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VASCULAR BIOLOGY
An important role of lymphatic vessel activation in limiting acute inflammation
Reto Huggenberger,1 Shoib S. Siddiqui,1 Daniela Brander,1 Stefan Ullmann,1 Kathrin Zimmermann,1 Maria Antsiferova,2
Sabine Werner,2 Kari Alitalo,3 and Michael Detmar1
1Institute
of Pharmaceutical Sciences and 2Institute of Cell Biology, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland;
and 3Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
In contrast to the established role of
blood vessel remodeling in inflammation,
the biologic function of the lymphatic
vasculature in acute inflammation has
remained less explored. We studied 2 established models of acute cutaneous inflammation, namely, oxazolone-induced
delayed-type hypersensitivity reactions
and ultraviolet B irradiation, in keratin
14-vascular endothelial growth factor
(VEGF)-C and keratin 14-VEGF-D transgenic mice. These mice have an expanded network of cutaneous lymphatic
vessels. Transgenic delivery of the lymphangiogenic factors VEGF-C and the
VEGFR-3 specific ligand mouse VEGF-D
significantly limited acute skin inflammation in both experimental models, with a
strong reduction of dermal edema. Expression of VEGFR-3 by lymphatic endothelium was strongly down-regulated at
the mRNA and protein level in acutely
inflamed skin, and no VEGFR-3 expression was detectable on inflamed blood
vessels and dermal macrophages. There
was no major change of the inflammatory
cell infiltrate or the composition of the
inflammatory cytokine milieu in the inflamed skin of VEGF-C or VEGF-D transgenic mice. However, the increased network of lymphatic vessels in these mice
significantly enhanced lymphatic drainage from the ear skin. These results provide evidence that specific lymphatic
vessel activation limits acute skin inflammation via promotion of lymph flow from
the skin and reduction of edema formation. (Blood. 2011;117(17):4667-4678)
Introduction
Acute and chronic inflammatory processes are associated with
the growth and/or enlargement of blood and lymphatic vessels.1
Indeed, vascular remodeling is a hallmark of a plethora of
inflammatory diseases, such as chronic airway inflammation,
rheumatoid arthritis, inflammatory bowel disease, and the chronic
inflammatory skin disease psoriasis.2-5 We previously identified an
important role of the blood vasculature and in particular of vascular
endothelial growth factor (VEGF)-A in the promotion of acute and
chronic inflammatory reactions in different experimental skin
inflammation models.6-11 Recently, we found that activation of
VEGFR-3 had a potent anti-inflammatory effect in a mouse model
of psoriasis.12 Conversely, inhibition of VEGFR-3 significantly
prolonged edema and inflammation during chronic bacterial airway
inflammation, in chronic inflammatory arthritis, and in chronic skin
inflammation.3,12,13 However, it has also been reported that the
lymphatic vasculature plays an active role in promoting corneal
and kidney transplant rejections, in part by facilitating dendritic
cell transport to draining lymph nodes.14,15 Furthermore, the
inflamed lymphatic endothelium might actively modulate immune
responses.16,17 Together, these results indicate an important role of
blood vessel angiogenesis in sustaining inflammation, whereas the
functional role of the lymphatic vasculature in acute inflammation
has remained less explored.
The cutaneous lymphatic vasculature is involved in the afferent
immune response and also maintains tissue fluid homeostasis.18-20
Among the VEGF family members, VEGF-C and VEGF-D are the
best described lymphangiogenic factors to date. Their receptor,
VEGFR-3, is mainly expressed on the lymphatic endothelium in the
adult.21 Transgenic overexpression of VEGF-C or of VEGF-D in
keratinocytes under control of the keratin 14 (K14) promoter leads
to the increased formation of lymphatic vessels in the skin, whereas
mice that overexpress a soluble VEGFR-3 under control of the
K14 promoter show edema and lack lymphatic vessels in the skin.22-24
To investigate the biologic role of lymphatic vessels in acute
inflammation, we first induced acute delayed-type hypersensitivity
(DTH) reactions in K14-VEGF-C and K14-VEGF-D transgenic
(Tg) mice and compared the course of skin inflammation with
that observed in wild-type mice. During DTH reactions, antigenpresenting cells take up the antigen in the skin and migrate to the
regional lymph nodes where they interact with T helper cells to
initiate the inflammatory reaction.25 We then investigated the acute
inflammatory reactions induced by a single irradiation of the skin
of transgenic and wild-type mice with a physiologically relevant
dose of ultraviolet B (UVB; 200 mJ/cm2 ⬃ 3 minimal erythema
doses7). Antigen-presenting cells are dispensable for UVB-induced
inflammation, and exposure to UVB light might even suppress
DTH reactions.26 We further investigated VEGFR-3 expression,
lymphatic and blood vessel morphology, inflammatory cell infiltration, and lymphatic drainage function during acute inflammation
and compared those parameters with noninflamed skin. Overall,
our studies reveal that an expanded lymphatic network limits acute
skin inflammation and reduces dermal edema formation, without
major effects on the inflammatory cell infiltration and the production of inflammatory cytokines.
Submitted October 28, 2010; accepted February 14, 2011. Prepublished online
as Blood First Edition paper, March 1, 2011; DOI 10.1182/blood-2010-10-316356.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
4667
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4668
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
HUGGENBERGER et al
Methods
Mouse models of acute skin inflammation
The generation of K14-VEGF-C Tg mice that express human VEGF-C under
control of the K14 promoter and of K14-VEGF-D Tg mice that express mouse
VEGF-D has been described previously.22,23 K14-VEGF-C and K14-VEGF-D
Tg mice (both FVB genetic background) were bred and housed in the animal
facility of ETH Zurich. The mice were genotyped as previously described.22,23
FVB wild-type littermates were used as controls. All experiments were initiated
when the mice were between 6 and 8 weeks of age. Experiments were performed
in accordance with animal protocols approved by the local veterinary authorities
(Kantonales Veterinäramt Zürich).
Acute DTH reactions and UVB irradiation of the skin of mice were
performed as described in supplemental Data (available on the Blood Web
site; see the Supplemental Materials link at the top of the online article).
Mice were killed 2 days after oxazolone challenge or UVB irradiation, and ear and back skin samples were embedded in optimal cutting
temperature compound (Sakura Finetek). Other ear and back skin samples
were stored in RNAlater solution (Applied Biosystems) for subsequent
RNA and protein extractions. All experiments were performed at least twice
with comparable results.
Immunofluorescence
Tissues were embedded in optimal cutting temperature compound, frozen
on liquid nitrogen, and 7-␮m cryostat sections were cut. Specimens were
placed on glass slides, air dried, and fixed with acetone for 2 minutes at
⫺20°C. After rehydration with 80% methanol at 4°C, phosphate-buffered
saline (PBS), and PBS with 12% bovine serum albumin, the specimens
were incubated with the respective antibodies. Standard hematoxylin and
eosin stainings were performed, and immunofluorescence was performed as
described,9,12 using the following antibodies: anti–mouse LYVE-1 (Angiobio),
anti–mouse CD31 (BD Biosciences), anti–mouse MECA-32 (BD Pharmingen), anti–mouse VEGFR-3 (AF743; R&D Systems), and biotin anti–
mouse CD11b (BD Biosciences PharMingen). Alexa488- or Alexa594coupled secondary antibodies and Hoechst 33342 were purchased from
Invitrogen.
Image acquisition and preparation
All digital images were examined on an Axioshop 2 mot plus microscope (Carl
Zeiss), equipped with an AxioCam MRc camera and a Plan-APOCHROMAT
10⫻/0.45 (for blood and lymphatic vessel analysis) and a Plan-NEOFLUAR
20⫻/0.50 objective (for all figure preparations; both Carl Zeiss). Images were
acquired using Axio-Vision software Version 4.7.1 (Carl Zeiss). All figures
were prepared using Adobe Photoshop CS4 extended Version 11.0.2.
