International Immunology

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International Immunology, Vol. 13, No. 5, 695-704, May 2001
© 2001 Japanese Society for Immunology

Skin antigens in the steady state are trafficked to regional lymph nodes by transforming growth factor-ß1-dependent cells

Hiroaki Hemmi^1,10, Miya Yoshino^1,2, Hidetoshi Yamazaki¹, Makoto Naito³, Tomonori Iyoda⁴, Yoshiki Omatsu⁵, Susumu Shimoyama⁵, John J. Letterio⁷, Toru Nakabayashi⁸, Hisashi Tagaya¹, Toshiyuki Yamane¹, Minetaro Ogawa⁶, Shin-Ichi Nishikawa⁶, Kazuo Ryoke², Kayo Inaba⁵, Shin-Ichi Hayashi¹ and Takahiro Kunisada^1,9

¹ Department of Immunology, School of Life Science, and
² Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan
³ Second Department of Pathology, Niigata University School of Medicine, Niigata 951-8510, Japan
⁴ Laboratory of Immunobiology, Graduate School of Science,
⁵ Graduate School of Biostudies and
⁶ Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8502, Japan
⁷ Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-5055, USA
⁸ Department of Internal Medicine, Health Science University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan
⁹ Department of Hygiene, Gifu University, School of Medicine, Gifu 500-8705, Japan

Correspondence to: S.-I. Hayashi

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Antigen capturing in the skin and antigen trafficking into regional lymph nodes (LN) initiate immune responses. In this study, employing melanin granule (MG) as an easily traceable antigen in two mouse strains that carried steel factor or hepatocyte growth factor transgenes and had melanocytosis in the epidermis or in the dermis respectively, we investigated the mechanism of antigen trafficking from the skin. MG captured in the epidermis or dermis accumulated in the regional LN, but not other tissues. Only in alymphoplastic mice did MG-laden cells pass through the lymphatics and reached many tissues. Since inflammatory regions were not observed in the skin of either type of transgenic mouse, our developmental system enables us to investigate constitutive capturing and trafficking of insoluble antigens in the steady state. Both dendritic cells and macrophages were laden with MG in the regional LN. To determine which cells traffic antigens to the LN, we prepared double mutants that carried the transgenes and lacked transforming growth factor (TGF)-ß1, since mice lacking TGF-ß1 are reported to be deficient of Langerhans cells. Few MG were observed in the regional LN of these double-mutant mice. We also showed that signaling via macrophage colony stimulating factor receptor or Flt3/Flk2 is not essential for development of the cells for this antigen trafficking. These results indicate that antigens in the epidermis and dermis in the steady state are trafficked into regional LN only by TGF-ß1-dependent cells, which may be a dendritic cell lineage.

Keywords: autoantigen, dendritic cell, hepatocyte growth factor (Hgf), Langerhans cell, melanin granule, steel factor (Mgf)

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The skin, which consists of the epidermis and underlying dermis, is the main barrier against organisms and the external environment. These tissues contain cell populations that play critical roles in immune responses (1,2). Antigens captured in the skin are subsequently trafficked into lymphoid tissues that drain through afferent lymphatics. Finally, they become located in paracortical T cell areas and are presented to lymphocytes (3–6).

Dendritic cells (DC) are known be critical for activating immune responses and inducing tolerance. DC consist of two cell lineages which are derived from myeloid and lymphoid precursors. Genetic evidence for separate subsets of DC development has come from the studies of gene knockout mice. Mice carrying a mutant Ikaros gene are only deficient in lymphoid DC (7). On the other hand, myeloid DC are further characterized by requirements for transforming growth factor (TGF)-ß1 (gene designation: Tgfb1) and a transcription factor, RelB. Tgfb1 null mice show a complete lack of Langerhans cells (LC), the epidermal contingent of myeloid DC, but normally contain other DC subsets (8–11). In contrast, mice lacking the relB gene are deficient in myeloid DC, but LC are normally present (12).

