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Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have been studying the biological roles of a macrophage endogenous C-type lectin, MMGL. MMGL is a 42,000 Mr type II transmembrane glycoprotein containing a single calcium-dependent carbohydrate recognition domain with specificity for galactose/N-acetylgalactosamine at its carboxyl terminus (7, 8). MMGL was originally detected on tumoricidal peritoneal macrophages (7), and involvement in the tumor cell recognition and tumoricidal activity of macrophages has been reported (9, 10, 11). In addition, MMGL was found on tumor-infiltrated macrophages within the lung metastatic lesions produced by experimental metastasis of mouse ovarian tumor cells (12). Using a specific mAb produced in our laboratory (13), we revealed that MMGL-positive cells were widely distributed, but that the distribution was restricted to connective tissue (12, 14). In skin, dermal macrophages strongly express MMGL, whereas epidermal Langerhans cells are devoid of its expression (14). Therefore, we thought that MMGL could be used as an ideal marker to distinguish dermal macrophages from Langerhans cells in tissue environments.
In the present study we investigated whether dermal macrophages contribute to the induction of contact hypersensitivity using unmanipulated mice. We attempted to directly investigate lymphatic cell trafficking of dermal macrophages during the sensitization phase. We used an anti-MMGL mAb to distinguish Langerhans cell migrants and dermal macrophage migrants in regional lymph nodes. Using various conditions for epicutaneous sensitization, we investigated the relationship between efficiency of sensitization and MMGL-positive cell migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female, specific pathogen-free CD-1 (ICR) and BALB/c mice were purchased from SLC Japan (Shizuoka, Japan).
Reagents
Biotin-conjugated mAb mouse anti-rat 
and 
light
chains (anti-/), Triton-X-100, FITC, streptomycin, aprotinin,
pepstatin A, leupeptin, poly-L-lysine, and PMSF were
purchased from Sigma (St. Louis, MO); DMEM was obtained from Nissui
Pharmaceutical (Tokyo, Japan); FCS was purchased from
BioWhittaker (Walkersville, MD); the bicinchoninic acid protein
assay kit was obtained from Pierce (Rockford, IL); SDS-PAGE protein
reference standards (phosphorylase b, BSA, aldolase, and
carbonic anhydrase) were obtained from Daiichi Pure Chemicals (Tokyo,
Japan); BSA fraction V was purchased from Seikagaku (Tokyo, Japan);
acetone, dimethylformamide (DMF), DMSO, ethanol, ethyl acetate, dibutyl
phthalate, olive oil, penicillin, and collagenase (Clostridium
histolyticum) were obtained from Wako Pure Chemical (Tokyo,
Japan); horseradish peroxidase-conjugated goat anti-rat IgG(H+L)
and alkaline phosphatase-conjugated streptavidin were purchased from
Zymed (South San Francisco, CA); Histomark Red was obtained from
Kirkegaard & Perry (Gaithersburg, MD); DNase I (grade II, bovine
pancreas), polyclonal sheep anti-digoxigenin Fab fragments,
alkaline phosphatase-labeled anti-digoxigenin Fab fragments, and
digoxigenin-3-O-methyl-carbonyl-
-aminocaproic
acid-N-hydroxysuccinimide ester were obtained from
Boehringer Mannheim (Mannheim, Germany); 5 (and
6)-(((4-chloromethyl)benzoyl)amino)- tetramethylrhodamine (CMTMR)
was obtained from Molecular Probes (Eugene, OR); SDS, paraformaldehyde,
and glutaraldehyde were purchased from Nacalai Tesque (Kyoto, Japan);
biotin-conjugated anti-mouse Thy 1.2 was obtained from Becton
Dickinson (San Jose, CA); Cy5-conjugated mouse anti-rat IgG(H+L)
was purchased from Jackson ImmunoResearch (West Grove, PA);
FluoreLink-Ab Cy3.5 labeling kit was obtained from Amersham (Aylesbury,
U.K.); and microbeads goat anti-rat IgG(H+L) was obtained from
Miltenyi (Bergisch Gladbach, Germany). Preparation of culture
supernatant of rat hybridoma cell lines producing mAb against MMGL (mAb
LOM-14; IgG2b and mAb LOM-8.7; IgG2a) in DMEM containing 4.5 g/l
glucose, 10% FCS, penicillin (100 U/ml), and streptomycin (100
µg/ml) and preparation of purified mAb LOM-14 were described
previously (13).