Computer-assisted morphometric analyses
Immunofluorescence stains of ear and back skin sections for CD31⫹/
LYVE-1⫹ lymphatic vessels and for MECA-32⫹ blood vessels27 were
examined on an Axioskop 2 mot plus microscope (Carl Zeiss), equipped
with an AxioCam MRc camera and a Plan-APOCHROMAT 10⫻/0.45
and a Plan-NEOFLUAR 20⫻/0.50 objective (Carl Zeiss). Images of
5 individual fields of view were acquired per section using AxioVision
software, Version 4.7.1 (Carl Zeiss). Computer-assisted analyses of digital
images were performed using the IP-LAB software (Scanalytics) as
described.10,12 The average number of CD31⫹/LYVE-1⫹ lymphatic vessels
and MECA-32⫹ blood vessels per millimeter epidermal basement membrane and the average size of vessels were determined in the area between
cartilage and stratum corneum. In back skin, the vessels were quantified in a
defined area, protruding 200 ␮m into the dermis from the basement
membrane. The results are expressed as vessel number per millimeter
epidermal basement membrane and not as vessel number per area, because
the formation of inflammatory edema (increase in area) would confound the
vessel number if it were calculated per area. To quantify CD11b⫹ cell
numbers per millimeter epidermal basement membrane, images of 3 or
4 individual fields of view were acquired per sample (covering the entire field of
view, between the cartilage backbone and the epidermis in the ear, and in an area
250 ␮m distant from the basement membrane in back skin samples).
Isolation of dermal lymphatic endothelial cells by FACS
Isolation, RNA extraction, and amplification of sorted lymphatic endothelial cells were performed as described in supplemental Data.
Quantitative real-time RT-PCR
Total cellular RNA was isolated from mouse ears or back skin using a
TissueLyser, stainless steel beads, and the RNeasy Mini Kit (all from
QIAGEN), and was treated with RQ1 RNase-free DNase (Promega). A total
of 1 ␮g RNA was used to synthesize cDNA using the High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems). The expression of mouse
CCL2, CXCL2, interferon-␥, interleukin-1␤ (IL-1␤), S100A8, S100A9, tumor
necrosis factor-␣, and VEGFR-3 was investigated by TaqMan or SYBR
Green real-time reverse-transcribed polymerase chain reaction (RT-PCR)
using the AB 7900 HT Fast Real-Time PCR System (Applied Biosystems)
and quantified using the 2⫺⌬⌬Ct method.28 The probes and primers for
CCL2 (Mm99999056_m1), CXCL2 (Mm00436450_m1), interferon-␥
(Mm00801778_m1), IL-1␤ (Mm99999061_mH), S100A8 (Mm00496696_g1),
S100A9 (QuantiTect; QT00105252), tumor necrosis factor-␣ (QuantiTect;
QT00104006), and VEGFR-3 (Mm00433337_m1) were predesigned. Each
reaction was multiplexed (TaqMan) or run (SYBR Green) with ␤-actin
(reference sequence NM_007393.1 or QT01136772; all Applied Biosystems or QIAGEN) as a reference gene, and all data were normalized based
on the expression levels of ␤-actin; N ⫽ 3 to 10 per group.
ELISA
Skin lysates were obtained from ear skin of wild-type, K14-VEGF-C, and
K14-VEGF-D Tg mice at 2 days after oxazolone challenge or UVB
irradiation (n ⫽ 5-7 per group), and of untreated wild-type, K14-VEGF-C,
and K14-VEGF-D Tg mice (n ⫽ 3 per group). Tissues were homogenized in lysis buffer (150mM NaCl, 50mM Tris, pH 7.5) with a protease
inhibitor cocktail (Roche Diagnostics). Homogenates were centrifuged
for 10 minutes at 14 000g. Supernatants were stored at ⫺80°C until
assayed. The fluorescence-activated cell sorting (ELISA) kits for CCL2 and
IL-1␤ were purchased from RayBiotech, the VEGF-A Quantikine ELISA
kit from R&D Systems. The absorbance was measured with an Infinite
M200 microplate reader (Tecan). Protein levels were normalized per
milligram of tissue.
Lymph flow assessment using Evans blue dye
A total of 3 ␮L of 1% Evans blue dye solution in PBS (Sigma-Aldrich) was
injected into the ear skin of anesthetized wild-type (n ⫽ 10), K14-VEGF-C
(n ⫽ 5), and K14-VEGF-D Tg (n ⫽ 5) mice without inflammation, or into
noninflamed back skin of wild-type (n ⫽ 6) and K14-VEGF-C Tg mice
(n ⫽ 7) using a Hamilton syringe. After 16 hours, the mice were killed.
Evans blue dye was extracted from the ears or from 6-mm punch biopsies of
the back skin by incubating them at 55°C for 5.5 hours in formamide
(Fluka). The background-subtracted absorbance was measured with an
Infinite M200 microplate reader by measuring at 620 nm and 740 nm. The
concentration of dye in the extracts was calculated from a standard curve of
Evans blue in formamide and is presented as absolute amount of dye that
remained in the ear skin or back skin punch biopsy.
In addition, wild-type (n ⫽ 20), K14-VEGF-C (n ⫽ 10), and K14-VEGF-D
(n ⫽ 9) Tg mice were sensitized and challenged with oxazolone solution or
irradiated with 200 mJ/cm2 UVB light. Then, 32 hours after oxazolone
challenge or UVB irradiation, the mice were anesthetized and injected into
the ear skin with Evans blue dissolved in PBS as described in the previous
paragraph. Evans blue was extracted 16 hours after injection of the dye
using formamide. The absorbance was measured as described in the
previous paragraph.
Measurement of vascular leakage
Oxazolone sensitized and challenged wild-type (n ⫽ 4) and K14-VEGF-C
Tg (n ⫽ 5) mice were anesthetized as described in supplemental Data and
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BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
were injected into the tail vein with 1% Evans blue dye solution in PBS at
2 days after oxazolone challenge. After 30 minutes, the mice were killed
and Evans blue was extracted by incubating the ears at 55°C for 24 hours in
formamide. The background-subtracted absorbance was measured as
described in “Lymph flow assessment using Evans blue dye,” and the
concentration of Evans blue in formamide was indicated.
LYMPHANGIOGENESIS IN ACUTE SKIN INFLAMMATION
4669
mal hyperplasia, inflammatory cell infiltration, and dermal edema
at 2 days after irradiation (Figure 1E). In contrast, dermal edema
was markedly less pronounced in K14-VEGF-C and K14-VEGF-D
Tg mice (Figure 1E).
Acute skin inflammation induces lymphatic and
blood vessel remodeling
Statistical analyses
Statistical analyses were performed using SPSS, Version 16.0 software
or the statistical functions of Excel 2002 (Microsoft Corporation). Data are
shown as mean ⫾ SD or ⫾ SEM as indicated and were analyzed with a
2-tailed, unpaired Student t test. When more than 2 groups were compared,
analysis of variance was applied and the individual groups were compared using
the Tukey-HSD post-hoc test. Homogeneity of variances was assessed using the
Levene test, and normalized distribution was assessed using Q-Q plots. Differences were considered statistically significant when P was ⱕ .05.
