, blood vessels of different
caliber representing different segments of circulatory tree were
consistently D6-negative (eg, Figure 5D
). Isotype control antibody
showed no specific staining in any of the tissues examined (not shown).
However, in the gut one anti-D6 clone out of four tested, weakly
immunostained additional structures, eg, the enterocytes lining the
lumen (not shown). The lack of the epithelial immunoreactivity by all
of the other anti-D6 monoclonal antibody clones tested (all stained the
afferent lymphatics) strongly suggests that enterocyte staining may be
because of cross-reactivity with an unrelated epitope. All of the
observations were on normal tissue from at least three different
individuals; D6-immunoreactive lymphatics were also seen in a section
from an acute appendicitis specimen (Figure 5G)
. Anti-D6 antibodies
failed to reveal the presence of D6-immunoreactive ECs in paraffin
sections of heart, kidney, liver, skeletal muscle, brain, cerebellum,
pancreas, prostate, and thyroid, although in some tissues (eg, lung,
liver, and placenta) D6 immunoreactivity was seen in cells that were
not lymphatic ECs. The identity of these cells is being investigated.
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D6 Immunoreactivity in Secondary Lymphoid Organs
Because of the presence of D6-immunoreactive channels within the
lymphoid tissue of the appendix, we also examined sections from other
secondary lymphoid organs (ie, tonsils, spleen, and lymph nodes), for
the presence of D6-immunoreactive structures. The parafollicular areas
of the tonsils contained abundant D6-immunoreactive sinus-like channels
(Figure 6, A and B)
; HEVs were
D6-negative (Figure 6A)
. The white pulp of spleen contained no
D6-immunoreactive structures, whereas in one out of four different
spleens tested, D6-immunoreactive vessels were observed in the red pulp
(Figure 6C)
.
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. Again, no
blood vessel ECs were D6-immunoreactive in any secondary lymphoid
tissue, including cells lining HEVs. Using the anti-podoplanin
antibodies on adjacent sections from lymph nodes and tonsils revealed
that D6-immunoreactive vessels also expressed
podoplanin-immunoreactivity (Figure 6, E and G
, and not shown), but
additionally, there were lymphatic sinuses detected that reacted with
the anti-podoplanin antibodies only (not shown). D6 Is Expressed by a Subset of Vascular Tumors
There has been considerable debate concerning the origin and
differentiation of the aberrant ECs seen in malignant vascular tumors,
ie, whether they are blood vessel EC-like or lymphatic EC-like.
Recently, the lymphatic EC markers podoplanin and VEGFR-3 have been
shown in some vascular tumors.1,2,4,35
As
D6-immunoreactive ECs are exclusively lymphatic, we reasoned that D6
immunoreactivity might be expressed in the vascular tumors that show
lymphatic origin or differentiation. As an initial assessment, we
screened a panel of 15 vascular tumors predominantly from the skin for
the expression of D6 immunoreactivity, and by using adjacent sections,
for the expression of podoplanin immunoreactivity. As shown in Table 1
, there was considerable variation in
the presence of these two markers. Examples of vascular tumors stained
with the D6 antibodies are shown in Figure 7
. In general, those tumors that
expressed podoplanin also displayed, on a variable proportion of their
cells, D6 immunoreactivity. The same 11 tumor samples showed some
immunoreactivity with both antibodies, although in only three of these
cases D6 immunoreactivity was present in almost all of the cells of the
tumor (Figure 7
; A, B, and D): podoplanin immunoreactivity was
expressed on 100% of the tumor cells in these three cases plus a
further four samples (Figure 7, E and F
, and Table 1
). In fact,
podoplanin immunoreactivity was consistently seen to be expressed on a
higher percentage of the tumor cells than D6. Also, the intensity of
the staining with the D6 antibodies varied between samples: six samples
showed intense immunoreactivity, three stained only weakly, and two
tumors contained populations of cells that expressed different levels
of D6 immunoreactivity (Table 1)
. These results further support the
observations made by others,1,2,4,35
showing that vascular
tumors often express markers of lymphatic ECs. However, there is
considerable heterogeneity between vascular tumor samples with respect
to the distribution and levels of expression of these markers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using in situ binding assays we have demonstrated previously that a receptor for pro-inflammatory ß-chemokines is expressed on lymphatic ECs in the skin.22 This receptor exhibited ligand-binding specificity akin to that observed with the ß-chemokine receptor D6. Monoclonal antibodies raised against the receptor, and antisense D6 RNA probes, specifically stained ECs in the skin that were also stained, in serial sections, with antibodies against the lymphatic EC marker podoplanin. D6-immunoreactive lymphatic ECs were also seen in the mucosa and other wall layers of the gut, in afferent lymphatic vessels entering lymph nodes and in lymphatic sinuses within secondary lymphoid tissue. We did not observe D6 immunoreactivity on ECs lining blood vessels of any caliber in any of the sections tested. The specificity of D6 expression described here is intriguing and, even in the absence of functional data, suggests a role for this molecule in lymphatic EC biology, possibly regulating leukocyte trafficking through these vessels, or the development and growth of lymphatic channels themselves.