Labeling of Abs
Sheep anti-digoxigenin Fab fragments (1 mg) were conjugated
with Cy3.5 using the FluoreLink-Ab Cy3.5 labeling kit according to the
manufacturer’s instructions. Digoxigenin labeling of mAb LOM-14 was
conducted by incubation of the purified mAb (1 mg) with
digoxigenin-3-O-methyl-carbonyl--aminocaproic
acid-N-hydroxysuccinimide ester (44 µg dissolved in 22
µl DMSO) in 0.1 M sodium borate (pH 8.8) for 3 h at 25°C.
Sensitization and elicitation of contact hypersensitivity reaction
The method of contact sensitization was based on those of earlier studies (15, 16) with modifications. Mouse forelimbs were shaved using a small animal clipper, and 80 µl of FITC solution (0.5%, w/v) dissolved in a solvent was epicutaneously applied to the shaved forelimbs. The solvents used for sensitization are as follows: acetone/dibutyl phthalate (AD; 1/1), 100% ethanol, acetone/olive oil (4/1), 30% SDS in water, ethyl acetate, DMF, and DMSO. On day 6, the baseline ear thickness (0 h) of each animal was measured using a dial thickness gauge. Mice were challenged by applying 20 µl of 0.5% FITC solution in AD on the outer surface of the right auricle, and ear thickness was measured after 24 h. The left auricle was left untreated as a control. Ear swelling was defined as follows: [(ear thickness of the right ear at 24 h) - (ear thickness of the right ear at 0 h)] - [(ear thickness of the left ear at 24 h) - (ear thickness of the left ear at 0 h)].
Detection of MMGL in lymph node extracts
Brachial lymph nodes were removed from anesthetized mice and homogenized in Dulbecco’s modified PBS (DPBS; containing 0.91 mM CaCl2 and 0.49 mM MgCl2) containing 1% Triton X-100, 0.02% NaN3, 0.1 µM aprotinin, 1 µM pepstatin A, 1 µM leupeptin, and 1 mM PMSF (lysis buffer) using a Potter-Elvehjem homogenizer. They were then extracted for 1 h on ice (1 ml of lysis buffer/100 mg organ wet weight). The homogenates were centrifuged at 100,000 x g for 30 min, and the supernatants were collected. The protein concentration in the organ lysates was assessed using a bicinchoninic acid protein assay kit. Proteins in the lysate were separated by SDS-PAGE (10% gel) under nonreducing conditions and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a Milli Blot-SDE system (Millipore). The membrane was treated in 10 mM sodium phosphate and 0.15 M NaCl (pH 7.2; PBS) that contained 2% normal goat serum and 3% BSA for 18 h at 4°C to block nonspecific Ab binding. The membrane was subsequently incubated with mAb LOM-14 (1/10 dilution of culture supernatant in PBS containing 0.2% Tween-20) for 90 min at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rat IgG(H+L) diluted at 1/1000 in PBS/0.2% Tween-20 for 90 min at room temperature. The binding of mAb was visualized using ECL Western blotting detection reagent and Hyperfilm ECL (Amersham, Arlington Heights, IL).
Immunohistochemistry for light microscopy
MMGL-positive cells were immunohistochemically detected on
frozen sections of lymph nodes using mAb LOM-14 (1/10), biotinylated
mAb mouse anti-rat / (1/100), and alkaline
phosphatase-streptavidin (1/100) as described previously (12). In some
experiments, biotin-conjugated anti-mouse Thy 1.2 (1/100) and
alkaline phosphatase-streptavidin were used to detect T lymphocytes.
