Lymphatic dysfunction impairs antigen-specific immunization, but augments tissue swelling following contact with allergens.

Abstract

The lymph transports tissue-resident dendritic cells (DCs) to regional lymph nodes (LNs), having important roles in immune function. The biological effects on tissue inflammation following lymphatic flow obstruction in vivo, however, are not fully known. In this study, we investigated the role of the lymphatic system in contact hypersensitivity (CHS) responses using k-cyclin transgenic (kCYC(+/-)) mice, which demonstrate severe lymphatic dysfunction. kCYC(+/-) mice showed enhanced ear swelling to both DNFB and FITC, as well as stronger irritant responses to croton oil compared with wild-type littermates. Consistently, challenged ears of kCYC(+/-) mice exhibited massive infiltrates of inflammatory cells. In contrast, DC migration to regional LNs, drainage of cell-free antigen to LNs, antigen-specific IFN-γ production, and lymphocyte proliferation were impaired during the sensitization phase of CHS in kCYC(+/-) mice. Transfer experiments using lymphocytes from sensitized mice and real-time PCR analysis of cytokine expression using challenged ear revealed that ear swelling was enhanced because of impaired lymphatic flow. Collectively, we conclude that insufficient lymphatic drainage augments apparent inflammation to topically applied allergens and irritants. The findings add insight into the clinical problem of allergic and irritant contact dermatitis that commonly occurs in humans with peripheral edema of the lower legs.

Full text

Page 1

Lymphatic Dysfunction Impairs Antigen-Specific
Immunization, but Augments Tissue Swelling
Following Contact with Allergens
Makoto Sugaya1,2, Yoshihiro Kuwano1,2, Hiraku Suga1,2, Tomomitsu Miyagaki1,2, Hanako Ohmatsu1,
Takafumi Kadono1, Hitoshi Okochi2, Andrew Blauvelt3, Kunihiko Tamaki1and Shinichi Sato1
The lymph transports tissue-resident dendritic cells (DCs) to regional lymph nodes (LNs), having important
roles in immune function. The biological effects on tissue inflammation following lymphatic flow obstruction
in vivo, however, are not fully known. In this study, we investigated the role of the lymphatic system in contact
hypersensitivity (CHS) responses using k-cyclin transgenic (kCYCþ/?) mice, which demonstrate severe
lymphatic dysfunction. kCYCþ/?mice showed enhanced ear swelling to both DNFB and FITC, as well as
stronger irritant responses to croton oil compared with wild-type littermates. Consistently, challenged ears of
kCYCþ/?mice exhibited massive infiltrates of inflammatory cells. In contrast, DC migration to regional LNs,
drainage of cell-free antigen to LNs, antigen-specific IFN-g production, and lymphocyte proliferation were
impaired during the sensitization phase of CHS in kCYCþ/?mice. Transfer experiments using lymphocytes from
sensitized mice and real-time PCR analysis of cytokine expression using challenged ear revealed that ear
swelling was enhanced because of impaired lymphatic flow. Collectively, we conclude that insufficient
lymphatic drainage augments apparent inflammation to topically applied allergens and irritants. The findings
add insight into the clinical problem of allergic and irritant contact dermatitis that commonly occurs in humans
with peripheral edema of the lower legs.
Journal of Investigative Dermatology (2012) 132, 667–676; doi:10.1038/jid.2011.349; published online 10 November 2011
INTRODUCTION
Lymphedema is a condition caused by damaged lymphatics
resulting in accumulation of lymph fluid and tissue swelling.
It is common in the legs of older individuals and in the arms
of women following breast cancer surgery. Lymphedema
is associated with a number of complications, including
infections with bacteria and fungi. In rare cases, lymphedema
may be complicated by the development of angiosarcoma
(Stewart and Treves, 1948; Ruocco et al., 2001), squamous
cell carcinoma (Epstein and Mendelsohn, 1984; Furukawa
et al., 2002), and lymphoma (d’Amore et al., 1990; Dargent
et al., 2005). These phenomena may be due to reduced tissue
immune surveillance secondary to lymphatic dysfunction.
Indeed, lymphatic vessels are critical for transporting
tissue-resident dendritic cells (DCs), as well as interstitial
fluid to the lymph nodes (LNs), having important roles in
immunity against infectious agents and malignancy (Kaplan
et al., 2005). Thus far, very little is known about how immune
cells traffic and how immune responses may be altered in the
setting of lymphatic dysfunction.
Contact hypersensitivity (CHS) is an experimental model
for the study of antigen-specific, T-cell-mediated immune
responses (Macher and Chase, 1969). CHS responses
comprise: (1) a sensitization phase, when an antigen is first
presented to naive T cells in the regional LNs, and (2) an
elicitation phase, when antigen-specific memory T cells get
activated and release cytokines that attract other inflamma-
tory cells to the exposed site, dilate cutaneous blood vessels,
and cause dermal edema (Hopkins and Clark, 1995). It is
widely accepted that antigen-presenting cells migrate to LNs
and present antigens to naive T cells in the sensitization
phase. It is unknown how lymphatic dysfunction affects
CHS responses.
Specific markers for lymphatic endothelium have been
reported, such as vascular endothelial growth factor receptor-
3 (VEGFR-3) (Jussila et al., 1998; Dupin et al., 1999),
podoplanin (Breiteneder-Geleff et al., 1999), and lymphatic
vessel endothelial hyaluronan receptor-1 (Hong et al., 2004).
Specific identification of lymphatic endothelial cells had led
& 2012 The Society for Investigative Dermatology
www.jidonline.org667
ORIGINAL ARTICLE
Received 7 February 2011; revised 23 August 2011; accepted 4 September
2011; published online 10 November 2011
1Department of Dermatology, Faculty of Medicine, University of Tokyo,
Tokyo, Japan;2Department of Regenerative Medicine, Research Institute,
National Center for Global Health and Medicine, Tokyo, Japan and3Oregon
Medical Research Center, Portland, Oregon, USA
Correspondence: Makoto Sugaya, Department of Dermatology, Faculty of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan. E-mail: sugayam-der@h.u-tokyo.ac.jp
Abbreviations: CHS, contact hypersensitivity; DC, dendritic cell; kCYCþ/?,
k-cyclin transgenic; LN, lymph node; MHC, major histocompatibility
complex; PE, phycoerythrin; VEGFR-3, vascular endothelial growth factor
receptor-3; WT, wild type