In the epidermis, antigen-capturing and -trafficking cells are thought to be LC (3,13–15). LC have the capacity to take up antigens and lose this capacity after maturation in response to various stimuli, including exposure to contact sensitizers, bacteria or UV light (16–18). Recently, Randolph et al. demonstrated that inflammatory monocytes other than LC carried phagocytosed particles to lymph nodes (LN) and differentiated into DC, and antigen trafficking was affected in mice with reduced numbers of monocytes resulting from macrophage colony stimulating factor (M-CSF) deficiency (19). It was reported that signaling via a ligand for a receptor protein tyrosine kinase, Flt3/Flk2, influenced DC growth (20). Chemokine signals have also been reported to play important roles in DC migration (13,21,22). Precursors of myeloid DC share characteristics with monocytes circulating in the bloodstream, and differentiation of DC from bone marrow cells is induced in the presence of granulocyte macrophage colony stimulating factor (GM-CSF), IL-4 and/or tumor necrosis factor (TNF)- in culture (23,24). Although vigorous efforts have been made to further classify DC according to their signaling requirements and the molecules they express, the heterogeneity of DC subsets has not been fully clarified (3,15,25–30).

We have developed two hyperpigmented transgenic (Tg) lines of mice, one carrying a steel factor (SLF; gene designation: Mgf) cDNA (31) and one carrying a hepatocyte growth factor (HGF; gene designation: Hgf) cDNAs (32): both of which are driven by the human cytokeratin 14 (K14) promoter (33,34). Mgf-Tg mice had dramatically increased numbers of melanocytes in the epidermis, whereas Hgf-Tg mice had increased numbers of melanocytes mainly in the dermis. In the present study, by tracing the location of melanin granules (MG), the mechanisms of antigen capturing in the skin and antigen trafficking into regional LN were examined. In the steady state, MG captured at the epidermis or dermis were trafficked only to regional LN. Only alymphoplastic (aly/aly) mice trafficked antigens to other tissues. The relation of signaling via Flt3/Flk2 and c-Fms (a receptor for M-CSF) to antigen trafficking was also examined by injection of antagonistic antibodies (35,36). Moreover, using LC-deficient Tgfb1^–/– mice crossed with Mgf-Tg and Hgf-Tg mice, we show that in the steady state, antigens in both epidermis and dermis are trafficked only by TGF-ß1-dependent cells, which might be LC and/or their equivalents. Our mouse models of melanocytosis provide new systems for answering questions about the dynamics of antigen transport in vivo.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Mice
Tg mice carrying full-length murine Mgf and human Hgf cDNAs driven by the human K14 promoter region, which is expressed in keratinocytes, were produced by microinjection methods as described (33,34). C57BL/6-Mgf-Tg/+ (Mgf-Tg, designated #Tg1-1) and C57BL/6-Hgf-Tg/+ (Hgf-Tg, designated #Tg43) mice were maintained in the Animal Research Center, Faculty of Medicine, Tottori University. ALY/Nsc-Jcl-aly/aly (aly/aly) (37) and C57BL/6 mice were purchased from Japan Clea (Tokyo, Japan). Mice carrying aly/aly Mgf-Tg and aly/aly Hgf-Tg were produced from matings of the respective double heterozygotes. Mice with the aly/aly genotype were identified by their lack of LN. 129/Sv-Tgfb1^–/– mice were obtained from the National Institutes of Health (Bethesda, MD) (8), and maintained and mated in the Animal Research Center, Faculty of Medicine, Tottori University.

Antibody injection
An antagonistic rat mAb against mouse Flt3/Flk2 (A2F10: 50 mg/kg) (35) was injected i. p. every 3 days from the day of birth until the 35th day. Anti-mouse c-Fms antibody (AFS98: 200 µg/mouse) (36) was injected daily from the day of birth until the 29th day. On day 36 (anti-Flt3/Flk2) or day 30 (anti-c-Fms), LN (cervical, axillary and inguinal) were prepared and the number of MG-laden cells/field (10 fields/mouse) was counted.

Preparation of tissue sections and immunohistochemistry
Mice were sacrificed under deep anesthesia with ether. Regional LN (cervical, axillary and inguinal), mesenteric LN, Peyer's patches, trunk skin, liver, kidney and spleen were fixed with 4% paraformaldehyde, washed overnight with PBS, dehydrated with a graded series of ethanol (70, 90 and 100% twice) and embedded in polyester wax (BDH, Poole, UK). Sections (7 µm thick) were cut, and unstained and immunohistochemically stained sections were prepared. Samples of these tissues were also prepared for electron microscopy.

Light and electron microscopic analyses
To examine the distribution of MG-laden cells, tissue sections were deparaffinized and prepared as unstained specimens for examination by light microscopy. For electron microscopic analysis, tissues were diced into 1–2 mm³ pieces, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 and then in 1.5% glutaraldehyde for 2 h, and post-fixed in 1.0% osmium tetroxide for 2 h. After dehydration in a graded series of ethanol, the specimens were processed through propylene oxide and embedded in Epon 812 (E. Fullan, Latham, NY). Ultra-thin sections were cut with diamond knives, stained with uranyl acetate and lead citrate, and examined with an electron microscope (H-800; Hitachi, Tokyo, Japan).