The Ab binding was histochemically detected using HistoMark Red, and
the cell nucleus was counterstained in Mayer’s hematoxylin. The
sections were observed under a microscope and photographed (Olympus,
Tokyo, Japan). As a negative control, normal rat serum (1/50) was used
instead of the first Ab. In immunofluorescence experiments, lymph nodes
embedded in OCT compound (Miles, Elkhart, IN) were directly frozen in
liquid nitrogen. Cryostat sections (10 µm thick) were picked up on
poly-L-lysine-coated slides and were used unfixed.
Nonspecific binding sites were blocked in a blocking solution (2%
normal goat serum and 3% BSA in DPBS) for 10 min at 25°C. The
sections were incubated with digoxigenin-conjugated mAb LOM-14 (1/100
dilution in the blocking solution) for 1 h at 20°C, fixed in 2%
paraformaldehyde/0.1 M sodium phosphate (pH 7.0), and then incubated
with Cy3.5-conjugated sheep Fab anti-digoxigenin (1/1000 dilution
in 3% BSA/DPBS). In some experiments, the sections were incubated with
mAb LOM-14 (1/10 culture supernatant diluted in the blocking solution),
fixed in 1% paraformaldehyde/0.1 M sodium phosphate (pH 7.0), and then
incubated with Cy5-conjugated mouse anti-rat IgG(H+L) (1/250
dilution in 3% BSA/DPBS). After each incubation or fixation, the
sections were washed twice gently in DPBS. After the final wash, the
sections were mounted in Vectashield (Vector Laboratories, Burlingame,
CA) and were observed using a confocal microscope (MRC-1024, Bio-Rad,
Herts, U.K.) equipped with a krypton/argon laser. A total of at
least 16 mice were used per condition for microscopic and
immunohistochemical observations, and experiments were repeated at
least five times.
Flow cytometry
Brachial lymph nodes were obtained from ICR mice that had been epicutaneously treated with FITC on the forelimb skin 24 h earlier. Single cell suspensions of lymph nodes from individual mice were prepared by cutting tissue using teasing needles in PBS containing 0.1% BSA and 0.1% NaN3. Cells were washed and resuspended in the same buffer at 106 cells/ml, and they were analyzed on an EPICS Elite flow cytometer (Coulter, Miami, FL) using gates of forward and side light scatter to collect signals of cell-associated fluorescence. Cells with signals of >200 channels (linear scale) were arbitrarily assigned to the bright fluorescence population. A total of 106 cells were analyzed.
Isolation of MMGL-positive cells from skin
BALB/c mouse shaved skin from the abdomen, fore- and hindlimbs, and dorsum was cut by scissors into 2-mm squares. The skin fragments were incubated in 50 ml of 0.1% collagenase and 0.01% DNase in sterile DMEM/Ham’s F-12 medium (Nissui Pharmaceutical, Tokyo, Japan) at 37°C for 2 h with continuous stirring. The tissue digests were passed through three layers of nylon mesh to remove tissue fragments, and the cells were centrifuged at 1000 rpm for 10 min. The cells (108) were suspended in 0.9 ml of 0.1% BSA/DPBS, and 100 µl of culture supernatant of anti-MMGL mAb LOM-8.7 was added. After incubation for 30 min at 4°C, the cells were washed twice in 0.1% BSA/DPBS and resuspended in 0.8 ml of 0.1% BSA/DPBS, and then 200 µl of goat anti-rat IgG(H+L) microbeads were added. The suspension was incubated for 15 min at 8°C, and then positive selection was conducted using an RS+ column with a magnetic cell sorter I (Miltenyi). Cells retained in the column were recovered by washing the column outside the magnetic field. An aliquot of cell suspension was subjected to cytocentrifugation and was immunohistochemically stained using digoxigenin-conjugated mAb LOM-14 plus alkaline phosphatase anti-digoxigenin Ab (1/100 dilution). Positive reaction was detected using HistoMark Red as described for frozen sections.