Page 2

to a variety of important studies on structure and function of
lymphatic vessels in both normal and disease states. We
recently generated transgenic mice expressing the Kaposi’s
sarcoma-associated herpesvirus latent-cycle gene, k-cyclin,
under the control of the VEGFR-3 promoter (Sugaya et al.,
2005). In Kaposi’s sarcoma, this viral gene is expressed
by lymphatic endothelial cells and probably contributes to
edema within lesions (Davis et al., 1997; Reed et al., 1998).
Interestingly, most k-cyclin transgenic (kCYCþ/?) mice
developed progressive accumulation of chylous pleural fluid.
In skin, dermal edema was detected by magnetic resonance
imaging (Sugaya et al., 2005). In addition, lymphatic drainage
of injected contrast dyes was markedly impaired in transgenic
mice. Using these mice, we investigated the role of the
lymphatic system in CHS responses in this study.
RESULTS
Augmented ear swelling in kCYCþ/?mice
We first investigated whether CHS responses were impaired
in kCYCþ/?mice, which demonstrate markedly impaired
lymphatic drainage (Sugaya et al., 2005). When the mice
were sensitized with 0.5 or 0.1% DNFB, ear swelling
was significantly augmented in kCYCþ/?mice compared
with wild-type (WT) mice (Figure 1a and b). Similar results
were obtained when we used FITC (Figure 1c), showing that
different antigens could induce augmented ear swelling in
kCYCþ/?mice. Nonimmunized mice and FITC-challenged
mice that had been sensitized with DNFB did not show CHS
responses (Figure 1a and data not shown).
Enhanced cellular infiltration in the challenged ear of
kCYCþ/?mice
We also evaluated CHS responses histopathologically. There
were no differences between ears from kCYCþ/?mice and
WT mice before treatment (Figure 1d). Ear swelling and
cellular infiltration 24hours after challenge with either DNFB
(Figure 1d) or FITC (data not shown) were prominent in
kCYCþ/?mice compared with WT mice. Edema and dilated
vessels in the ear from kCYCþ/?mice suggested impaired
lymphatic flow in these mice. There were more infiltrating
cells, such as mononuclear cells, eosinophils, and major
histocompatibility complex (MHC) class IIþDCs in kCYCþ/?
mice compared with WT mice (Figure 1e).
Impaired migration of skin-derived DCs into draining LNs
of kCYCþ/?mice during the sensitization phase of CHS
To elucidate the mechanism of augmented ear swelling
in kCYCþ/?mice, each step involved in the generation
of CHS responses was examined. We first studied migration
of antigen-bearing DCs from skin to regional LNs. Untreated
epidermal sheets contained equal numbers of DCs (WT,
752±24/mm2vs. kCYCþ/?, 806±43/mm2, n¼5). The
shape and distribution of epidermal DCs (Langerhans cells)
were similar in WT and kCYCþ/?mice (Figure 2a). To count
draining DCs in LNs, inguinal LN cells were harvested 24 or
48hours after applying 0.5% FITC on shaved abdominal skin.
Inguinal LN cells from untreated mice were also obtained.
After applying FITC, the number of DCs and antigen-bearing
DCs in draining LNs in WT mice increased, as expected
(Figure 2b and c). In contrast, migration of antigen-bearing
DCs in kCYCþ/?mice was almost completely abrogated.
Similar results were obtained when DCs were labeled by
anti-CD11c mAb (data not shown). The results suggest that
DCs cannot migrate from the skin to draining LNs when
lymphatic flow is impaired. We detected almost no antigen-
bearing DCs in the spleen after sensitization in both WT and
kCYCþ/?mice (data not shown). Interestingly, the numbers
of DCs in kCYCþ/?mice were significantly decreased
compared with WT mice without stimuli (Figure 2c), which
suggests that lymphatic dysfunction in kCYCþ/?mice may
decrease the steady-state migration of DCs (Ruedl et al.,
2000; Henri et al., 2001; Ohl et al., 2004).
Impaired proliferation of lymphocytes in draining LNs of
kCYCþ/?mice during the sensitization phase of CHS
We next examined the proliferation of lymphocytes in
draining LNs. There were almost no gross and histological
differences in the thymus, spleen, and peripheral LNs
between WT and kCYCþ/?mice without any stimulus
(Sugaya et al., 2005). Inguinal LNs were harvested before
and after sensitization with 0.5% DNFB. The total numbers of
draining LN cells increased after sensitization in both WT and
kCYCþ/?mice, although the increase was less remarkable
in the latter (Figure 3a). The numbers of CD4þT cells, CD8þ
T cells, and B cells were also examined. Each cell type
increased in number following sensitization in both types of
mice (Figure 3a). Increases, however, were less remarkable in
kCYCþ/?mice when compared with WT mice, especially for
CD8þT cells and B cells. We next examined correlations
between frequencies of DCs and those of CD4þT cells,
CD8þT cells, and B cells in the draining LNs. As expected,
frequencies of DCs strongly correlated with those of CD8þ
T cells and B cells (Figure 3b), both of which are reported
to be involved in CHS responses (Kehren et al., 1999; Wang
et al., 2000; Larsen et al., 2007; Watanabe et al., 2007).
On the other hand, frequencies of CD4þT cells negatively
correlated with those of DCs, which might reflect the relative
increase of other cell populations. Interestingly, MHC class II
expression on B cells significantly correlated with the
numbers of B cells in draining LNs, suggesting activation
of B cells in the draining LNs during the sensitization phase of
CHS responses (Figure 3b).
Impaired CHS responses in kCYCþ/?mice after removal
of sensitized ear
Some topically applied antigens can be carried along by
lymphatic flow and not be cell associated, and then picked
up by resident DCs or B cells within draining LNs (Allenspach
et al., 2008; Lee et al., 2009). We next examined the effects
of mechanical blockade of antigen drainage to LNs on CHS
responses. Sensitized ears were removed 1, 24, or 48hours
after application of 0.5% DNFB. CHS responses were
completely abrogated when sensitized ears were removed
1hour after painting in both WT and kCYCþ/?mice
(Figure 4a and b). When ears were removed 24hours after
sensitization, WT mice showed CHS responses comparable
668Journal of Investigative Dermatology (2012), Volume 132
M Sugaya et al.
Response to Allergen in Lymphatic Dysfunction