Results
Activation of lymphatic vessels reduces
edema formation during acute skin inflammation
We first investigated the biologic role of lymphatic vessels in
2 different models of acute skin inflammation, using K14-VEGF-C and
K14-VEGF-D Tg mice that are characterized by a dense lymphatic
vessel network in their skin.22,23 K14-VEGF-C and K14-VEGF-D
Tg mice and their wild-type littermates were either subjected to
DTH reactions, induced by sensitization with oxazolone solution
and, 5 days later, challenged on both sides of the ears by topical
application of oxazolone,9 or were irradiated with a physiologic
dose of UVB light, an established model of non–immune-mediated
acute inflammation.29
We found that K14-VEGF-C mice showed significantly less
pronounced inflammatory ear swelling during the first 3 days after
oxazolone challenge, compared with their wild-type littermates
(Figure 1A). Inflammatory ear swelling was also significantly
lower in K14-VEGF-D Tg mice at days 2, 4, and 5 after challenge
(Figure 1A). Because the increased preexisting lymphatic vessel
network might have modulated the sensitization phase in the
DTH model, we next irradiated mice with 200 mJ/cm2 UVB light,
as a second model of acute skin inflammation. We found that the
increase in ear thickness that occurs within 24 to 48 hours after
UVB irradiation as a result of inflammation was significantly lower
in both K14-VEGF-C and K14-VEGF-D Tg mice compared with
wild-type littermates, and remained lower until day 8 (Figure 1B).
Because we observed the strongest reduction of ear thickness in
the DTH model after 2 days (K14-VEGF-C, 19%, P ⱕ .001;
K14-VEGF-D, 13%, P ⱕ .001); and because there were major
differences in ear thickness also at 2 days after UVB irradiation
(K14-VEGF-C, 31%, P ⱕ .001; K14-VEGF-D, 14%, P ⱕ .01), we
performed all further morphologic characterizations at this time
point. Hematoxylin and eosin stains of inflamed ear tissue sections
obtained from wild-type mice showed the typical epidermal
hyperplasia at 2 days after oxazolone challenge compared with
noninflamed wild-type mice (Figure 1C-D). Furthermore, there
was a marked inflammatory cell infiltration and dermal edema. In
contrast, dermal edema and epidermal thickening were markedly
less pronounced in K14-VEGF-C and in K14-VEGF-D Tg mice
(Figure 1D). In general, the reduction of swelling appeared to be
more pronounced in K14-VEGF-C Tg mice than in K14-VEGF-D
Tg mice. Similarly, hematoxylin and eosin stains of sections from
UVB-irradiated mouse ears of wild-type mice showed mild epider-
Because we observed a strongly reduced edema formation in the
inflamed skin of K14-VEGF-C and K14-VEGF-D Tg mice and
because fluid drainage represents a major function of lymphatic
vessels, we next investigated whether there might be changes in the
lymphatic vasculature of these mice. Computer-based morphometric analyses of immunofluorescence stains for the lymphatic
marker LYVE-1 revealed that untreated K14-VEGF-C mice had a
significantly increased number (2.0-fold, P ⱕ .01) and average size
(2.8-fold, P ⱕ .05) of cutaneous CD31⫹/LYVE-1⫹ lymphatic vessels compared with their wild-type littermates (Figure 2A-C).
K14-VEGF-D Tg mice also showed an increase in the average size
(1.5-fold, P ⱕ .05) of lymphatics, although these changes were less
pronounced than in VEGF-C Tg mice (Figure 2B-C). During acute
skin inflammation, 2 days after oxazolone challenge, the average
lymphatic vessel size was significantly increased in wild-type,
K14-VEGF-C, and K14-VEGF-D Tg mice, compared with their
noninflamed corresponding genotypes (Figure 2B-C): 334 ␮m2
versus 1179 ␮m2, P ⱕ .001 (noninflamed vs inflamed wild-type);
919 ␮m2 versus 3065 ␮m2, P ⱕ .05 (noninflamed vs inflamed
K14-VEGF-C); and 514 ␮m2 versus 2044 ␮m2, P ⱕ .01 (noninflamed vs inflamed K14-VEGF-D), whereas their numbers were
not further increased over those observed in noninflamed skin
(Figure 2A). Interestingly, although there was less edema in acutely
inflamed K14-VEGF-C and K14-VEGF-D Tg mice and although
the size of lymphatic vessels was already larger in the normal skin
of these mice than in wild-type mice, the relative fold increase of
lymphatic vessel size was comparable in all genotypes (Figure 2B):
3.5-fold in wild-type mice, 3.3-fold in K14-VEGF-C Tg mice,
and 4.0-fold in K14-VEGF-D Tg mice. Similarly, untreated
K14-VEGF-C and K14-VEGF-D Tg mice had a significantly
increased number (K14-VEGF-C 3.2-fold, P ⱕ .001; K14-VEGF-D
1.6-fold, P ⱕ .05) and average size (K14-VEGF-C 1.9-fold,
P ⱕ .01; K14-VEGF-D 1.3-fold, P ⱕ .05) of lymphatic vessels
in their back skin, compared with their wild-type littermates
(Figure 2D-E). At 2 days after UVB irradiation, the average
lymphatic vessel size was significantly increased in wild-type,
K14-VEGF-C, and K14-VEGF-D Tg mice, compared with their
noninflamed corresponding genotypes (Figure 2E): 107 ␮m2 versus
214 ␮m2, P ⱕ .01 (noninflamed vs inflamed wild-type); 207 ␮m2
versus 446 ␮m2, P ⱕ .01 (noninflamed vs inflamed K14-VEGF-C);
and 140 ␮m2 versus 298 ␮m2, P ⱕ .001 (noninflamed vs inflamed
K14-VEGF-D), whereas their numbers were not further increased
over those observed in noninflamed skin (Figure 2D).
We next investigated morphologic changes of cutaneous blood
vessels, using immunostains for the blood vessel marker MECA32.27 We found that, in contrast to the lymphatic vasculature,
the number and size of blood vessels were not significantly
different in the untreated, noninflamed skin of K14-VEGF-C and
of K14-VEGF-D Tg mice, compared with their wild-type littermates (Figure 3A-B). There was a slight increase in the number of
blood vessels in mice of all genotypes at 2 days after oxazolone
challenge (wild-type, 1.2-fold, P ⫽ .08; K14-VEGF-C, 1.3-fold,
P ⱕ .05; K14-VEGF-D, 1.4-fold, P ⱕ .05), whereas the average
size of cutaneous blood vessels was significantly increased in
mice of all genotypes (Figure 3B; wild-type, 1.6-fold, P ⱕ .01;
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HUGGENBERGER et al
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
Figure 1. Lymphatic vessel activation reduces edema
during acute skin inflammation. (A) K14-VEGF-C
(F, n ⫽ 7), K14-VEGF-D (F, n ⫽ 8) Tg mice, and
their wild-type littermates (䡺, n ⫽ 12) were painted
with 2% oxazolone and challenged, 5 days later, with
1% oxazolone on the ears. The ear thickness of
K14-VEGF-C and K14-VEGF-D Tg mice was significantly reduced compared with wild-type controls at the
indicated time points. (B) K14-VEGF-C (F, n ⫽ 7),
K14-VEGF-D (F, n ⫽ 5) Tg mice, and their wild-type
littermates (䡺, n ⫽ 11) were irradiated with 200 mJ/cm2
UVB light, and the ear thickness was measured
using calipers. The ear thickness of K14-VEGF-C and
K14-VEGF-D Tg mice was significantly reduced compared with wild-type controls until day 8 after UVB
irradiation. (A-B) Data are mean ⫾ SEM. *P ⱕ .05.
**P ⱕ .01. ***P ⱕ .001. (C-E) Hematoxylin and eosin
stains of untreated mouse ears (C), at day 2 after
oxazolone challenge (2-day oxa, D) or UVB irradiation
(2-day UVB, E) revealed reduced edema in inflamed
K14-VEGF-C and K14-VEGF-D Tg mice, compared
with inflamed skin of wild-type mice. One ear-half is
shown. Bars represent 100 ␮m.
K14-VEGF-C, 1.3-fold, P ⱕ .05; K14-VEGF-D, 1.4-fold, P ⱕ .001).