Immunohistochemical analysis
Sliced sections were stained with antibodies directed against the following antigens: murine DEC-205, CD11c and I-A^b for detection of the DC lineage, and Mac-1, F4/80 and FA11 for the macrophage lineage. After de-paraffinization and blocking of endogenous peroxidase in 0.3% hydrogen peroxide/methanol, the sections were blocked with Block Ace on ice for 20 min and incubated overnight at 4°C with anti-E-cadherin (10 µg/ml, ECCD-2), anti-DEC-205 (20 µg/ml, NLDC-145) (38), anti-CD11c (20 µg/ml, HL3; PharMingen, San Diego, CA), anti-I-A^b (PharMingen), anti-Mac-1 (10 µg/ml, M1/70; PharMingen), anti-F4/80 (10 µg/ml) (39) or anti-FA11 (a hybridoma supernatant) (40) antibodies. The sections were washed 3 times with 0.02% Tween 20/PBS at 4°C for 10 min, incubated with fluorescence-labeled secondary antibodies or biotinylated antibodies and streptavidin–FITC (Kirkegaard & Perry, Gaithersburg, MD).

Flow cytometry
Regional LN were homogenized and used to prepare single-cell suspensions in RPMI 1640 containing 5% FBS. Cells were blocked on ice with heat-inactivated normal rabbit serum for 10 min and then stained with mAb against CD11c, CD1, Mac-1, B220, DEC-205, I-A^b, CD40, CD54, CD80, CD86 or F4/80 (PharMingen). The stained cells were analyzed using an Epics XL flow cytometer or sorted using an Epics Elite cell sorter (Coulter, Hialeah, FL).

RT-PCR
To determine whether melanocytes were present in the regional lymph nodes, total RNAs from 100 mg of freshly prepared LN and trunk skins of both Mgf-Tg and Hgf-Tg mice were isolated, and each sample was reverse transcribed using Reverse TraAce (Toyobo, Osaka, Japan). The DNA fragments were amplified from the cDNAs by PCR. The oligonucleotide primers used in this PCR were as follows: Trp2: 5'-GGACCGGCCCCGACTGTAATC-3', and 5'-CATCCCGTTGCGTTTCCTGAGTA-3'; Hprt: 5'-GTAATGATCAGTCAACGGGGGAC-3' and 5'-CCAGCAAGCTTGCAACCTTAACCA-3'. PCR thermal cycling consisted of one incubation at 94°C for 3 min; 36 cycles of 94°C for 45 s, 58°C for 60 s and 72°C for 90 s; and finally one incubation at 72°C for 3 min.

	Results

Top Abstract Introduction Methods Results Discussion References

 
MG-capturing cells in the skin of Mgf-Tg and Hgf-Tg mice
In normal mice, melanocytes are generally present only in hair follicles and are rare in the epidermis or dermis. Dramatic accumulation of melanocytes was observed in Mgf-Tg (33,41) and Hgf-Tg mice (34) (Fig. 1A). In this study, we took advantage of the MG produced by melanocytes as an antigen distributed in skin regions. Since melanocytosis in Mgf-Tg mice was restricted to the epidermis, we generally did not investigate MG-capturing macrophages or DC in the dermis (Fig. 1A and B). In contrast, melanocytosis in Hgf-Tg mice was preferentially observed in the dermis and MG-capturing macrophages were frequently observed in their dermis; however, epidermal LC in the Hgf-Tg mice did not capture MG (Fig. 1B). DEC-205⁺ LC in Mgf-Tg and Hgf-Tg mice were located in the basal and suprabasal parts of the epidermis at a similar interval to that in normal littermates (Fig. 1B). In some experiments, we observed slightly reduced numbers of MHC class II-expressing DC in the epidermal sheet from Mgf-Tg skin (Fig. 1C).