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to mice whose ears were not removed, whereas kCYCþ/?
mice showed almost no CHS responses. These results suggest
that drainage of adequate antigen to induce normal CHS
responses, either in free form or within migratory DCs, occurs
within 24hours in WT mice, as previously described
(Turk and Stone, 1963), whereas this time range is not long
enough to induce CHS in kCYCþ/?mice with severe
lymphatic dysfunction. In contrast, both WT and kCYCþ/?
mice whose ears were removed 48hours after sensitization
showed similar CHS responses as those without removal of
14
12
10
TG sensitized
WT sensitized
TG nonsensitized
WT nonsensitized
Time after elicitation (days)
8
ΔEar thickness (x10–2 mm)
ΔEar thickness (x10–2 mm)
ΔEar thickness (x10–2 mm)
**
*
6
2
2
2
1
0
4
4
3
435
5
6
7
8
9
10
11
**
0
16
14
** **
12
10
8
6
2
4
0
16
14
12
10
20
18
8
6
4
2
0
16
14
12
10
20
18
8
6
4
2
0
1
WT
WT
0 Hours
24 Hours
Mononuclear cellsEosinophilsDendritic cells
50
45
40
35
30
25
20
15
Cells/HPF
Cells/HPF
Cells/HPF
10
5
0
WT TG
0
*
*
*
WT
0.1% DNFB
TG
TG
TG
0.5% DNFB
WT TG
WT TG
24
(Hours)
WT TG
0
WT TG
24
(Hours)
WT TG
0
WT TG
24
(Hours)
Figure 1. Augmented contact hypersensitivity (CHS) responses in k-cyclin transgenic (kCYCþ/?) mice. (a) Mice sensitized with 0.5% DNFB or nonimmunized
mice were challenged with 0.25% DNFB. TG, transgenic; WT, wild type. (b) Mice were sensitized with 0.5 or 0.1% DNFB. Ear thickness was measured 24hours
after challenge. (c) Mice were sensitized with 0.5% FITC; n¼10 for each condition. *Po0.05; **Po0.01. (d) Hematoxylin and eosin (H&E) staining of sections
from ears of wild-type (WT) and kCYCþ/?mice before (0hours) and 24hours (24hours) after elicitation (scale bar¼100mm). Prominent dermal edema and
dilated vessels (arrows) in the ear from kCYCþ/?mice. Representative pictures from 10 mice per group. (e) The numbers of mononuclear cells, eosinophils,
and dermal major histocompatibility complex (MHC) class IIþdendritic cells (DCs) per ?400 high-power fields (HPFs; n¼5). *Po0.05.
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M Sugaya et al.
Response to Allergen in Lymphatic Dysfunction