Cell tracer experiments
MMGL-positive cells (1–5 x 107 cells/ml) isolated from skin were suspended in 1 ml of DMEM/Ham’s F-12 medium-10% FCS, and 6 µl of CMTMR (1 mg/ml in DMSO) was added. After incubation for 15 min at 37°C, cells were washed and resuspended in PBS at 5 x 107 cells/ml. An aliquot of the labeled cells (106 cells in 20 µl) was intradermally injected into the shaved skin of a forelimb of a BALB/c mouse. After 16 h, mice were epicutaneously treated on the forelimb skin over the site of cell injection with 80 µl of FITC solution (0.5%, w/v) in AD or AD alone, or they received no treatment. After 22 h, brachial lymph nodes were collected, and frozen sections of the lymph nodes were immunohistochemically examined using mAb LOM-14 and Cy5-conjugated anti-rat IgG followed by examination under a confocal microscope as described above ("Immunohistochemistry for light microscopy" section). Tissue sections prepared from the forelimb skin, where CMTMR-labeled cells were injected, were examined under a fluorescence microscope (Olympus).
Statistical analyses
Student’s t test, the z test for proportions, Spearman’s rank correlation test, and Kendall’s rank correlation test were used. The methods used for specific analyses are specified in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the specificity of FITC as an Ag in our experimental
conditions, mice were sensitized by epicutaneous application on
forelimb skin. After 6 days, mice were challenged by epicutaneous
application on ear, and then ear swelling was measured at 24 h
(Fig. 1
). Mice sensitized with FITC
dissolved in AD followed by challenge with FITC in AD developed
substantial ear swelling. By contrast, mice sensitized with FITC in AD
followed by challenge with AD alone and mice sensitized with AD alone
followed by treatment with FITC in AD produced only marginal responses.
Mice sensitized with AD alone followed by challenge with AD did not
produce any response compared with untreated mice. These results
demonstrate that FITC plays a major role as an antigenic substance in
these experimental conditions. A time-course study revealed that ear
swelling had peaked at 16–24 h after challenge. There was a transient
peak of the ear swelling response (50–60% of the maximal swelling) at
2–4 h. At 48 h, the ear swelling was decreased by 80% relative
to the amount found at 24 h. Since FITC was applied on only one
side of the auricle, swelling was only evident on the treated half of
the auricle by microscopic observation. At this site, extensive
vasodilatation was observed (data not shown).
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We investigated whether epicutaneous sensitization with Ag affects
the number and distribution of MMGL-positive cells in draining lymph
nodes (Fig. 2). In normal mouse lymph
nodes, MMGL-positive cells were mainly detected in the medulla,
subcapsular sinus, and interfollicular sinus (17). A scattered
distribution of MMGL-positive cells with dendritic morphology was
occasionally seen in the T cell area, especially at the border between
T and B cell areas (Fig. 2d). Twenty-four hours after
epicutaneous application of FITC dissolved in AD on forelimb skin, a
marked increase in the number of MMGL-positive cells was observed
within the T cell area of brachial lymph nodes, especially around the
border between T and B cell areas (Fig. 2a). The
localization of MMGL-positive cells was also recognized by comparative
observation of serial sections stained with anti-Thy 1.2 mAb, which
revealed localization of T lymphocytes (Fig. 2c). This is
also confirmed by comparing their position relative to that of high
endothelial venules, which are present in the T cell area (Fig. 2b). Unexpectedly, solvent alone (AD) produced an effect
similar to that of AD containing FITC, indicating that the solvent
makes an important contribution to the increase in MMGL-positive cells
in the T cell area (Fig. 2e). Cytochemical control did not
produce specific staining, regardless of the treatments, and a
representative result (treated with AD) is shown in Fig. 2f.
A time-course study revealed that the increase in MMGL-positive cells
was initially observed 8 h after sensitization and had peaked at
20–24 h. At 48 h after sensitization, the number of MMGL-positive
cells had returned to the control level. Typically, the number of
MMGL-positive cells in the T cell area (border to the lymphoid
follicles) was scored using the criteria shown in the footnote to Table I as follows: 4 h, ±; 8 h, +;
12 h, ++; 24 h, +++; and 48 h, ±.