View larger version (96K): [in this window] [in a new window]   Fig. 1. Melanocytosis in the epidermis of Mgf-Tg and the dermis of Hgf-Tg mice, and accumulation of MG in the regional LN. (A and B) Transversal trunk skin sections from 8-week-old Mgf-Tg, Hgf-Tg and control mice were stained with an FITC-labeled anti-E-cadherin (E-cad.) antibody (A) for epidermis and with an anti-DEC-205 antibody (B) for LC cells. Arrows indicate LC cells in the epidermis (B). Bars represent 50 (A) and 20 (B) µm. (C) Epidermal sheets were stained with anti-I-Ab (MHC II) antibody. Bars represent 10 µm. (D) Blackened LN in the Mgf-Tg lymphoid tissues were only detected in the cervical (Cer.), axillary (Axi.) and inguinal (Ing.) LN, but not mesenteric (Mes.) LN, and Peyer's patches (PP). LN were prepared from Mgf-Tg mice (lower) and normal littermates (upper). Results similar to those observed in Mgf-Tg mice were also observed in the Hgf-Tg mice.

 
SLF is known to function as not only a melanocyte growth factor, but also a mast cell growth factor (31). Although some Mgf-Tg lines which produced a large amount of SLF developed mastocytosis in the skin (33), the Mgf-Tg #1-1 line used here did not show any symptoms of mastocytosis or inflammatory responses by histopathological analyses (Fig. 1A). Using anti-CD11c antibody combined with several antibodies against Mac-1, B220, DEC-205, I-A^b (MHC II), CD40, CD54, CD80 or CD86, we also ascertained that the content of cell lineages in the Mgf-Tg LN was comparable to that in non-Tg control littermates (Fig. 2).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Flow cytometric analyses of regional LN cells in Mgf-Tg and control littermate mice. Using anti-CD11c antibody combined with antibodies against several cell markers, the indicated staining was performed. No significant difference was detected in the profiles.

 
Detection of MG-laden cells in skin regional LN
To determine where MG in the skin are trafficked, we investigated the lymphoid tissues of 8-week-old Mgf-Tg and Hgf-Tg mice. Regional (cervical, axillary and inguinal) LN of skin sites were blackened and MG-laden cells were detected there but not in other tissues (thymus, spleen, liver, kidneys, lung, mesenteric LN, Peyer's patches or other non-skin regional LN) (Fig. 1D). Almost all MG trafficked from the epidermis in Mgf-Tg mice or dermis in Hgf-Tg mice were trapped in the regional LN.

To confirm these observations, which are genetically unable to produce LN, aly/aly mice were crossed with Mgf-Tg or Hgf-Tg mice and double mutants, aly/aly Mgf-Tg or aly/aly Hgf-Tg mice respectively, were obtained. These mice displayed melanocytosis in the same skin regions as the respective parental Tg mouse strain. MG-laden cells in both types of double-mutant mice were observed in the white pulp as well as the red pulp of the spleen, in liver sinusoids and peri-bile ductal regions, where mononuclear cells infiltrated in aly/aly mice, and in perivascular mononuclear cell-infiltrated regions of the lung (Table 1 and Fig. 3). Lack of LN leads MG-laden cells passing through lymphatics and the thoracic duct into the bloodstream, and MG may then reach the spleen, liver and lung. These findings indicate that MG do not enter blood vessels directly and also suggest that MG may not directly drain to lymphatics without being captured.

View this table: [in this window] [in a new window]   Table 1. Distribution of MG-laden cells in alymphoplastic mice

 

View larger version (82K): [in this window] [in a new window]   Fig. 3. Detection of MG in spleen, liver and lung of aly/aly Mgf-Tg double-mutant mice. Tissue sections of spleen, liver and lung from aly/+ Mgf-Tg and aly/aly Mgf-Tg mice. MG (arrows) were detected in the double-mutant mice, but not heterozygous littermates. Similar results were also observed in aly/aly Hgf-Tg tissues. Bars represent 25 µm for aly/+ Mgf-Tg spleen, 10 µm for aly/+ Mgf-Tg liver and 50 µm for others.