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sensitized ears (data not shown). Quantitative analysis of
infiltrating cells, including mononuclear cells, eosinophils,
and neutrophils, showed similar results with skin thickness
measurements (Figure 4c). kCYCþ/?mice showed almost the
same response as WT mice when sensitized ears were not
removed, which was quite different from the ear thickness
model (Figure 1). This prompted us to further investigate
whether augmented ear swelling was from enhanced
immunization or solely from impaired drainage following
elicitation.
Impaired lymphatic drainage in the elicitation phase of CHS
enhances apparent inflammation in kCYCþ/?mice
Severe lymphatic dysfunction induced impaired migration of
skin DCs, as well as free antigen drainage to regional LNs,
which could not explain augmented ear swelling in kCYCþ/?
mice. Therefore, we adoptively transferred sensitized lym-
phocytes and challenged transplanted mice to investigate
components in the elicitation phase of CHS. WT mice
transferred with sensitized lymphocytes from either WT or
kCYCþ/?mice showed minimal ear swelling (Figure 5a).
In contrast, kCYCþ/?
mice transferred with sensitized
lymphocytes either from WT or kCYCþ/?mice showed
enhanced ear swelling. No ear swelling was detected when
sensitized lymphocytes were not transferred to mice. In
addition, kCYCþ/?mice showed stronger irritant responses
compared with WT mice following application of croton oil
(Figure 5b), suggesting that impaired drainage was critically
important for augmented ear swelling in kCYCþ/?mice.
We next examined antigen-specific IFN-g production and
proliferative T-cell responses. IFN-g enzyme-linked immuno-
spot assay revealed more IFN-g-producing cells in inguinal
LNs in WT mice compared with kCYCþ/?mice (Figure 5c).
Almost no spots were detected in cell suspensions from
unsensitized mice or from cells not restimulated with
antigen. Antigen-specific proliferative responses were also
much higher in WT mice compared with kCYCþ/?mice
(Figure 5d), suggesting decreased immunization in the setting
of lymphatic dysfunction. Moreover, we assessed IFN-g,
tumor necrosis factor-a, CXCL9 (chemokine (C-X-C motif)
ligand 9), and CXCL10 (chemokine (C-X-C motif) ligand 10)
mRNA expression in challenged ears, all of which were
reported to be strongly associated with CHS responses
(Goebeler et al., 2001; Ogawa et al., 2010). Surprisingly,
ears of kCYCþ/?mice 24hours after challenge contained
significantly lower amounts of IFN-g, tumor necrosis factor,
CXCL9, and CXCL10 mRNAs compared with WT mice
(Figure 5e). These results strongly suggested that augmented
ear swelling of kCYCþ/?mice was mainly due to retention of
infiltrating cells and fluid within inflamed tissue.
DISCUSSION
In this study, we have demonstrated that transgenic mice with
severe lymphatic dysfunction have enhanced ear swelling.
Although migration of skin-derived DCs and establishment of
antigen-specific T cells were impaired in the sensitization
phase, defects in drainage of accumulated inflammatory cells
and fluid in the elicitation phase dominated and resulted in
augmented ear swelling overall. The results of this study
provide insight into the immunopathological basis of contact
dermatitis and venous stasis dermatitis commonly observed
in humans with peripheral lymphedema of the lower legs.
We were surprised to see that ear swelling was signifi-
cantly augmented in kCYCþ/?mice compared with WT mice
WT
WT
24 Hours
48 Hours
1.2 x 105
1.0 x 105
Cells/LN
0.8 x 105
0.6 x 105
0.4 x 105
0.2 x 105
0
WT
0
TG
*
1.4 x 105
FITC
**
*
*
MHC class II
TG
TG
WT
24
MHC class II+ cells
TG
WT
48
TG WT
0
TGWT
24(Hours)
FITC + MHC class II+ cells
TG
WT
48
TG
103
103
102
102
101
101
100
103
102
101
100
100
103
102
101
100
103
103
102
102
101
101
100
103
102
101
100
100
103
102
101
100
0.69%
0.03%
0.01%
0.64%
0.01%
03
01 02
04
03
01 02
04
03
0102
04
0.77%
03
0102
04
1.64%
Figure 2. Impaired migration of skin-derived dendritic cells (DCs) into
draining lymph nodes (LNs) of k-cyclin transgenic (kCYCþ/?) mice.
(a) Epidermal sheets were stained with phycoerythrin (PE)-conjugated
anti-I-A/I-E mAb. Representative pictures from four mice per group (scale
bar¼100mm). TG, transgenic; WT, wild type. (b, c) Inguinal LN cells were
harvested 24 or 48hours after applying 0.5% FITC on shaved abdomen
(n¼5). (b) Representative data plots by flow cytometry are shown.
(c) The numbers of major histocompatibility complex (MHC) class IIþcells
and FITCþMHC class IIþcells were examined at the indicated times.
*Po0.05.
670Journal of Investigative Dermatology (2012), Volume 132
M Sugaya et al.
Response to Allergen in Lymphatic Dysfunction