Lymphocytes and DCs leave the peripheral tissues and migrate via the afferent lymphatics into the draining lymph nodes. This requires leukocytes to find the afferent lymphatics, enter them, and rapidly advance through them. Although traditionally all three of these migration steps have been considered passive, they may be influenced by chemokines. The ability of several ß-chemokines to bind D6 on the lymphatic ECs may enable or disable the chemokine control of leukocyte trafficking through the afferent lymphatics. One can envisage a number of possible functions for D6 on lymphatic vessels in lymphocyte and DC trafficking. The range of potential alternatives is particularly broad because currently the cellular function of D6 is unclear. However, the promiscuity, nonsignaling status and EC expression of D6 make it tempting for us to group it together with DARC into a functionally related subclass of chemokine receptors.
We are considering the following functional outcomes for D6-chemokine interaction on the lymphatic EC surface that may be relevant for leukocyte trafficking.
First, the chemokines may be sequestered and neutralized by D6 ("D6 the chemokine sink" hypothesis). This function is reminiscent of the suggested role of erythrocyte DARC.27 Pro-inflammatory ß-chemokines that bind to D6 would be neutralized, but other non-D6-binding chemokines such as SLC (R. Nibbs, unpublished observation), a chemokine that can be expressed by lymphatic ECs36 and likely to be involved in constitutive lymphocyte and DC trafficking,9 would remain free to interact with their target cells. It is also possible that neutralization of the inflammatory ß-chemokines by D6 within and around the lymphatic vessels prevents the chemokine-induced firm adhesion of leukocytes to the lymphatic ECs. An anti-adhesion mechanism of this type may allow the passive lymph flow to carry the leukocytes that entered lymphatics into the lymph node.
Second, the chemokines may remain fully functional on D6 binding and be presented to leukocytes ("D6 the chemokine presenting molecule" hypothesis), a role also suggested for DARC on venular ECs.17 This putative function for D6 would allow tissue-derived pro-inflammatory ß-chemokines to drive leukocyte migration into the lymph node (assuming the expression of the appropriate chemokine receptors by the pertinent cell populations) and thus contribute to the immune processing of the inflammation. Conversely, firm adhesion of D6 ligand-responsive leukocytes to lymphatic ECs may actually prevent their migration into the lymphatic vessels and retain them in the tissue. Only those cells no longer responsive to D6 ligands, such as mature DCs, would be able to enter the lymph nodes via the D6-expressing lymphatics.
Third, D6 may influence the transport of chemokines across the lymphatic ECs ("D6 the chemokine transporter" hypothesis). This may be analogous with chemokine transcytosis in abluminal to luminal direction by venular ECs17 that allows tissue-derived chemokines to cross the endothelium, a function possibly involving DARC.17 Recently, the transport of chemokines from skin via lymphatics into the draining lymph node has been described,37 and chemokines are readily detectable in the afferent lymphatics of larger experimental animals.38 It is tempting to speculate that these chemokines form a component of the afferent lymph that can alter the structure or function of the draining node.39 Indeed, intravital microscopy has shown that, at least in the case of secondary lymphoid tissue chemokine, lymphatic chemokine transport allows for the "remote control" of cell recruitment into the lymph node via the HEV, as the peripheral tissue-derived SLC can be presented on the surface of these cells.37 Although SLC, the chemokine used in this study, does not bind D6, the demonstrable ability of several pro-inflammatory chemokines to rapidly reach the lymph node after their intradermal injection (A. Rot, unpublished observation) may involve D6-mediated chemokine transport. Conversely, it is also possible that in its putative role as a "chemokine sink" (see above), D6 inhibits the lymphatic transport of the chemokines, thus preventing their "remote control" of lymph node function. A careful analysis of the lymphatic transport of D6-binding and -nonbinding chemokines to the lymph node may provide support for these alternative functions of D6.
Although we can speculate about D6 functions on afferent lymphatics, it is more difficult to discuss the putative function of D6 expressed on the sinuses and channels within tonsils and lymph nodes, as the molecular mechanism of leukocytes trafficking through these, eg, in the process of exiting the secondary lymphoid tissues, has not been thoroughly investigated.
Extensive experimental work and a combination of in vivo and in vitro approaches will be required to establish which of the several alternative hypotheses described above best reflects the function of D6 on the lymphatic ECs.