 
Characterization of MG-laden cells in skin regional LN
To characterize the cell lineage of MG-laden cells in skin regional LN, we performed immunohistostaining and electron-microscopic analyses. In both Mgf-Tg and Hgf-Tg mice, MG-laden cells were mainly located in the marginal sinus, paracortical T cell areas and medullary cords of skin regional LN (Hgf-Tg: Fig. 4A; Mgf-Tg: data not shown). Some DEC-205⁺, CD11c⁺ or I-A⁺ cells, which might be from the DC lineage, and FA11⁺ or F4/80⁺ macrophage-like cells carrying MG were present in the tissue sections (Fig. 4B) and cytospun cells (data not shown). Further analysis was performed by electron microscopy. Typical DC were loaded with small amounts of MG and macrophages carried large amounts of MG (Mgf-Tg: Fig. 4C; Hgf-Tg: data not shown). Since we did not find any difference between LN from Mgf-Tg and Hgf-Tg mouse strains, the representative data were presented in Fig. 4.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Location and characterization of MG-laden cells in the regional LN of Tg mice. (A) Low magnification of tissue section of Hgf-Tg inguinal LN. The bar represents 200 µm. Similar results were observed in Mgf-Tg LN. (B) In the Mgf-Tg (left), and Hgf-Tg (right) inguinal LN, some DEC-205+ DC (upper) and FA11+ macrophages (lower) were laden with MG. Bars represent 20 µm. (C) Electron-microscopic analysis of MG-laden cells in the Mgf-Tg inguinal LN. Typical DC (upper) and macrophage (lower) loaded with MG in the cytoplasm. Similar results were also observed in Hgf-Tg LN.

 
We also analyzed LN cells using FACS. Sorted I-A^b- and CD11c-positive cells or Mac-1- and F4/80-positive cells in the Mgf-Tg LN appeared morphologically to be DC and macrophages respectively, and some of them were laden with MG in their cytoplasm (data not shown). Semi-quantitative RT-PCR analysis showed that compared with skin tissues, LN expressed <1/1000 of the mRNA for the tyrosinase-related protein 2 (Trp2) gene, a marker of melanocyte lineage cells (42). This result rules out the possibility that MG-bearing cells in the LN are melanocytes (Fig. 5).

View larger version (25K): [in this window] [in a new window]   Fig. 5. No detection of melanocyte lineage cells in Mgf-Tg LN. RT-PCR for Trp2 and Hprt gene expression in inguinal LN or trunk skin from Mgf-Tg mice was performed with (1–6) or without (RT–) reverse transcriptase. cDNA for templates was diluted for detection of the Hprt gene. The same concentration of cDNA from the LN (lane 1) and skin (lane 2), and 1/10 (lane 3), 1/100 (lane 4), 1/1000 (lane 5) and 1/10,000 (lane 6) dilutions of the cDNA from skin were subjected to PCR amplification specific for Trp2 and Hprt transcripts.

 
If MG continuously trafficking from skin sites were trapped in the regional LN, MG-laden cells might increasingly accumulate in the LN as the age of the mice increased. The numbers of MG-laden cells in regional LN of Mgf-Tg mice were counted at various times after birth. The number of MG-laden cells increased with increasing age, indicating that MG in the skin were constantly trafficked to the regional LN and accumulated in situ. MG-laden cells in regional LN were morphologically classified by microscopy and their numbers were counted. The number of MG-laden macrophage-like cells showed a linear increase with increasing age of the mice. The number of MG-laden DC-like cells also increased linearly up to 28 days (Fig. 6). We detected MG-laden cells even at 3 days of age, at which time the microarchitecture of LN has not yet been constructed (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Accumulation of MG-laden cells in the regional LN. The numbers of MG-laden DC-like and macrophage-like cells in the inguinal LN of Mgf-Tg mice were counted under the microscope. A field was 0.0589 mm2 and 10 fields were counted for each sample.

 
No significant effects were observed on antigen trafficking by injection of antibodies against Flt3/Flk2 or c-Fms
As both DC and macrophages were loaded with MG in the LN, we could not identify which cells captured MG in the skin and trafficked to regional LN. Development of DC and macrophages has been reported to be influenced by signaling via Flt3/Flk2 (20,43) and c-Fms (19,36,44) respectively. To determine whether the signaling via these receptors influenced antigen capturing and/or trafficking from skin sites to LN, antagonistic antibodies were repeatedly injected into Mgf-Tg mice from the newborn stage, and the effect of the antibodies was investigated by counting the MG-laden cells in regional LN (Table 2). We found the following effects of the antibodies: anti-Flk3/Flk2 antibody injection reduced I-A⁺ CD11c⁺ DC in the spleen (0.80 ± 0.31% in non-treated mice versus 0.32 ± 0.13% in anti-Flk3/Flk2 antibody-injected mice, P = 0.0169); and anti-c-Fms antibody injection resulted in a significant reduction of peritoneal macrophages, shortened limbs and osteopetrotic bones, features similar to those observed in op/op mice, which lack functional M-CSF (data not shown) (44,45). LN contain M-CSF-dependent and -independent macrophages, and the DC lineages in op/op mice are relatively normal (46,47). After the antibody injections, we counted MG-laden cells in the regional LN, and we observed no significant differences between the LN of non-treated mice and mice treated with either antibody (Table 2). These results indicate that blockage of the signaling mediated by Flt3/Flk2 or c-Fms does not affect MG capturing or trafficking to regional LN.