Page 5

(Figure 1). Migration of antigen-bearing DCs to the regional
LNs is believed to be important for CHS responses, especially
in the sensitization phase. Therefore, augmented ear swelling
in kCYCþ/?mice prompted us to investigate whether DCs
could migrate to draining LNs regardless of impaired
lymphatic flow. Migratory DCs move actively via interactions
between CCL21 (chemokine (C-C motif) ligand 21) and
CCR7 (chemokine (C-C motif) receptor 7) (Saeki et al., 1999;
1.2 x 107
Total lymphocytes CD8+T cells
CD4+T cells B cells
1.0 x 107
1.0 x 106
1.5 x 106
2.0 x 106
2.5 x 106
3.0 x 106
0.5 x 106
**
**
**
**
*
0.8 x 107
Cells/LN
Cells/LN
Cells/LN
Cells/LN
0.6 x 107
0.4 x 107
0.2 x 107
0
0
0
1.0 x 106
1.0 x 106
1.5 x 106
2.0 x 106
2.0 x 106
2.5 x 106
3.0 x 106
3.0 x 106
4.0 x 106
5.0 x 106
6.0 x 106
0.5 x 106
0
80
70
50
40
40
30
30
25
35
20
20
15
r = 0.87
P < 0.01
DC (%)
DC (%)B cells (%)
B cells (%)
MHC class II (MFI)
CD4+ T cells (%)
CD8+ T cells (%)
r = 0.64
P < 0.01
r = 0.50
P < 0.01
r = –0.82
P < 0.01
10
0
10
5
0
30
25
35
35
20
15
10
5
5
0
30
30
25
2535
20
20
15
15
10
105
0
0
0 0.511.522.5
00.511.522.5
DC (%)
00.51 1.522.5
60
WT TG
0
WT TG
48
WT TG
96 (Hours)
WT TG
0
WT TG
48
WT TG
96 (Hours)
WT TG
0
WT TG
48
WT TG
96 (Hours)
WT TG
0
WT TG
48
WT TG
96 (Hours)
**
**
*
*
*
Figure 3. Impaired proliferation of lymphocytes in the draining lymph nodes (LNs) of k-cyclin transgenic (kCYCþ/?) mice. Inguinal LN cells were harvested 0,
48, or 96hours after applying 0.5% DNFB on shaved abdominal skin. The cells were stained for FITC-conjugated anti-CD8, FITC-conjugated anti-B220,
phycoerythrin (PE)-conjugated anti-I-A/I-E, PE-conjugated anti-CD11c, and PE-conjugated anti-CD4 mAbs and were analyzed by flow cytometry (n¼5).
(a) Time course of cell numbers of each population in the draining LNs. *Po0.05; **Po0.01. (b) Correlations between frequencies of dendritic cells (DCs) and
those of CD4þT cells, CD8þT cells, and B cells in the draining LNs, and correlation between frequencies of B cells and major histocompatibility complex
(MHC) class II expression on B cells. MFI, mean fluorescence intensity.
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Ohl et al., 2004). Lymphatic retention in our transgenic mice
almost completely blocked migration of skin DCs to draining
LNs (Figure 2). Abrogation of a chemotactic gradient might
explain the impaired DC migration.
Once antigen-bearing DCs reach draining LNs, prolif-
eration of antigen-specific lymphocytes commences. The
numbers of lymphocytes in draining LNs increased 3 to
5 days following topical antigen exposure (Macatonia et al.,
1987; Tomei et al., 2009). The transgenic mice in this study
also showed increases in draining LN lymphocytes at similar
time points, although the degree was less remarkable than
in WT mice (Figure 3a). This suggests that immune responses,
although less strong, occur within LNs of kCYCþ/?mice
after sensitization. Proliferation of CD8þT cells and B cells
was impaired in those mice (Figure 3a). CHS responses
are largely mediated by CD8þT cells (Kehren et al., 1999;
Wang et al., 2000), but B cells are also activated and
involved in CHS responses (Larsen et al., 2007; Watanabe
et al., 2007). Activation of CD8þT cells and B cells seems
to be mainly mediated by migratory DCs (Macatonia et al.,
1987). Consistently, frequencies of DCs strongly correlated
with those of CD8þT cells and B cells (Figure 3b). As very
few numbers of migratory DCs in LNs of kCYCþ/?mice were
observed before 96hours after sensitization (Figure 2 and
data not shown), this may explain why we observed impaired
numbers of CD8þT cells and B cells. On the other hand,
proliferation of CD4þ
T cells was not so impaired in
transgenic mice (Figure 3a), suggesting that these cells may
be activated during CHS in a DC-independent manner.
MHC class II expression on B cells increased in WT mice,
suggesting activation of B cells during CHS, whereas MHC
class II expression on B cells remained low in kCYCþ/?mice.
Interestingly, MHC class II expression on B cells significantly
correlated with the numbers of B cells in draining LNs
(Figure 3b).
Migratory DCs are mainly composed of epidermal
Langerhans cells and dermal DCs (Ohl et al., 2004).
The roles of Langerhans cells in CHS responses are now
WT
50
40
Mononuclear cells
*
30
Cells/HPF
Cells/HPF
Cells/HPF
0
0
2
4
6
10
8
10
20
TGWT
No
sensitization
No
sensitization
Ear cut
after 1 hour
Ear cut
after 1 hour
Ear cut
after 24 hours
Ear cut
after 24 hours
*
*
**
Eosinophils
No cut
No cut
TG
TGWTTGWTTG WT
TG
WT
No
sensitization
Ear cut
after 1 hour
Ear cut
after 24 hours
No cut
TG
WT
TG
WT
TG
WT
TG WT
No
sensitization
Ear cut
after 1 hour
Ear cut
after 24 hours
No cut
TGWT TGWT TG WT
TG
WT
No
sensitization
Ear cut
after 1 hour
Ear cut
after 24 hours
No cut
TG
WT
TG
WT
TG
WT
120
Neutrophils
80
100
40
40
25
35
45
50
30
Skin thickness (x10–2 mm)
60
20
0
Figure 4. Impaired contact hypersensitivity (CHS) responses in k-cyclin transgenic (kCYCþ/?) mice after removal of sensitized ears. (a) Sensitized ears were
removed 1 or 24hours after the application of 0.5% DNFB. For some mice, ears were not removed as positive controls (no cut). Nonsensitized mice were used
as negative controls (no sensitization). CHS responses were elicited by applying 20ml of 0.25% DNFB onto shaved back skin. Sections from back skin 24hours
after challenge were stained for hematoxylin and eosin (H&E). Representative pictures from five mice per group (scale bar¼100mm). TG, transgenic;
WT, wild type. (b) Skin thickness was measured for each condition (n¼5). **Po0.01. (c) The numbers of mononuclear cells, eosinophils, and neutrophils
per ?400 high-power fields (HPFs; n¼5). *Po0.05.
672 Journal of Investigative Dermatology (2012), Volume 132
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Response to Allergen in Lymphatic Dysfunction