However, there was no significant difference in blood vessel number
and size between wild-type, K14-VEGF-C, and K14-VEGF-D Tg
mice at 2 days after oxazolone challenge (Figure 3A-B). Furthermore, there was also no difference in blood vessel number and
blood vessel size between all genotypes at 2 days after UVB
irradiation (Figure 3C-D).
Down-regulation of VEGFR-3 during acute skin inflammation
To investigate whether the chronic release of the lymphangiogenic
factors VEGF-C or VEGF-D in the Tg mice affects the expression
of their common receptor VEGFR-3 on lymphatic endothelium, we
next investigated VEGFR-3 expression by real-time RT-PCR,
fluorescence-activated cell sorting (FACS), and immunofluorescence stains. We found that VEGFR-3 mRNA levels were significantly increased in untreated ear and back skin of K14-VEGF-C
and K14-VEGF-D Tg mice compared with their wild-type littermates (Figure 4A-B): ear skin (K14-VEGF-C, 5.52-fold, P ⱕ .001;
and K14-VEGF-D, 2.78-fold, P ⱕ .001) and back skin (K14-VEGF-C,
5.44-fold, P ⱕ .001; and K14-VEGF-D, 2.75-fold, P ⱕ .001). The
VEGFR-3 transcript levels were significantly reduced 2 days
after oxazolone challenge in all types of mice (Figure 4A;
wild-type, 4.06-fold, P ⱕ .001; K14-VEGF-C, 3.47-fold, P ⱕ .001;
K14-VEGF-D, 4.24-fold, P ⱕ .001). A similar down-regulation
was seen 2 days after UVB irradiation in mice of all genotypes (Figure 4B; wild-type, 2.52-fold, P ⱕ .001; K14-VEGF-C,
9.30-fold, P ⱕ .001; K14-VEGF-D, 5.33-fold, P ⱕ .001).
The differences observed at the mRNA levels in samples
obtained from total mouse skin might have at least in part been the
result of the different amount of lymphatic vessels in these samples,
and possibly to the different extent of immune cell infiltration in the
inflamed situation. Thus, we next performed FACS analyses in
mice to specifically assess VEGFR-3 expression on lymphatic
endothelial cells. CD31⫹/podoplanin⫺/CD45⫺ cells represent
blood vascular endothelial cells, whereas CD31⫹/podoplanin⫹/
CD45⫺ cells represent lymphatic endothelial cells (Figure 4C).
Importantly, we found, by real-time RT-PCR, that the VEGFR-3
transcript levels in lymphatic endothelial cells of wild-type mice
were 4.86-fold (P ⱕ .001) lower at 2 days after oxazolone challenge than in noninflamed skin (Figure 4D).
In agreement with these findings, double immunofluorescence
analyses for the expression of VEGFR-3 and LYVE-1 revealed a
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BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
LYMPHANGIOGENESIS IN ACUTE SKIN INFLAMMATION
4671
Figure 2. Enlargement of cutaneous lymphatic vessels during acute inflammation. (A-B) Quantitative image analyses of CD31⫹/LYVE-1⫹ lymphatic vessels in the ear
skin of mice revealed a significantly increased number per millimeter basement membrane (BM, A) and size (B) of lymphatic vessels in untreated K14-VEGF-C Tg mice,
compared with untreated wild-type mice (n ⫽ 3 mice per group). The lymphatic vessel size was also increased in untreated K14-VEGF-D Tg mice, compared with untreated
wild-type mice (n ⫽ 3 mice per group). The average number of lymphatic vessels in wild-type mice was not significantly different at 2 days after oxazolone challenge (2-day
oxa) compared with untreated wild-type mice. The lymphatic vessel number was also not significantly different between untreated and oxazolone-challenged K14-VEGF-C and
K14-VEGF-D Tg mice, respectively (A; wild-type, n ⫽ 10; K14-VEGF-C, n ⫽ 5; K14-VEGF-D, n ⫽ 5). At 2 days after oxazolone challenge, the average size of lymphatic
vessels was significantly increased in K14-VEGF-C, K14-VEGF-D Tg, and wild-type mice, compared with untreated mice of the same genotype (B). (C) Representative images
of CD31⫹/LYVE-1⫹ lymphatic vessels (green) in the ear skin. CD31⫹/LYVE-1⫺ structures represent blood vessels (red). The positive staining of LYVE-1 in the stratum corneum
in panel C is unspecific. Bars represent 100 ␮m. (D-E) The average lymphatic vessel number (D) and size (E) were significantly increased in the back skin of untreated
K14-VEGF-C and K14-VEGF-D Tg mice, compared with untreated wild-type mice. At 2 days after UVB irradiation (2-day UVB), lymphatic vessel size (E), but not lymphatic
vessel number (D), was significantly increased in K14-VEGF-C, K14-VEGF-D, and wild-type mice compared with untreated mice of the same genotype. (A-B,D-E) Data are
mean ⫾ SD. ‡P ⱕ .05. ‡‡P ⱕ .01. ‡‡‡P ⱕ .001. ns indicates not significant versus untreated wild-type. *P ⱕ .05. **P ⱕ .01. ***P ⱕ .001. ns indicates not significant versus
untreated mice (untreated wild-type vs 2-day oxa/2-day UVB wild-type; untreated VEGF-C vs 2-day oxa/2-day UVB VEGF-C; untreated VEGF-D vs 2-day oxa/2-day UVB
VEGF-D).
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HUGGENBERGER et al
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
Figure 3. Enlargement of blood vessels during
acute skin inflammation. (A-B) Immunofluorescence
analyses for the blood vessel-specific marker MECA-32
and subsequent morphometric quantification showed a
significant increase in blood vessel number per millimeter basement membrane (BM) in K14-VEGF-C and
K14-VEGF-D Tg mice at 2 days after oxazolone challenge (2-day oxa), compared with untreated mice (A).
The blood vessel size was also increased in all 3 groups
of mice at 2 days of oxazolone challenge, compared
with untreated mice of the same genotype (B). There
was no significant difference in blood vessel number
(A,C) or blood vessel size (B,D) at 2 days after
oxazolone challenge or UVB irradiation (2-day UVB)
between K14-VEGF-C, K14-VEGF-D Tg, and wild-type
mice. (A-B) Quantification of ear skin. (C-D) Quantification of back skin. (A-D) Data are mean ⫾ SD. *P ⱕ .05.
**P ⱕ .01. ***P ⱕ .001. ns indicates not significant versus untreated mice of the same genotype.
pronounced down-regulation of VEGFR-3 protein on LYVE-1⫹
lymphatic vessels in wild-type mice at 2 days after oxazolone
challenge, compared with noninflamed skin (Figure 4E). In addition, VEGFR-3 protein was almost completely absent on LYVE-1⫹
lymphatic vessels in wild-type mice 2 days after UVB irradiation,
whereas lymphatic vessels express VEGFR-3 in noninflamed skin of
wild-type mice (Figure 4F). VEGFR-3 expression was also absent on
cutaneous MECA-32⫹ blood vessels and CD11b⫹ cells in normal and
inflamed wild-type mice (data not shown). Furthermore, K14-VEGF-C
and K14-VEGF-D Tg mice showed a pronounced down-regulation of
VEGFR-3 on LYVE-1⫹ lymphatic vessels 2 days after oxazolone
challenge or UVB irradiation, compared with noninflamed transgenic
littermates (supplemental Figures 1-2).