View this table: [in this window] [in a new window]   Table 2. No significant effects were observed on antigen trafficking by injections of antibodies against Flt3/Flk2 or c-Fms

 
TGF-ß1-dependent cells trafficked skin MG into the regional LN
Next, we produced Mgf-Tg and Hgf-Tg mice lacking TGF-ß1. Tgfb1^–/– mice have been reported to lack LC (9,48). If double mutants, Mgf-Tg Tgfb1^–/– and/or Hgf-Tg Tgfb1^–/– mice, contained MG-laden cells in skin regional LN, it would imply that cells of some lineage other than the LC lineage mainly contribute to antigen trafficking. Heterozygous Tgfb1^+/– mice carrying Mgf-Tg or Hgf-Tg contained MG-laden cells normally in their LN (Fig. 7). However, we rarely detected MG-laden cells in skin regional LN of Mgf-Tg Tgfb1^–/– or Hgf-Tg Tgfb1^–/– mice (Fig. 7). As treatment with rapamycin abrogates the fatal wasting syndrome of Tgfb1^–/– mice, similar experiments were performed using rapamycin-treated mice (9) and comparable results were obtained. The double-mutant mice showed melanocytosis in their skin like Mgf-Tg Tgfb1^+/– and Hgf-Tg Tgfb1^+/– mice, and the distributions of MG were also comparable to those in the respective strains. Some LN from double-mutant mice were enlarged, as reported in Tgfb1^–/– mice (Fig. 7) (9). F4/80⁺ macrophages in the dermis and in the LN were present normally in both types of double-mutant mice (data not shown). The results clearly indicate that only TGF-ß1-dependent cells, which are thought to represent one of the DC lineages, contribute to antigen trafficking from the epidermis and dermis. When these experiments were repeated in double-mutant mice, we sometimes detected a few macrophage-like cells laden with a small amount of MG in regional LN. Since these mice had dermatitis, inflammative reactions appear to have occurred regularly, allowing antigens to pass through blood vessels and lymphatics, as indicated by the fact that these rare cases were accompanied by MG distributed to not only the regional LN but also the spleen and liver.

View larger version (109K): [in this window] [in a new window]   Fig. 7. Lack of MG-laden cells in the regional LN of Tgfb1–/– Tg mice with melanocytosis. Inguinal LN and their tissue sections from 18-day-old Mgf-Tg- or Hgf-Tg-bearing Tgfb1 null mice and respective Tgfb1+/– littermates. Few MG-laden cells were detected in Tgfb1–/–-carrying Mgf-Tg and Hgf-Tg mice. Bars represent 1 mm for upper panels and 10 µm for lower panels.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Initiation of immune responses starts from capturing of antigens, trafficking them into lymphoid organs and presenting them to immunocompetent cells. The classical question of which cells capture antigens in skin sites and traffic them to regional LN still remains to be answered. The present study demonstrated that in the `steady state' in which no inflammatory response is induced, antigens are captured by DC and macrophages in the skin, and the LN contain DC and macrophages which are laden with antigens, but it is only TGF-ß1-dependent cells that traffic them from the skin.

It has been reported that DC and macrophages function to traffic antigens from peripheral non-lymphoid tissues via the lymphatics and/or bloodstream into lymphoid tissues (49,50). A majority of studies which have investigated antigen trafficking by soluble antigen painting, injection of antigens into skin sites or transplantation of skin grafts, might have induced the `active state' accompanied by contact hypersensitivity, injured vessels or induction of inflammation (16–18,51–55). Our transgenic mouse models for melanocytosis enable us to investigate constitutive capturing and trafficking of insoluble antigens in the steady state.

MG-laden cells in Mgf-Tg and Hgf-Tg LN were DC and macrophages, and they accumulated increasingly in the LN for 28 days according to the age of the mice. However, comparison of the numbers in 28-day- and 3-month-old mice showed that the number of macrophages loaded with MG increased linearly, but not the number of DC (Fig. 6). The Tg mice lacking the Tgfb1 gene have few MG-laden cells in the LN, indicating that MG in the skin were constantly trafficked by TGF-ß1-dependent cells, which are thought to be DC-lineage. This implies that MG loaded in macrophages in the regional LN should be trafficked by DC. TGF-ß1-dependent DC traffic MG into LN, and in the LN, DC may transfer MG to macrophages. Since the life span of DC is known to be relatively short (2), macrophages in LN may phagocytose DC that contain MG.