Page 7

controversial (Bennett et al., 2005; Kaplan et al., 2005; Teoh
et al., 2009). Furthermore, it has been revealed that some
antigens are presented by DCs residing in LNs (Allenspach
et al., 2008; Lee et al., 2009). Therefore, very few antigen-
bearing DCs within LNs of kCYCþ/?mice prompted us to
investigate the role of free antigen directly carried into the
draining LNs. Results revealed that 24hours was long enough
for the drainage of antigens, either in free form or within
migratory DCs, in WT mice, whereas 48hours was necessary
for kCYCþ/?mice (Figure 4). These results were comparable
**
**
**
**
16
14
12
10
8
ΔEar thickness (x10–2 mm)
Number of IFNγ-SFC per 106 cells
ΔEar thickness (x10–2 mm)
4
6
Donor
WT
DNBS
TNBS
–
DNBS
TNBS
–
DNBS
TNBS
–
DNBS
TNBS
–
DNBS
TNBS
–
DNBS
TNBS
–
DNBS
TNBS
–
DNBS
TNBS
–
**
WT
6 Hours 24 Hours
WT
↓↓↓↓
WT
WT
WT
TG
TG
TG
TG
Sensitized (–)Sensitized (+)
WTTG
TG
TG
2
0
12
10
8
4
6
2
0
25
** **
** **
**
**
(AU)
0.7
0.6
0.5
0.4
0.2
0.3
0.1
0
*
* *
*
20
15
10
5
0
WT
TG
WTTG
Sensitized (–)Sensitized (+)
WT TG
WTTGWT TG
WT TGWT
TG
IFN-γ
TNF-α
CXCL9CXCL10
**
0.0035
0.0030
0.0025
Ratio to GAPDH
Ratio to GAPDH
Ratio to GAPDH
Ratio to GAPDH
0.0015
0.0010
0.0005
0
0.0020
0.035
0.030
0.025
0.020
0.040
0.015
0.010
0.005
0.002
0.004
0.006
0.008
0.010
0.012
0
0
0.0030
0.0025
0.0015
0.0010
0.0005
0
0.0020
**
**
Recipient
Figure 5. The elicitation phase is critical for augmenting contact hypersensitivity (CHS) responses in k-cyclin transgenic (kCYCþ/?) mice. (a) Inguinal lymph
node (LN) cells from DNFB-sensitized mice were adoptively transferred intravenously. Recipient mice were elicited with 0.25% DNFB 24hours after the
transfer. Ear swelling responses were measured 24hours after challenge. (b) Croton oil was applied to the mouse ear. After 6 and 24hours, changes in ear
thickness were measured (n¼10). (c) Enzyme-linked immunospot (ELISPOT) assay using inguinal LN cells from sensitized and nonsensitized mice. (d) BrdU
proliferation assay using inguinal LN cells from sensitized and nonsensitized mice. (e) Ears challenged with DNFB were harvested and RNA was obtained.
Quantitative reverse transcription-PCR (RT-PCR) was performed for IFN-g, tumor necrosis factor-a (TNF-a), CXCL9 (chemokine (C-X-C motif) ligand 9), and
CXCL10 (chemokine (C-X-C motif) ligand 10). *Po0.05; **Po0.01. One representative result from two independent experiments with triplicates (a, c–e).
AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SFC, spot-forming cell; TG, transgenic; TNBS, trinitrobenzene sulfonic acid;
WT, wild type.
www.jidonline.org673
M Sugaya et al.
Response to Allergen in Lymphatic Dysfunction