Differential effects of VEGF-C and VEGF-D on
inflammatory cytokine expression
We next investigated whether the reduced edema formation in
the inflamed skin of K14-VEGF-C and K14-VEGF-D Tg mice
was associated with reduced expression of acute inflammation markers. TaqMan- or SYBR Green-based real-time RT-PCR
of total mouse ear and back skin revealed that the inflammation markers S100A8, S100A9, tumor necrosis factor-␣, IL-1␤,
interferon-␥, CCL2, and CXCL2 were all significantly upregulated at 2 days after oxazolone challenge or UVB irradiation, respectively (Figure 5A-B). Interestingly, K14-VEGF-C and
K14-VEGF-D Tg mice did not show reduced transcript levels
of inflammation markers in the skin at 2 days after oxazolone
challenge or UVB irradiation, compared with wild-type mice,
despite strongly reduced edema (Figure 5A-B). The transcript
levels of S100A8, S100A9, and CXCL2 were increased in
K14-VEGF-C Tg mice at 2 days after UVB irradiation (Figure 5B),
compared with wild-type mice. By contrast, protein levels of IL-1␤
and VEGF-A (assessed by ELISA), which were both increased at
2 days after oxazolone challenge, were slightly reduced in
K14-VEGF-C Tg mice compared with challenged wild-type mice
(Figure 5C; IL-1␤, P ⱕ .05; VEGF-A, P ⱕ .01), whereas no such
changes were seen in K14-VEGF-D Tg mice (Figure 5C). The
reduction of IL-1␤ and VEGF-A proteins was not associated with
reduced vascular permeability in K14-VEGF-C Tg mice compared
with wild-type mice at 2 days after oxazolone challenge
(15.1 ⫾ 1.2 ␮g Evans blue/mL formamide vs 16.4 ⫾ 1.9 ␮g Evans
blue/mL formamide; wild-type vs VEGF-C). The levels of all
3 proteins analyzed were not different between UVB irradiated Tg
and wild-type mice, although they were all significantly upregulated compared with noninflamed mice (Figure 5C).
Immunofluorescence and computer-assisted image analyses of
the monocyte/granulocyte marker CD11b in the skin revealed that
there was a strong infiltration of dermal CD11b⫹ cells 2 days after
oxazolone challenge compared with untreated mice (Figure 6A,C).
This increase was not reduced in the skin of K14-VEGF-C
and K14-VEGF-D Tg mice at 2 days after oxazolone challenge
compared with wild-type littermates (Figure 6A,C). Indeed,
there were even more dermal CD11b⫹ cells in the ear skin of
K14-VEGF-C Tg mice (Figure 6A,C; 291 ⫾ 13 cells in wild-type
mice vs 436 ⫾ 30 cells in K14-VEGF-C Tg mice, P ⱕ .001). The
number of CD11b⫹ cells was also significantly increased 2 days
after UVB irradiation (Figure 6B,D). However, there was no
difference in the number of infiltrated CD11b⫹ cells between mice
of all genotypes 2 days after UVB irradiation (Figure 6B,D).
Increased lymph flow in VEGF-C and VEGF-D Tg mice
We next investigated whether enhanced lymphatic drainage might
have contributed to the reduced ear thickness and edema formation
in K14-VEGF-C and K14-VEGF-D Tg mice. To this end, we used
Evans blue dye that was intradermally injected into the noninflamed ear skin of wild-type, K14-VEGF-C, or K14-VEGF-D Tg
mice. Evans blue is specifically taken up by the lymphatic
vasculature after intradermal injection.30 The extraction of Evans
blue from the mouse ear, 16 hours after the injection, revealed that
K14-VEGF-C and K14-VEGF-D Tg mice had significantly less
Evans blue remaining in their noninflamed ear skin than wild-type
mice (Figure 7A; VEGF-C, 34%, P ⱕ .001; VEGF-D, 25%, P ⱕ .01),
indicating an enhanced lymphatic clearance function. Because
there are studies indicating that K14-VEGF-C Tg mice have
retrograde filling of their initial lymphatics,31 we additionally
analyzed the lymph drainage from the back skin of untreated
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BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
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Figure 4. Down-regulation of VEGFR-3 during acute skin inflammation. (A-B) TaqMan-based real-time RT-PCR analyses were performed on whole ear and back skin
extracts after 2 days of oxazolone challenge (2-day oxa; n ⫽ 6-10 per group) or UVB irradiation (2-day UVB; n ⫽ 5-8 per group) and in untreated K14-VEGF-C, K14-VEGF-D
Tg, and wild-type mice (n ⫽ 3-5 per group). VEGFR-3 was significantly up-regulated in untreated K14-VEGF-C and K14-VEGF-D Tg mouse ear (A) and back skin
(B) compared with untreated wild-type mice. VEGFR-3 was significantly down-regulated at 2 days of oxazolone challenge (A) or UVB irradiation (B) in all 3 groups of mice
compared with untreated mice of the same genotype. (C-D) Single-cell suspensions from the ear of normal and oxazolone challenged mice were analyzed by FACS.
(C) CD31⫹/CD45⫺ cells represent endothelial cells, whereas CD31⫹/podoplanin⫹/CD45⫺ cells are lymphatic endothelial cells. (D) Cutaneous lymphatic endothelial cells from
inflamed ears of wild-type mice (2 days after oxazolone challenge) showed a 5-fold decrease of VEGFR-3 mRNA transcript levels compared with lymphatic endothelial cells
from untreated mice. (A-B,D) Data are mean ⫾ SD. ‡‡‡P ⱕ .001 versus untreated wild-type. ***P ⱕ .001 versus untreated mice of the same genotype. (E-F) Double
immunofluorescence analyses of VEGFR-3 (red) and LYVE-1 (green) stains demonstrated that VEGFR-3 was strongly down-regulated on LYVE-1⫹ lymphatic vessels at
2 days after oxazolone challenge (E) or UVB irradiation (F) in the ear and back skin of wild-type mice (arrows). The positive staining of LYVE-1 in the stratum corneum (E) is
unspecific. Bars represent 100 ␮m.
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HUGGENBERGER et al
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
Figure 5. Inflammation marker expression in the skin during acute inflammation. (A-B) Real-time RT-PCR analyses were performed using RNAs from whole ear and
back skin harvested 2 days after oxazolone challenge (2-day oxa; n ⫽ 6-10 per group) or UVB irradiation (2-day UVB; n ⫽ 5-8 per group) and from skin of untreated
K14-VEGF-C, K14-VEGF-D Tg, and wild-type mice (n ⫽ 3-5 per group). All inflammation markers shown were significantly up-regulated in K14-VEGF-C, K14-VEGF-D Tg, and
wild-type mouse ear (A) and back skin (B) 2 days after oxazolone challenge (A) or UVB irradiation (B), compared with untreated wild-type or transgenic mice. S100A8, S100A9,
and CXCL2 mRNA levels were slightly increased in K14-VEGF-C Tg mice 2 days after UVB irradiation compared with irradiated wild-type mice (B). (C) ELISA analyses of ear
lysates showed significantly increased levels of IL-1␤, VEGF-A, and CCL2 at 2 days after oxazolone challenge or UVB irradiation in the ear skin of K14-VEGF-C, K14-VEGF-D
Tg, and wild-type mice compared with untreated mice. The protein levels of IL-1␤ and VEGF-A were slightly but significantly reduced in the ear skin of oxazolone challenged
K14-VEGF-C Tg mice compared with oxazolone challenged wild-type mice. (A-C) Data are mean ⫾ SD. *P ⱕ .05 versus oxazolone challenged or UVB irradiated wild-type
mice. **P ⱕ .01 versus oxazolone challenged or UVB irradiated wild-type mice.
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BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
LYMPHANGIOGENESIS IN ACUTE SKIN INFLAMMATION
4675
Figure 6. Inflammatory cell infiltration during acute
skin inflammation. (A-D) Immunofluorescence analyses for the monocyte/granulocyte marker CD11b and
subsequent computer-based quantification showed a
significantly increased number of dermal CD11b⫹ cells
per millimeter basement membrane (BM) 2 days
after oxazolone challenge (2 days oxa, A,C; ear skin) or
UVB irradiation (2 days UVB, B,D; back skin) in
K14-VEGF-C, K14-VEGF-D Tg, and wild-type mice,
compared with untreated mice of the same genotype.