Recently, Moodycliffe et al. reported that the signaling via CD40 regulated the migration of antigen-bearing DC and speculated that TNF- production by mast cells might play a critical role in this event (56). They also showed that the activation induced phenotypic changes of DC in the regional LN. Although we did not detect any mast cell accumulation in Mgf-Tg skin, SLF may stimulate mast cells and thereby activate antigen-trafficking even in the steady state. However, as shown in Fig. 2, LN in Mgf-Tg mice contained comparable phenotypes of cells with normal littermates. Moreover, the fact that Hgf-Tg mice also showed similar antigen trafficking rules out the possibility that in Mgf-Tg mice, mast cell accumulation or activation results in antigen trafficking in the steady state.

The possibility still remains that uncaptured MG directly flow into dermal lymphatics, although this is not likely because melanosomes are large granules (>0.5 µm in diameter). Since MG were found in skin regional LN and not in other LN or tissues, MG are not likely to enter blood vessels in the steady state. This observation is consistent with a recent study (19). Some Mgf-Tg Tgfb1^–/– and Hgf-Tg Tgfb1^–/– mice, which had dermatitis, were found to contain small amounts of MG-laden cells in the LN. These mice also had MG in the spleen and liver. This implies that inflammatory events may induce MG draining to blood vessels and lymphatics directly or activate antigen trafficking by TGF-ß1-independent cells such as macrophages.

Lack of functional M-CSF in op/op mice reduces the number of skin macrophages by up to 20% compared to the number in normal littermates but does not reduce the number of DC (46,47,57). Randolph et al. showed that op/op mice have reduced numbers of monocytes, which differentiate into DC in LN (19). They observed a very limited or no inflammatory reaction in op/op mice at the site of antigen injection. In contrast, although Mgf-Tg mice treated with anti-c-Fms antibody had a reduced number of macrophages and a lack of osteoclasts, like op/op mice (36,58), the number of MG-laden cells in skin regional LN was not decreased, indicating that signaling via c-Fms does not contribute to antigen capturing or trafficking. Tgfb1^–/– mice have not been reported to have deficiencies of macrophages, lymphoid DC or myeloid DC except for LC (9,59). The discrepancies about the effects of M-CSF signaling might need to be addressed to clarify the difference of the active state versus the steady state. It might be the case that the antigen-trafficking cells are totally different in the active and steady states.

Maraskowsky et al. have demonstrated that administration of Flt3L dramatically increased the number of mature DC in vivo (20,43). We showed here that blockage of the signaling via Flt3/Flk2 by injection of anti-Flt3/Flk2 antibody into Mgf-Tg mice did not affect antigen capturing or trafficking from the epidermis. There may be DC lineage cells with the potential to respond to Flt3L; however, under in vivo conditions, signaling via Flt3/Flk2 is not essential for development of DC responsible for antigen trafficking. Alternatively, TGF-ß1-dependent cells may belong to a different lineage in Flt3L-responsive cells (8,28).

We would like to propose the following model of the antigen-trafficking process in the skin (Fig. 8). Myeloid DC precursors (#1 and #1' in Fig. 8) migrate into the dermis from post-capillary venules. Some of the cells whose differentiation depends on the presence of TGF-ß1, migrate into the epidermis or remain in the dermis and differentiate to LC (#2) or DC (#4) respectively. LC in the epidermis capture antigens and re-migrate into the dermis (#3) and subsequently enter into lymphatics. In the dermis, DC (#4 and #6) and macrophages (#5) differentiate from myeloid precursors, and capture antigens, but only TGF-ß1-dependent cells (#4) enter into lymphatics and reach regional LN.

View larger version (36K): [in this window] [in a new window]   Fig. 8. A hypothetical mechanism of antigen trafficking from the skin in the steady state. Myeloid precursor cells migrate from post-capillary venules. Both TGF-ß1-dependent and -independent cells differentiate into antigen-capturing cells, but only the former traffic antigens to regional LN (See text). Shaded cells have the capacity to capture antigens.