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to our previous study in which we used injected dye and
lymphangiograms to assess lymphatic flow (Sugaya et al.,
2005). Free antigens can travel much faster than antigen-
bearing DCs. It was reported that fluorescent DCs in the
draining LNs were detectable 30minutes after skin painting
with FITC, which appears to be too early for skin DCs to
reach LNs (Saeki et al., 1999). Fluorescent B cells were also
detected 1 day following sensitization. Taken together,
resident DCs or B cells in LNs may take up cell-free antigen
flowing through lymphatic vessels and may be involved in
establishing memory T cells.
Consistent with impaired DC migration and LN lympho-
cyte proliferation, antigen-specific IFN-g production and
proliferative responses were decreased in kCYCþ/?mice
(Figure 5c and d). Cytokine mRNA expression in the
challenged ears from transgenic mice were also significantly
decreased (Figure 5e). It was surprising to see a discrepancy
between ear thickness and cytokine expression. Our results
reveal that ear thickness does not necessarily reflect the
degree of immune reaction within tissue. Irritant dermatitis
induced by croton oil, which does not need prior sensitiza-
tion, represents a nonspecific response to foreign antigen.
Enhanced irritant dermatitis in kCYCþ/?mice points to the
importance of lymphatic vessels to clear fluid and infiltrating
cells from inflamed skin. Not only do lymphocytes and DCs
use lymphatics, but erythrocytes are collected through
lymphatic vessels as well (Kissenpfennig et al., 2005). We
previously showed that erythrocytes were detected in dilated
lymphatic vessels in tagged ears of kCYCþ/?mice (Sugaya
et al., 2005). During inflammation, tissue fluid drainage can
be increased by X10-fold (Flessner et al., 1983; Fischer et al.,
1996). Impaired lymphatic system in kCYCþ/?mice, which
do not show clinical symptoms of skin disease in the absence
of skin inflammation, cannot adequately manage the increase
in tissue fluid and cells during inflammation. Defects in the
drainage of accumulated inflammatory cells and fluid in the
elicitation phase of CHS leads to augmented ear swelling,
despite impaired DC migration during the sensitization
phase of CHS. Thus, our findings point to a dominant role
for lymphatic drainage in clearing inflammatory cells from
tissue and in resolving tissue inflammation following the
onset of cutaneous inflammation. These findings are also
clinically relevant in that they provide mechanistic insight
into the problem of allergic contact dermatitis and venous
stasis dermatitis, which frequently occur in the lower legs of
individuals with severe lymphedema.
Although VEGFR-3 is mainly expressed on lymphatic
endothelial cells, other cell types such as corneal DCs,
murine macrophages, and B-cell chronic lymphocytic
leukemia cells can express VEGFR-3 (Hamrah et al., 2003;
Bairey et al., 2004; Stepanova et al., 2007). When we
previously analyzed transgene expression, kCYC mRNA
signals localized to karyomegalic lymphatic endothelial cells
lining vessels positive for VEGFR-3 and podoplanin (Sugaya
et al., 2005). Expression of the kCYC transgene in different
cell types, however, cannot be completely ruled out, and thus
may have a role in the functional changes observed in our
experiments. Other mouse lymphedema models or studies
using human tissues or cells would be necessary to address
this issue.
MATERIALS AND METHODS
Mice
FVB/N mice were purchased from Clea Japan (Tokyo, Japan).
kCYCþ/?mice were generated as previously described (Sugaya
et al., 2005). All mice were free of pathogenic bacteria and viruses.
All experiments were conducted using mice between 6 and
14 weeks of age. All studies and procedures were approved by the
Animal Committee of National Center for Global Health and
Medicine.
Sensitization and elicitation of CHS
CHS responses were induced either with DNFB or FITC, as
previously described (Watanabe et al., 2007). A volume of 50ml of
0.5 or 0.1% DNFB was painted onto shaved abdominal skin on day
0, and CHS was elicited by applying 0.25% DNFB on the left ear on
day 5. CHS responses to FITC were induced by applying 0.5% FITC
to shaved abdominal skin. After 5 days, CHS reactions were elicited
by applying FITC solution. For all CHS experiments, baseline ear
thickness was determined with a spring-loaded caliper. Ear swelling
responses were measured at 24hours after elicitation and the change
in ear thickness from baseline measurement was computed. Each ear
was measured three times by a researcher who was blind to the
results and the mean of these values was used. Croton oil was used
to elicit irritant contact dermatitis. A volume of 15ml of 2.0% croton
oil was painted on the left ear. After 6 and 24hours, the change
in ear thickness from baseline was measured as described.
DNFB, FITC, and croton oil were purchased from Sigma-Aldrich
(St Louis, MO).
Removal of sensitized ear
To evaluate the effects of delay in antigen draining to LN during CHS
responses, 20ml of 0.5% DNFB was applied on the left ear on day 0.
Ears were removed 1, 24, or 48hours after the application.
CHS responses were elicited by applying 0.25% DNFB on the
shaved back skin on day 5. The back skin was removed 24hours
after the challenge and was assessed histologically. Skin thickness
was histologically measured.
Histological examination
Ear or back skin samples were fixed in 4% formalin and embedded
in paraffin. Sections, 4mm thick, were cut and stained with
hematoxylin and eosin. Skin thickness was histologically measured.
The numbers of mononuclear cells and eosinophils were counted in
10 random grids under magnification of
fields and averaged. In some experiments, ear skin samples were
snap-frozen, cut into 5-mm-thick cryostat sections, and fixed in
acetone. These sections were then stained with phycoerythrin
(PE)-conjugated anti-I-A/I-E mAb. The numbers of dermal MHC
class IIþDCs were counted. Each section was examined indepen-
dently by two investigators in a blinded manner.
?400 high-power
Quantitative reverse transcription-PCR to assess cytokine
production in the challenged ears
The ears of WT and kCYCþ/?mice were challenged with DNFB as
described above. After 24hours, the ears were harvested and RNA
674 Journal of Investigative Dermatology (2012), Volume 132
M Sugaya et al.
Response to Allergen in Lymphatic Dysfunction