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To confirm the effect of epicutaneous sensitization on the
molecular level, we investigated whether the amount of MMGL in lymph
node lysates changes upon sensitization. It has been demonstrated that
MMGL can be detected in lymph node detergent extracts by SDS-PAGE and
immunoblot analyses using mAb LOM-14 (14). Draining lymph node lysates
of known protein amount were compared for the signals representing MMGL
with or without epicutaneous sensitization (Fig. 3). Signals representing MMGL were
markedly increased upon sensitization not only with FITC in AD but also
with AD alone. These results are consistent with the
immunohistochemical observations of lymph nodes. The apparent m.w. of
MMGL in lymph node lysates was consistent with that determined in our
previous studies (13, 14).
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In the initiation phase of DTH, Langerhans cells are known
to migrate to the T cell area of draining lymph nodes to exert Ag
presentation activity (3). After epicutaneous application of FITC, such
migrants are visible as fluorescent cells in lymph nodes. To
investigate whether FITC was incorporated within MMGL-positive cells
that were transiently increased in lymph nodes upon sensitization,
frozen sections of brachial lymph nodes obtained 24 h after
sensitization were stained using mAb LOM-14 and were observed using a
confocal microscope for two-color fluorescence (Fig. 4, a and b). The
cell-associated fluorescein signals were detected in the T cell area.
MMGL-positive cells were also detected in the vicinity of the
fluorescein-positive cells; however, the majority of MMGL-positive
cells were devoid of the fluorescein label. A minor population of
MMGL-positive cells (7 ± 2%) was positive for fluorescein (Table I
). The fluorescein signals that appeared not to be associated with
cells were also observed around the interfollicular sinus (Fig. 4a). These signals may be due to molecular transport of
FITC-labeled substances along reticular fibers.
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The migration of Langerhans cells was also demonstrable by flow
cytometric analysis. Single cell suspension prepared from brachial
lymph nodes 24 h after epicutaneous application of FITC/AD on the
forelimb skin (Fig. 5a) was
compared with that from untreated lymph nodes (Fig. 5c). The
number of events representing cells that contained significant FITC
label (as defined in Materials and Methods) was 223 of
106 cells analyzed for the FITC/AD condition compared with
39 events/106 cells for untreated lymph nodes. The latter
number presumably reflects background signals due to noise. These data
also provide evidence, in addition to the confocal microscopic
observation, of significant migration of FITC-labeled cells into the
draining lymph nodes upon epicutaneous application of FITC/AD. The
results of flow cytometric analyses are summarized in Table I.
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AD has been used as an optimal solvent for sensitization with FITC
(3, 15, 16). Since AD did not work as an immunogen by itself (Fig. 1),
it is conceivable that AD has some adjuvant effects on sensitization.
Since AD by itself had activity inducing a transient increase in
MMGL-positive cells in the lymph node T cell area (Fig. 2), this
activity could be related to the adjuvant effects. To investigate this
possibility, FITC was dissolved in a variety of solvents, and the
degree of sensitization as well as the effect of increasing
MMGL-positive cells in lymph nodes were compared upon sensitization.
The degree of sensitization monitored by ear swelling was variable when
solvents were changed (Table I). When both APCs (FITC-positive cells)
and MMGL-positive cells were abundant in lymph nodes (AD), the maximal
response was obtained. Even though substantial numbers of
FITC-presenting cells or non-cell-associated FITC-modified molecules
were seen in the T cell area, when MMGL-positive cells were not
substantially increased in the T cell area (acetone/olive oil), or when
they decreased in the medulla of the lymph nodes (ethyl acetate and
DMF), the degree of sensitization was at relatively low levels. When
SDS was used as the vehicle, FITC-presenting cells were seldom seen in
lymph nodes (Table I and Fig. 5b), whereas a significant
increase in MMGL-positive cells was observed in lymph nodes. Despite
the apparent lack of the migration of cells containing FITC,
significant sensitization was obtained. When DMSO was used as a
solvent, sensitization to FITC did not take place. In this case,
neither a migration of FITC-positive cells nor an increase in
MMGL-positive cells in the lymph nodes was observed.
To compare the relationship between the efficiency of sensitization and
the appearance of MMGL-positive cells in lymph nodes, the data in Table I were rescored as follows: 0 for ±, 1 for +, 2 for ++, and 3 for +++.