K14-VEGF-C Tg mice had even more infiltrated dermal
CD11b⫹ cells in their ear skin 2 days after oxazolone
challenge compared with oxazolone challenged wildtype mice (A,C). The hair follicle sebaceous glands are
stained red (C-D) because of endogenous biotin.
(A-D) Data are mean ⫾ SD. ***P ⱕ .001. ns indicates
not significant versus oxazolone challenged or UVBirradiated wild-type mice. Bars represent 100 ␮m (C),
50 ␮m (D).
K14-VEGF-C Tg versus wild-type mice. There was significantly
less Evans blue dye remaining in the back skin of K14-VEGF-C Tg
mice than in wild-type mice 16 hours after injection of the dye
(0.71 ⫾ 0.36 ␮g vs 2.01 ⫾ 0.52 ␮g; VEGF-C vs wild-type;
P ⱕ .001). We also assessed whether there are still differences of
lymph flow between Tg and wild-type mice during acute skin
inflammation. Interestingly, we found that K14-VEGF-C and
K14-VEGF-D Tg mice still drained significantly faster after UVB
irradiation than irradiated wild-type mice (Figure 7B; VEGF-C,
44%, P ⱕ .001; VEGF-D, 22%, P ⱕ .05). There was no difference
in lymph draining capacity between all types of mice during acute
oxazolone challenge (Figure 7C).
Discussion
In this study, we found that specific promotion of lymphatic vessel
function limits acute skin inflammation in mice, without major
effects on the composition of the inflammatory infiltrate and the
tissue cytokine milieu. We used 2 independent experimental models of
acute inflammation, namely, induction of cutaneous DTH reactions
and UVB irradiation of the skin. The DTH model is an immunebased model where antigen-presenting cells are thought to be
essential to initiate the inflammatory reaction.32 Because modulation of lymphatic vessel function in this model might potentially
also have affected the induction phase of acute inflammation via
modulation of the transport of antigen-presenting cells to the
draining lymph nodes, we also performed all studies in parallel in a
second acute inflammation model (ie, UVB irradiation of the skin).
In this model, a single exposure to UVB irradiation (290-320 nm
wavelength) induces inflammatory skin alterations that include
erythema, vascular hyperpermeability, dilation of dermal blood
vessels, and epidermal hyperplasia without the requirement for
prior antigen sensitization.7,25
In both models, VEGF-A probably represents the major driver
of vascular remodeling and hyperpermeability, leading to edema
formation. The amount of VEGF-A is increased in the skin during
acute oxazolone-induced DTH reactions.9 Acute UVB irradiation
also increases cutaneous VEGF-A levels, and mice that overexpress VEGF-A are more sensitive to DTH reactions and UVB
irradiation than wild-type mice.7,9,33 Conversely, we previously
found that systemic blockade of VEGF-A reduces UVB-induced
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HUGGENBERGER et al
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
Figure 7. Increased lymph flow from the ear in K14-VEGF-C and K14-VEGF-D Tg mice. (A-C) Evans blue dye was intradermally injected into the ear of untreated or
inflamed K14-VEGF-C, K14-VEGF-D Tg, and wild-type mice and was extracted 16 hours after the dye injection. The total dye remaining in the ear skin is indicated.
Representative pictures before Evans blue extraction are shown at the bottom. (A) Untreated K14-VEGF-C (n ⫽ 5) and K14-VEGF-D (n ⫽ 5) Tg mice had significantly less
Evans blue remaining in their ear skin 16 hours after injection compared with wild-type mice (n ⫽ 10). (B) Evans blue was also injected 32 hours after UVB irradiation and
extracted 16 hours later. UVB irradiated K14-VEGF-C (n ⫽ 5) and K14-VEGF-D (n ⫽ 4) Tg mice showed a faster lymph flow than irradiated wild-type mice (n ⫽ 10). (C) There
was no difference in Evans blue clearance from the inflamed ear during acute inflammation after oxazolone challenge between all mice (wild-type, n ⫽ 10; K14-VEGF-C,
n ⫽ 5; K14-VEGF-D, n ⫽ 5). Data are mean ⫾ SD. *P ⱕ .05 versus wild-type mice. **P ⱕ .01 versus wild-type mice. ***P ⱕ .001 versus wild-type mice.
inflammation and vascular enlargement and that combined blockade of VEGFR-1 and VEGFR-2 partially reduced acute oxazolonemediated inflammation and vascular remodeling.7,9
In addition to VEGF-A, VEGF-C has also been implicated in
the inflammation-induced vascular remodeling, in particular regarding the lymphatic vasculature.18,34 Epidermal keratinocytes and
macrophages secrete both VEGF-A and VEGF-C, and they participate in lymphatic vessel remodeling during inflammation.5,35,36
Whereas VEGF-A binds to VEGFR-1 and VEGFR-2, VEGF-C
specifically binds to VEGFR-3 and, after proteolytic cleavage of
the propeptides, also binds and activates VEGFR-2.18 In contrast,
murine VEGF-D only activates VEGFR-3.37 Importantly, VEGFR-2
is expressed by both the lymphatic and the blood vascular
endothelium, whereas VEGFR-3 is mainly restricted to the lymphatic vasculature in the adult.21,38 In our studies, we used a genetic
approach (chronic transgenic overexpression of either VEGF-C or
of murine VEGF-D in the skin) to investigate the biologic effects of
an expanded lymphatic vascular network on acute inflammation.
Thus, VEGF-C derived from K14-VEGF-C Tg mice might theoretically also affect the blood vasculature, whereas mouse VEGF-D
specifically activates lymphatic vessels.22 However, in agreement
with a previous report,23 we did not detect any changes of the blood
vasculature in K14-VEGF-C Tg mice. Therefore, our findings that
Tg expression of VEGF-C more potently reduced edema formation
than Tg expression of murine VEGF-D were probably not caused
by effects on blood vessels but by the denser network of dermal
lymphatic vessels in K14-VEGF-C mice. The denser network of
lymphatics in K14-VEGF-C Tg mice might be caused by the
different binding affinities of VEGF-C (dissociation constant ⫽ 1.35 ⫻ 10⫺10M) and VEGF-D (dissociation constant ⫽ 8.9 ⫻ 10⫺8M) to VEGFR-3 or by potential copy number
differences.37,39
The expression of VEGFR-3 has been generally thought to be
restricted to the lymphatic vascular system with the exception of
corneal dendritic cells and some angiogenic blood vessels in
tumors, healing wounds, and during early embryonic development.38,40,41 Our current findings reveal that, in the setting of acute
skin inflammation in mice, VEGFR-3 is strongly down-regulated
(at the mRNA and the protein level) in inflamed, enlarged lymphatic
vessels and is completely absent from the blood vascular endothelium and from cutaneous CD11b⫹ monocytes/granulocytes. These
findings are in contrast to those recently reported in a model of
inflammatory peritonitis, where VEGFR-3 up-regulation on lymphatic endothelium was detected several days before the onset of
lymphangiogenesis.42 These differences might be explained by the
different models and organs studied. Moreover, the repeated
application of thioglycollate every 48 hours in the inflammatory
peritonitis model42 might rather reflect a chronic inflammation
setting, in contrast to the acute inflammation model (48 hours after
induction) of our study, which is characterized by lymphatic
enlargement but not by sprouting lymphangiogenesis. A potential
mechanism might be that down-regulation of Prox1 that also
occurs during acute skin inflammation, at least on the mRNA level
(data not shown), directly down-regulates VEGFR-3 expression on
the lymphatic endothelium by binding to the VEGFR-3 promoter.42-45 Our finding that down-regulation of the lymphaticspecific VEGFR-3 in acute inflammation was comparable in total
ear skin (4-fold) and in FACS-isolated lymphatic endothelial cells
(5-fold) further confirms its lymphatic-specific expression in the
skin. The higher expression of VEGFR-3 in the skin of noninflamed K14-VEGF-C and K14-VEGF-D Tg mice was probably
the result of the increased lymphatic vascular network in these mice
and not to a specific enhancement of VEGFR-3 expression on
lymphatic endothelium. There is additional evidence that lymphatic
function is reduced in acute skin inflammation via a downregulation of the VEGF-C/VEGFR-3 axis because epidermal VEGF-C
expression is also reduced after acute UVB irradiation.46 Moreover, specific inhibition of VEGFR-3 in K14-VEGF-A Tg mice
significantly increased edema formation after UVB irradiation,
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BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
LYMPHANGIOGENESIS IN ACUTE SKIN INFLAMMATION
which was reduced by injection of the VEGFR-3-specific mutant
VEGF-C156S.46,47
Several aspects of cutaneous inflammation could have been
modulated by the increased lymphatic network in K14-VEGF-C or
VEGF-D Tg mice, thereby limiting the inflammatory response.