 
MG-capturing F4/80⁺ macrophages were frequently observed in the Hgf-Tg dermis; however, epidermal LC in the Hgf-Tg epidermis did not capture MG (Fig. 1B). This result indicates that the precursors of LC in the dermis (cell #1 in Fig. 8) may not efficiently capture MG in the dermis, although Murphy and colleagues have reported that after human bone marrow engraftment, repopulating LC were pigmented (60). We rarely detected MG-capturing macrophages or DC in the dermis of Mgf-Tg mice, whose MG were only present in the epidermis (Fig. 1A and B), suggesting that on the way to re-entry to the dermis from the epidermis (cell #3 in Fig. 8), LC descendants might enter into lymphatics immediately. In Hgf-Tg Tgfb1^–/– dermis, MG-capturing macrophages were frequently observed, but did not traffic MG into LN (cells #5 and #6 in Fig. 8). Trafficking antigens in the steady state from the dermis as well as the epidermis is carried out only by TGF-ß1-dependent cells, suggesting that LC or their equivalent cells may be localized in the dermis.

An alternative possibility is not able to be ruled out that TGF-ß1 may be required for migration of macrophages (#5 in Fig. 8) or DC (#6 in Fig. 8) from the skin to the regional LN. However, we observed that the number of macrophages in the dermis of Mgf-Tg Tgfb1^–/– was comparable to that of normal littermates and macrophages captured MG in the dermis of Hgf-Tg Tgfb1^–/– mice (data not shown). It was reported that TGF-ß1 polarized DC precursors to generate LC-like cells in vitro and inhibited the expression of a chemokine receptor, CCR7 in DC, resulting in down-regulation of the DC migration in response to its ligand, SLC (28,61). Macrophage development has not been reported to be dependent on TGF-ß1. Therefore, significant reduction of MG-laden cells in Tgfb1^–/– LN might result from a deficiency of development of the cells for the antigen trafficking rather than that of DC or macrophage migration.

Since our Tg mice produce enormous amount of MG, it is unclear whether the capturing of excess MG does not induce LC or their equivalent cells to the active state. Phenotypic analysis of LN cells in Mgf-Tg mice did not indicate any circumstantial evidence of activation (Fig. 2), and the location and number of epidermal LC in the transversal skin sections of Mgf-Tg and Hgf-Tg mice were similar to those of normal littermates (Fig. 1B). However, in some experiments, we observed that an epidermal sheet from Mgf-Tg skin but not Hgf-Tg skin seemed to contain a slightly reduced number of MHC class II⁺ DC (Fig. 1C). Although Mgf-Tg epidermis contains a massive number of MG and the granules may abate the fluorescence used to detect LC, this matter must be carefully considered.

MG in the skin are continuously captured and trafficked only to regional LN in the steady state. This means that any molecules accessible to skin DC may also be continuously trafficked to regional LN and presented constitutively as self-antigens. In Tgfb1^–/– mice, which are subject to autoimmune disease (8,9), this process might be hampered. The present study provides a useful model mice for studying dynamic mechanisms of skin antigen capturing and trafficking not only in the active phase but also in the steady state, and will enable us to assess the relationship between constitutive antigen presentation and self-tolerance, and its failure resulting in autoimmune diseases.

	Acknowledgments

 
We thank Dr Shinsuke Taki (Chiba University) for his helpful discussions, Dr Mitsuo Oshimura (Tottori University) for sharing a cell sorter, and Dr Toshiyuki Hamaoka (Osaka University), Dr Kiyoshi Takatsu (Tokyo University), Dr Toru Nakano (Osaka University) and Dr. Ken-Ichi Yamamura (Kumamoto University) for their warm encouragement. We also acknowledge Dr Toshiyuki Shibahara and Dr Takashi Iwaki (Tottori University) for maintenance of mice, and Ms Toshie Shinohara for secretarial assistance. This study was supported by grants from the, Special Coodination Funds of the Ministry of Education, Culture, Sports, Science Technology, the Japanese Government, the Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd and the Osaka Foundation for Promotion of Clinical Immunology. T. Y. is a Research Fellow of the Japan Society for the Promotion of Science.

	Abbreviations

 

DC dendritic cell

GM-CSF granulocyte macrophage colony stimulating factor

HGF hepatocyte growth factor

K14 cytokeratin 14

LC Langerhans cell

LN lymph node

M-CSF macrophage colony stimulating factor

MG melanin granule

SLF steel factor/stem cell factor/mast cell growth factor

Tg transgenic

TGF transforming growth factor

TNF tumor necrosis factor

	Notes

 
¹⁰ Present address: Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan

Transmitting editor: K. Takatsu

Received 25 December 2000, accepted 13 February 2001.

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