Page 9

was obtained using the RNeasy Fibrous Tissue Mini Kit (QIAGEN,
Valencia, CA). Complementary DNA was synthesized using TaqMan
Reverse Transcription Reagents (Applied Biosystems, Foster City,
CA). Quantitative reverse transcription-PCR was performed as des-
cribed previously (Sugaya et al., 2006). Primers for mouse IFN-g,
tumor necrosis factor, CXCL9, CXCL10, and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) were as follows: IFN-g forward,
50-AGCAACAGCAAGGCGAAAA-30and reverse, 50-CTGGACCTGT
GGGTTGTTGA-30; tumor necrosis factor forward, 50-CCACCACGCT
CTTCTGTCTAC-30and reverse, 50-AGGGTCTGGGCCATAGAAC
T-30; CXCL9 forward, 50-TGGGCATCATCTTCCTGGAG-30
reverse, 50-CCGGATCTAGGCAGGTTTGA-30; CXCL10
50-CCCACGTGTTGAGATCATTG-30and reverse, 50-CACTGGGTAA
ACGGGAGTGA-30; GAPDH forward, 50-CGTGTTCCTACCCCCAAT
GT-30and reverse, 50-TGTCATCATACTTGGCAGGTTTCT-30.
and
forward,
Flow cytometry
Inguinal LNs were harvested at the described time after sensitization,
and cell suspensions were prepared by digesting tissues with
1mgml?1collagenase D (Sigma-Aldrich) and 0.2mgml?1DNase
(Sigma-Aldrich). Single-cell suspensions were stained for two-color
immunofluorescence analysis at 41C using FITC-conjugated anti-
CD8, FITC-conjugated anti-B220, PE-conjugated anti-I-A/I-E, PE-
conjugated anti-CD11c, and PE-conjugated anti-CD4 mAbs (BD
PharMingen, San Diego, CA) for 20minutes. Labeled cells were
analyzed on an EPICS XL flow cytometer (Beckman Coulter,
Fullerton, CA) with fluorescence intensity shown on a 4-decade
log scale. Positive and negative populations of cells were determined
using isotype-matched Abs (Southern Biotechnology, Birmingham,
AL) as controls for background staining. Mean fluorescence intensity
for MHC class II on B220þcells (B cells) was determined for each
experiment.
Adoptive transfer of sensitized LN cells
Donor mice were sensitized with 0.5% DNFB on day 0 as described
above. On day 5, inguinal LN cells were harvested and a mixture of
2–4?106cells in 200ml of phosphate-buffered saline was adoptively
transferred intravenously. After 24hours, mice were elicited with
0.25% DNFB and ear swelling responses were measured after
24hours.
IFN-c enzyme-linked immunospot assay
Inguinal LNs were harvested 5 days after DNFB sensitization. Cell
suspensions were restimulated in vitro by overnight culture with
mitomycin C-treated syngeneic spleen cells (106per well) from naive
mice in complete RPMI medium supplemented with 10% fetal calf
serum and containing a final concentration of 0.4mM DNBS. Control
cultures included cells cultured overnight in medium supplemented
with 0.2mM of the irrelevant hapten trinitrobenzene sulfonic acid, or
in medium alone. The number of IFN-g-producing cells was
determined using an enzyme-linked immunospot assay kit (R&D
systems, Minneapolis, MN). The number of IFN-g spot-forming cells
present in each well was counted using a microscope, and the results
were expressed as IFN-g spot-forming cells per 106cells.
Hapten-specific T-cell proliferation in vitro
Inguinal LNs were harvested 5 days after DNFB sensitization. Cell
suspensions were cocultured for 3 days with mitomycin C-treated
syngeneic spleen cells (106per well) from naive mice, which had
been previously incubated for 20minutes at 371C with 4mM DNBS,
2mM trinitrobenzene sulfonic acid, or medium only and washed in
complete medium before use. Cells were stained with BrdU for
16hours and reacted with anti-BrdU Ab peroxidase conjugate,
followed by peroxidase substrate using Cell Proliferation ELISA,
BrdU (Roche Applied Science, Basel, Switzerland). Sulfuric acid was
added to the solution to terminate enzyme activity. Optical densities
were measured at 450nm using a 550 microplate reader (Bio-Rad
Laboratories, Hercules, CA).
Statistics
All data are shown as mean valuesþSEM. Statistical analysis
between two groups was performed using the Mann–Whitney
U-test. Correlation coefficients were determined using Spearman’s
rank correlation test. The P-values of o0.05 were considered
statistically significant.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
We thank Kiyoko Nashiro for technical assistance. This study was supported
by grants from the Ministry of Education, Culture, Sports and Technology
in Japan, a grant for basic dermatological research from Shiseido, and a grant
from the Lydia O’Leary Memorial Foundation.
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