Then the differences between experimental and control conditions were
shown as an index in Table II. For
example, Table I indicates that MMGL-positive cell distribution in the
medulla was observed as +++ for the FITC/AD condition and ++ for the
control. The index is 3–2 = 1. Because the increase in the number
of cells in the deep T cell area was less dramatic (Table I), only the
values for T cell area (border), subcapsular sinus, and medulla were
taken into account, and the sum of these values was expressed as an
overall index (Table II). Table II also includes the rank order of the
overall index and the rank order of the DTH response (ear swelling).
Based on these calculations, the correlation between the MMGL-positive
cell increase in the lymph nodes and the DTH response was demonstrated
in Fig. 6. These results indicated that
DTH responses were proportional to the increase in MMGL-positive cells
in lymph nodes. Statistical significance of the correlation was
confirmed by either Spearman rank correlation analysis
(p < 0.05) or Kendall rank correlation
analysis (p < 0.02).
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Although the experiments demonstrated a transient increase in
MMGL-positive cells in the draining lymph nodes and the apparent
contribution of this increase to the sensitization process, there was
no evidence that MMGL-positive cells migrated from the dermis. The
epicutaneous labeling with FITC did not provide evidence supporting
this hypothesis (Fig. 4, a and b). Therefore, we
tried to isolate MMGL-positive cells from mouse skin to address this
question directly. BALB/c mouse skin was digested by collagenase, and
MMGL-positive cells were isolated by a magnetic cell sorter using the
anti-MMGL mAb LOM-8.7. Cells were immunohistochemically analyzed
using digoxigenin-conjugated mAb LOM-14, which binds to MMGL on an
epitope independent from that of mAb LOM-8.7, plus alkaline
phosphatase-labeled anti-digoxigenin Ab. Representative cells with
a positive reaction are shown in Fig. 4c. After collagenase
digestion, 5 x 107 cells were recovered per mouse,
and 2–3% of them were MMGL positive. After magnetic cell sorting,
0.75–1.25 x 106 cells were obtained per mouse, and
>90% of them were MMGL positive (Fig. 4c).
Migration of MMGL-positive cells into lymph nodes upon sensitization
MMGL-positive cells obtained from BALB/c mouse skin were labeled
with a cell tracer (CMTMR) and then they were intradermally injected
into recipient BALB/c mouse forelimb skin. Twenty-four hours after
treatment with FITC/AD (Fig. 4, d–g) or with AD alone (Fig. 4, h and i) at the site of injection,
CMTMR-positive cells were found in the T cell area of the brachial
lymph nodes (Fig. 4, e, g, and h).
These cells were also positive for MMGL, which was detected by mAb
LOM-14 plus Cy5-conjugated anti-rat IgG (Fig. 4, e–i).
Most of the CMTMR-positive cells did not contain FITC fluorescence upon
treatment with FITC/AD (Fig. 4, d, e, and
g). The results indicated that the CMTMR-positive cells
represent MMGL-positive cell migrants from the site of intradermal
injection. In the vicinity of CMTMR-positive cells in the sections,
MMGL-positive cells without CMTMR label were also observed (Fig. 4, h and i, small arrows). These cells presumably
represent migrants of host origin. Normal rat serum plus Cy5-conjugated
anti-rat IgG (cytochemical control) did not produce positive
signals (data not shown). Skin samples containing the site of injection
were also examined, and a good retention of the CMTMR fluorescence
within the cells was observed. Quantitative comparison using frozen
section samples revealed that similar numbers of CMTMR-positive cells
were detected upon treatment with FITC/AD or with AD alone (Fig. 7). On the other hand, CMTMR-positive
cells were not detected in untreated lymph nodes. These results
indicated that the migration was induced by epicutaneous sensitization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we demonstrated the contribution of dermal macrophages to the efficiency of contact sensitization using unmanipulated mice. Lymphatic trafficking of dermal macrophages was involved in the process. Our conclusion is based on the following observations. First, dermal macrophages express a macrophage lectin, MMGL, whereas epidermal Langerhans cells do not (14). Second, we found in the present study that the number of MMGL-positive cells in the draining lymph node T cell area increased transiently upon epicutaneous application of Ag (FITC). Because the majority of these cells were devoid of FITC fluorescence, the increased MMGL-positive population could be distinguished from Langerhans cell migrants. Third, the degree of the MMGL-positive cell increase in the lymph nodes was greatly influenced by the conditions of Ag application, especially by solvents used to dissolve the Ag. The efficiency of sensitization was also influenced by these conditions, and the increase in MMGL-positive cell number and the efficiency of sensitization were positively correlated. For optimal sensitization, the following requirements were recognized: 1) FITC-presenting cell (mainly of Langerhans-cell origin) migration, 2) MMGL-positive cell increase, and 3) lack of depletion of MMGL-positive cells from the medulla. Fourth, we successfully isolated MMGL-positive cells from mouse skin by positive selection using a mAb against MMGL, and we injected these cells into syngeneic recipient mice intradermally after labeling them with a fluorescent cell tracer. We observed the appearance of the labeled cells in the draining lymph nodes of the recipient mice upon epicutaneous sensitization.