Interestingly, we found that inflammatory cell infiltration and the
expression of molecular markers and mediators of inflammation
were largely unchanged in K14-VEGF-C and K14-VEGF-D Tg
mice, despite their reduced ear swelling compared with wild-type
littermates. These findings are in agreement with a recent report
that Tg VEGF-C reduced LPS-induced edema but did not reduce
inflammatory cell migration to the draining lymph node.48 Further
studies are needed to investigate whether the increased lymphatic
network in the Tg mice might have trapped inflammatory chemokines by the lymphatic-specific decoy receptor D6.49
Importantly, our findings that the clearance of intradermally
injected Evans blue from the ear skin was significantly faster in
both K14-VEGF-C and K14-VEGF-D Tg mice clearly indicate that
the increased preexisting lymphatic network and the increased
lymphatic drainage were largely responsible for the reduced edema
formation. The drainage-promoting function of VEGF-C/VEGF-D
stimulation in our model is in agreement with recent findings that
application of a soluble VEGFR-3 (which blocks both VEGF-C
and VEGF-D from reaching their receptor on lymphatic endothelium) decreased lymph flow in a model of bacterial inflammation,48
whereas genetic overexpression of soluble VEGFR-3 in the skin of
mice resulted in a lymphedema-like phenotype.24 Although not
assessed in this study, it would be of interest to specifically knock
out VEGFR-3 on adult lymphatic vessels and to compare the
course of induced inflammation with normal wild-type mice, to
further clarify the specific role of VEGFR-3 and its downregulation in the acute inflammatory process.
In conclusion, our study provides the first evidence that specific
lymphatic vessel activation limits acute skin inflammation via
promotion of fluid drainage from the skin and reduction of edema
formation, without major effects on inflammatory cell recruitment
and the inflammatory cytokine milieu. Therefore, the role of lymphatic
4677
vessels in acute and chronic skin inflammation might differ in that
lymphatic vessels also help resolving proinflammatory cells from
the site of inflammation in the latter.12 Our findings that increased
lymphatic function reduces inflammation-induced edema are indeed in line with the well-established clinical observation that
patients with impaired lymphatic function (eg, in congenital or
acquired lymphedemas of the extremities) are more prone to
develop inflammatory skin reactions.50
Acknowledgments
The authors thank Jeannette Scholl and Cornelius Fischer for
excellent technical assistance and Carlos Ochoa for help with the
animal studies.
This work was supported by the National Institutes of Health
(grant CA69184), Swiss National Science Foundation (grants
3100A0-108207 and 31003A-130627), Commission of the European Communities (grant LSHC-CT-2005-518178), Oncosuisse,
and Krebsliga Zurich (M.D.).
Authorship
Contribution: R.H. designed the research, performed experiments,
analyzed results, and wrote the manuscript; S.S.S., D.B., S.U., and
K.Z. performed experiments and analyzed results; M.A. performed
experiments; S.W. designed the research and analyzed results; K.A.
contributed material; and M.D. designed the research, analyzed
results, and wrote the manuscript.
Conflict-of-interest disclosure: K.A. is the Chairman of the
Scientific Advisory Council of Circadian Technologies. The remaining authors declare no competing financial interests.
Correspondence: Michael Detmar, Institute of Pharmaceutical
Sciences, Swiss Federal Institute of Technology, ETH Zurich,
Wolfgang Pauli-Str 10, HCI H303, CH-8093 Zurich, Switzerland;
e-mail: [email protected].
References
1. Halin C, Detmar M. Inflammation, angiogenesis,
and lymphangiogenesis. Methods Enzymol.
2008;445:1-25.
9.
2. Bainbridge J, Sivakumar B, Paleolog E. Angiogenesis as a therapeutic target in arthritis: lessons from oncology. Curr Pharm Des. 2006;
12(21):2631-2644.
3. Baluk P, Tammela T, Ator E, et al. Pathogenesis
of persistent lymphatic vessel hyperplasia in
chronic airway inflammation. J Clin Invest. 2005;
115(2):247-257.
10.
4. Danese S, Sans M, de la Motte C, et al. Angiogenesis as a novel component of inflammatory
bowel disease pathogenesis. Gastroenterology.
2006;130(7):2060-2073.
11.
5. Detmar M, Brown LF, Claffey KP, et al. Overexpression of vascular permeability factor/vascular
endothelial growth factor and its receptors in psoriasis. J Exp Med. 1994;180(3):1141-1146.
12.
6. Halin C, Tobler NE, Vigl B, Brown LF, Detmar M.
VEGF-A produced by chronically inflamed tissue
induces lymphangiogenesis in draining lymph
nodes. Blood. 2007;110(9):3158-3167.
13.
7. Hirakawa S, Fujii S, Kajiya K, Yano K, Detmar M.
Vascular endothelial growth factor promotes sensitivity to ultraviolet B-induced cutaneous photodamage. Blood. 2005;105(6):2392-2399.
8. Kajiya K, Hirakawa S, Detmar M. Vascular endothelial growth factor-A mediates ultraviolet B-
14.
induced impairment of lymphatic vessel function.
Am J Pathol. 2006;169(4):1496-1503.
Kunstfeld R, Hirakawa S, Hong YK, et al. Induction of cutaneous delayed-type hypersensitivity
reactions in VEGF-A transgenic mice results in
chronic skin inflammation associated with persistent lymphatic hyperplasia. Blood. 2004;104(4):
1048-1057.
Schonthaler HB, Huggenberger R, Wculek SK,
Detmar M, Wagner EF. Systemic anti-VEGF treatment strongly reduces skin inflammation in a
mouse model of psoriasis. Proc Natl Acad Sci
U S A. 2009;106(50):21264-21269.
Xia YP, Li B, Hylton D, Detmar M, Yancopoulos GD,
Rudge JS. Transgenic delivery of VEGF to mouse
skin leads to an inflammatory condition resembling
human psoriasis. Blood. 2003;102(1):161-168.
Huggenberger R, Ullmann S, Proulx ST,
Pytowski B, Alitalo K, Detmar M. Stimulation of
lymphangiogenesis via VEGFR-3 inhibits chronic
skin inflammation. J Exp Med. 2010;207(10):
2255-2269.
Guo R, Zhou Q, Proulx ST, et al. Inhibition of
lymphangiogenesis and lymphatic drainage via
vascular endothelial growth factor receptor 3
blockade increases the severity of inflammation
in a mouse model of chronic inflammatory arthritis. Arthritis Rheum. 2009;60(9):2666-2676.
Cursiefen C, Cao J, Chen L, et al. Inhibition of
hemangiogenesis and lymphangiogenesis after
normal-risk corneal transplantation by neutralizing VEGF promotes graft survival. Invest
Ophthalmol Vis Sci. 2004;45(8):2666-2673.