We currently favor a role for D6 in some aspect of chemokine-driven leukocyte trafficking. However, bearing in mind that chemokines mediate cellular chemotaxis, plus the observations that chemokines can induce EC migration and regulate angiogenesis,14,16,40,41 it cannot be excluded that D6 is involved in regulating lymphangiogenesis during development and/or tissue regeneration, or some other aspect of lymphatic EC biology, by outside-in signaling through D6 on ligand binding. Lymphangiogenesis is poorly understood, although a number of potential regulatory molecules have been identified:42-45 for example, the development of the lymphatic vasculature is aberrant in mice lacking the transcription factor Prox1,42 whereas the ligand for VEGFR-3, VEGF-C, induces lymphatic vessel hyperplasia when transgenically expressed in mouse skin45 and may play a role in lymphatic vessel regeneration in healing wounds.43 It remains to be seen if D6 and its chemokine ligands contribute to developmental or regenerative lymphangiogenesis.
We have shown here that D6 expression is restricted to subsets of lymphatic vessels. Although podoplanin is expressed on a large proportion of these vessels in a wide variety of tissues, D6 immunoreactivity is restricted to a selection of vessels in the gut, skin, and secondary lymphoid tissue. These results suggest phenotypic heterogeneity among lymphatic vessels, an observation not without precedent as the expression of certain adhesion molecules differs between lymphatic vessels.5-7 It is not known whether this reflects differences in the maturation status of the lymphatic ECs, or is a consequence of the environment in which an individual lymphatic vessel finds itself. It is interesting to note that examination of the murine D6 promoter sequence suggests that it is likely to be responsive to a range of inflammatory stimuli (R Nibbs, unpublished observation). Inflammation-regulated expression of D6 would be in keeping with the fact that the chemokines bound by D6 are expressed predominantly during inflammatory reactions. Lymphatic vessels draining inflamed sites, or tissues exposed to the external environment, may have different functional requirements in terms of molecule and/or cell movement, and this may be in part regulated by D6 presentation, sequestration, or transport of pro-inflammatory ß-chemokines. Finally, it is an interesting possibility that D6-immunoreactive lymphatic vessels in lymphoid tissue are the termini of D6-immunoreactive lymphatic vessels in the adjacent draining tissue, such that expression of this phenotype is present along an entire branch of the lymphatic vasculature. Inductive signals transported within the afferent lymph, akin to those that are required to maintain the properties of ECs lining HEVs,39 may likewise be necessary to maintain lymphatic vessel EC phenotypes. These questions need to be addressed by further experimentation.
Anti-D6 antibodies are likely to be of use in immunohistochemically identifying a subset of lymphatic ECs. We have also used them here to examine D6 immunoreactivity in vascular tumors. Our experiments with a small group of vascular tumors show 11 out of 15 with detectable D6 immunoreactivity. The same 11 samples also stained with the podoplanin antibodies. In most cases, within a tumor section, there was only expression of these lymphatic EC markers in a proportion of the cells, with podoplanin consistently expressed in a larger proportion of the cells than D6. Also, there was considerable variation in the expression level of D6 between and within tumor samples. Although D6 and podoplanin immunoreactivity may indicate that the tumor is derived entirely, or at least in part, from the lymphatic endothelium, similar experiments by others suggest that this simple interpretation may not be valid and that much ectopic transcription is seen in these tumors.1,2,4,35 VEGFR-3/Flt4 for instance, which is restricted to lymphatics in normal adult tissue, is reported to be widely expressed in vascular tumors that have a clear blood vessel EC origin.35 Its presence in vascular tumors is certainly not indicative of their derivation from lymphatic ECs, and in fact, VEGFR-3/Flt4 can be expressed by blood vessel ECs during development and tumor angiogenesis.46,47 Similarly, podoplanin expression in vascular tumors was often coincident with the expression of blood vessel EC markers.4 In each case, as with D6, expression of the marker was often seen only in a proportion of the tumor cells.1,2,4,35 These experiments suggest that rather than simply acting as an indicator of the source of the tumor, the expression of lymphatic EC markers may be because of the process of cellular transformation, or the cytokine milieu of the tumor, aberrantly up-regulating their expression. Whether lymphatic EC proteins are involved in the development of vascular tumors is uncertain, but the presence of D6 could conceivably alter a tumor’s response to locally produced chemokines, or affect leukocyte infiltration of the tumor. An examination of the role of D6 in vascular tumorigenesis awaits further insight into the biological function of this receptor.
In summary, we have shown that the ß-chemokine receptor, D6, can be expressed by lymphatic ECs and within a subset of vascular tumors. Further experiments will shed light on the function of D6 and how this impinges on the biology of the lymphatic vasculature and leukocyte trafficking.
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Supported by grants from the Cancer Research Campaign, United Kingdom (to R. J. B. N and G. J. G.); and the ICP Program of the Austrian Ministry for Research and Transport (to E. K. and D. M.).
Accepted for publication November 21, 2000.
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