One might argue that the CMTMR-positive cells in lymph nodes do not
represent cellular trafficking from dermis but represent in situ uptake
of fluorescent molecules that might be released from the labeled cells.
However, such a possibility is unlikely for the following reasons.
First, we used CMTMR as a cell tracer. CMTMR is trapped within cells in
the form of a thiol-conjugated product by the activity of cellular
glutathione S-transferase (20, 21). This label is much more
resistant to lipophilic environments than widely used lipophilic cell
tracers, such as DiI and PKH26. Actually, observation of skin samples
containing the site of injection revealed a good retention of the
fluorescence label within cells. Second, there was very low background
for CMTMR fluorescence in lymph nodes. The background was lower than
that produced by FITC. For example, a strong FITC fluorescence along
reticular fibers was observed. Third, if macrophages incorporated CMTMR
in situ, it is natural to expect macrophages also to have incorporated
FITC, because FITC-labeled molecules were much more abundant in the
lymph nodes after epicutaneous application of FITC. However,
CMTMR-positive cells were not positive for FITC fluorescence (Fig. 4, d, e, and g). For these reasons, we
concluded that CMTMR-labeled cells appearing in draining lymph nodes
represent migrants from the dermal site of injection.
One might also argue that the main reason for the increase in MMGL-positive cells in the T cell area is not cell migration of dermal macrophages but up-regulation of MMGL on resident macrophages and/or dendritic cells. We do not exclude the possibility that the induction or up-regulation of MMGL expression in some types of cells may be responsible for MMGL-positive cell increase in part. However, it should be noted that our results clearly demonstrated the presence of lymphatic trafficking of MMGL-positive cells that was inducible by epicutaneous sensitization. It should also be emphasized that Langerhans cell migrants did not acquire MMGL expression in the lymph node environments immediately. Because the present study allows us to examine the nature of Langerhans cell migrants that could be labeled with FITC, the questions remains as to whether MMGL expression can be induced on some resident dendritic cells in lymph nodes by signals mediated by soluble factors or by cell-cell interaction between macrophage migrants and the resident cells.
It has been reported that occlusion of afferent lymphatic vessels by surgical manipulation severely decreases the number of subcapsular sinus macrophages in lymph nodes (22, 23). Furthermore, the occlusion of afferent lymphatic vessels is known to result in the change in localization of macrophages (detected by mAb MOMA-1) from the subcapsular sinus to the T cell area of the lymph nodes in an early stage after the occlusion (23). These results suggest the possibility that lymph node macrophages are continuously supplied by lymphatic cellular trafficking, changing localization within the lymph nodes, and that this then results in their turnover. Alternatively, it is also possible that lymph node macrophages are dependent on factors provided by afferent lymphatic vessels. Although the latter possibility has not been ruled out, our present results not only appeared to be compatible with the former possibility, but provided new insights into the origin of lymph node macrophages in relation to lymphatic cell trafficking.