15. Kerjaschki D, Regele HM, Moosberger I, et al.
Lymphatic neoangiogenesis in human kidney
transplants is associated with immunologically
active lymphocytic infiltrates. J Am Soc Nephrol.
2004;15(3):603-612.
16. Podgrabinska S, Kamalu O, Mayer L, et al. Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac-1/
ICAM-1-dependent mechanism. J Immunol.
2009;183(3):1767-1779.
17. Kerjaschki D. Lymphatic neoangiogenesis in renal transplants: a driving force of chronic rejection? J Nephrol. 2006;19(4):403-406.
18. Cueni LN, Detmar M. New insights into the molecular control of the lymphatic vascular system
and its role in disease. J Invest Dermatol. 2006;
126(10):2167-2177.
19. Ruddle NH, Akirav EM. Secondary lymphoid organs: responding to genetic and environmental
cues in ontogeny and the immune response.
J Immunol. 2009;183(4):2205-2212.
20. Tammela T, Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise. Cell.
140(4):460-476.
21. Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the fms-like tyrosine kinase 4 gene
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
4678
BLOOD, 28 APRIL 2011 䡠 VOLUME 117, NUMBER 17
HUGGENBERGER et al
becomes restricted to lymphatic endothelium
during development. Proc Natl Acad Sci U S A.
1995;92(8):3566-3570.
22. Haiko P, Makinen T, Keskitalo S, et al. Deletion of
vascular endothelial growth factor C (VEGF-C)
and VEGF-D is not equivalent to VEGF receptor
3 deletion in mouse embryos. Mol Cell Biol. 2008;
28(15):4843-4850.
23. Jeltsch M, Kaipainen A, Joukov V, et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic
mice. Science. 1997;276(5317):1423-1425.
24. Makinen T, Jussila L, Veikkola T, et al. Inhibition
of lymphangiogenesis with resulting lymphedema
in transgenic mice expressing soluble VEGF
receptor-3. Nat Med. 2001;7(2):199-205.
25. Kripke ML. Ultraviolet radiation and immunology:
something new under the sun—presidential address. Cancer Res. 1994;54(23):6102-6105.
26. Takigawa M, Miyachi Y, Toda K, Yoshioka A.
Mechanisms of contact photosensitivity in mice:
IV. Antigen-specific suppressor T cells induced by
preirradiation of photosensitizing site to UVB.
J Immunol. 1984;132(3):1124-1129.
27. Keuschnigg J, Henttinen T, Auvinen K,
Karikoski M, Salmi M, Jalkanen S. The prototype
endothelial marker PAL-E is a leukocyte trafficking molecule. Blood. 2009;114(2):478-484.
28. Schmittgen TD, Livak KJ. Analyzing real-time
PCR data by the comparative C(T) method.
Nat Protoc. 2008;3(6):1101-1108.
29. Kumin A, Huber C, Rulicke T, Wolf E, Werner S.
Peroxiredoxin 6 is a potent cytoprotective enzyme in the epidermis. Am J Pathol. 2006;169(4):
1194-1205.
30. Liao S, Ruddle NH. Synchrony of high endothelial
venules and lymphatic vessels revealed by immunization. J Immunol. 2006;177(5):3369-3379.
31. Lohela M, Helotera H, Haiko P, Dumont DJ,
Alitalo K. Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in
mouse embryos but leads to persistent lymphatic
hyperplasia in adult tissues. Am J Pathol. 2008;
173(6):1891-1901.
32. Asherson GL, Zembala M. Contact sensitivity
in the mouse: IV. The role of lymphocytes and
macrophages in passive transfer and the mechanism of their interaction. J Exp Med. 1970;132(1):
1-15.
33. Yano K, Kajiya K, Ishiwata M, Hong YK,
Miyakawa T, Detmar M. Ultraviolet B-induced skin
angiogenesis is associated with a switch in the
balance of vascular endothelial growth factor and
thrombospondin-1 expression. J Invest Dermatol.
2004;122(1):201-208.
34. McDonald DM. Angiogenesis and remodeling of
airway vasculature in chronic inflammation. Am J
Respir Crit Care Med. 2001;164(10):S39-S45.
35. Cursiefen C, Chen L, Borges LP, et al. VEGF-A
stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via
macrophage recruitment. J Clin Invest. 2004;
113(7):1040-1050.
36. Kerjaschki D, Huttary N, Raab I, et al. Lymphatic
endothelial progenitor cells contribute to de novo
lymphangiogenesis in human renal transplants.
Nat Med. 2006;12(2):230-234.
37. Baldwin ME, Catimel B, Nice EC, et al. The specificity of receptor binding by vascular endothelial
growth factor-d is different in mouse and man.
J Biol Chem. 2001;276(22):19166-19171.
38. Partanen TA, Arola J, Saaristo A, et al. VEGF-C
and VEGF-D expression in neuroendocrine cells
and their receptor, VEGFR-3, in fenestrated blood
vessels in human tissues. FASEB J. 2000;14(13):
2087-2096.
39. Joukov V, Sorsa T, Kumar V, et al. Proteolytic processing regulates receptor specificity and activity
of VEGF-C. EMBO J. 1997;16(13):3898-3911.
40. Hamrah P, Chen L, Zhang Q, Dana MR. Novel
expression of vascular endothelial growth factor
receptor (VEGFR)-3 and VEGF-C on corneal
dendritic cells. Am J Pathol. 2003;163(1):57-68.
41. Tammela T, Zarkada G, Wallgard E, et al. Block-
ing VEGFR-3 suppresses angiogenic sprouting
and vascular network formation. Nature. 2008;
454(7204):656-660.
42. Flister MJ, Wilber A, Hall KL, et al. Inflammation induces lymphangiogenesis through upregulation of VEGFR-3 mediated by NF-␬B and
Prox1. Blood. 2010;115(2):418-429.
43. Hong YK, Harvey N, Noh YH, et al. Prox1 is a
master control gene in the program specifying
lymphatic endothelial cell fate. Dev Dyn. 2002;
225(3):351-357.
44. Pan MR, Chang TM, Chang HC, Su JL,
Wang HW, Hung WC. Sumoylation of Prox1 controls its ability to induce VEGFR3 expression and
lymphatic phenotypes in endothelial cells.
J Cell Sci. 2009;122(18):3358-3364.
45. Petrova TV, Makinen T, Makela TP, et al. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 2002;21(17):4593-4599.
46. Kajiya K, Sawane M, Huggenberger R,
Detmar M. Activation of the VEGFR-3 pathway
by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis. J Invest Dermatol. 2009;129(5):
1292-1298.
47. Kajiya K, Detmar M. An important role of lymphatic vessels in the control of UVB-induced
edema formation and inflammation. J Invest
Dermatol. 2006;126(4):919-921.
48. Kataru RP, Jung K, Jang C, et al. Critical role of
CD11b⫹ macrophages and VEGF in inflammatory
lymphangiogenesis, antigen clearance, and inflammation resolution. Blood. 2009;113(22):
5650-5659.
49. Jamieson T, Cook DN, Nibbs RJ, et al. The
chemokine receptor D6 limits the inflammatory
response in vivo. Nat Immunol. 2005;6(4):
403-411.
50. Harwood CA, Mortimer PS. Causes and clinical
manifestations of lymphatic failure. Clin Dermatol.
1995;13(5):459-471.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2011 117: 4667-4678
doi:10.1182/blood-2010-10-316356 originally published
online March 1, 2011
An important role of lymphatic vessel activation in limiting acute
inflammation
Reto Huggenberger, Shoib S. Siddiqui, Daniela Brander, Stefan Ullmann, Kathrin Zimmermann,
Maria Antsiferova, Sabine Werner, Kari Alitalo and Michael Detmar
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