One interesting point concerning MMGL-positive cell migration was that
this phenomenon was not only greatly influenced by the vehicles used to
dissolve Ag, but was produced by the vehicle alone (Figs. 2, 3, and 7
and Table I). TCRs are likely to recognize FITC determinants rather
than substances included in a vehicle such as AD, because the presence
of FITC was required to produce a DTH response in mice in this system
(Fig. 1). Thus, vehicles such as AD can be regarded as adjuvants during
sensitization, and one of the mechanisms of the adjuvant effects is
considered to be the ability to induce dermal macrophage migration.
The majority of MMGL-positive cells in the draining lymph nodes were
devoid of FITC fluorescence, suggesting that the majority of
MMGL-positive migrants may not serve as APCs (Fig. 4). What are the
roles of MMGL-positive cell migrants other than that of Ag
presentation? One possibility is that MMGL-positive cells could
cooperate with Ag-presenting dendritic cells through cytokine
production in the lymph node environments. The contribution of
cytokines that can be produced by macrophages, including TNF-
,
IL-1ß, and IL-12, to the sensitization phase has already been
proposed (18, 24, 25, 26). Another possibility is that MMGL-positive
macrophages could contribute to the maintenance of high endothelial
venules (HEV) and their expression of ligands for L-selectin, which are
required for lymphocyte recirculation into lymph nodes. In this case,
MMGL-positive macrophages would contribute to the sensitization phase
by enhancing the probability that circulating naive T lymphocytes will
encounter the APCs in the lymph nodes. The transient accumulation of
MMGL-positive cells in areas surrounding HEV (Fig. 2) may suggest such
a possibility. Experiments by others have also demonstrated that
occlusion of afferent lymphatic vessels results in the reduction of
functional HEV ligands (GlyCAM-1 and CD34) and MECA-79 epitope (a
determinant of peripheral lymph node addressin) expression on HEV as
well as in changes in HEV morphology (22, 23, 27). The occlusion of
afferent lymphatic vessels also decreased the number of lymph node
macrophages, suggesting that macrophages supplied by afferent
lymphatics may be responsible for the maintenance of HEV function (23).
In an in vitro study, lymph node subcapsular sinus macrophages appeared
to contribute to the maintenance of HEV adhesive function (28).
However, the question of whether some uncharacterized soluble factors,
rather than cellular components, might be responsible for the
maintenance of HEV function is still controversial (23, 27).
A remaining question is whether MMGL molecules themselves are involved in the process of migration of dermal macrophages. It would be interesting to know whether administration of anti-MMGL mAbs, which efficiently interfere with the binding of carbohydrate ligands (13), could block the migration of dermal macrophages and whether such treatment could affect the sensitization phase of contact hypersensitivity. We are in the process of studying whether anti-MMGL mAbs can block part of the process that is responsible for the migration of dermal macrophages.
In conclusion, we observed that epicutaneous sensitization produced a transient increase in MMGL-positive cells in the T cell area of the regional lymph nodes. The extent of the increase was not only greatly influenced by the conditions of sensitization, especially by the selection of vehicles used to dissolve Ag, but was also positively correlated with the efficiency of sensitization. Finally, we provided direct evidence that epicutaneous sensitization produces trafficking of MMGL-positive cells into regional lymph nodes.
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2 Current address: Department of Microbiology, University of Shizuoka School of Pharmaceutical Sciences, Yada, Shizuoka 422-8526, Japan.
3 Address correspondence and reprint requests to Dr. Tatsuro Irimura, Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail address:
4 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; MMGL, mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin; DMF, dimethylformamide; CMTMR, 5(and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; AD, acetone/dibutyl phthalate (1:1); DPBS, Dulbecco’s modified PBS; HEV, high endothelial venules.
Received for publication February 17, 1998. Accepted for publication August 27, 1998.
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is required for accumulation of dendritic cells in draining lymph nodes and for optimal contact sensitization. Immunology 84:31.[Medline]
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=
0.865, p = 0.03; by Kendall rank correlation
analysis: 
= 0.781, p = 0.01).