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International Immunology Advance Access originally published online on September 11, 2006
International Immunology 2006 18(11):1541-1548; doi:10.1093/intimm/dxl087
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Constant rate of steady-state self-antigen trafficking from skin to regional lymph nodes

Miya Yoshino1, Hidetoshi Yamazaki1,2, Leonard D. Shultz3 and Shin-Ichi Hayashi1

1 Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, 683-8503, Japan
2 Division of Genomics and Regenerative Biology, Department of Physiology and Regenerative Medicine, Institute of Medical Science, Mie University Graduate School of Medicine, 2-174, Edobashi, Tsu, 514-8507, Japan
3 The Jackson Laboratory, Bar Harbor, ME 04609, USA

Correspondence to: M. Yoshino; E-mail: myoshi{at}grape.med.tottori-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
It is suggested that dendritic cells (DCs) capture and present both foreign antigens such as components of pathogens as well as endogenous self-antigens. However, the magnitude of self-antigen trafficking to secondary lymphoid organs is still unclear. Here we show constitutive trafficking of self-antigens from skin to regional lymph nodes (LNs) quantitatively using a KRT14-Kitl transgenic mouse. This mouse model expresses the Kit ligand in keratinocytes, shows hyperpigmentation of the epidermis and exhibits constitutive accumulation of melanin granules (MGs) in skin regional LNs transported by Langerhans cells. Using an MG-solubilization technique, we revealed that 128 µg per week of MGs, a marker of self-antigens, accumulated in skin regional LNs and that the rate of accumulation was constant from 3 to 50 weeks. Activation markers such as CD40, CD54 and CD86 did not increase in the LNs, and abrogation of CD40 signaling did not affect the accumulation. Additionally, the total amount of MGs did not increase significantly following stimulation with intravenous LPS injections. These results suggest that the accumulation is not caused by inflammatory stimuli, and the steady-state trafficking of self-antigens is intrinsically maintained at a constant rate. Because the levels of self-antigens as well as the phenotype of these DCs are thought to be important in the strength of immune responses, the results may imply that the constant rate of trafficking of self-antigens is required for maintaining homeostatic conditions, such as self-tolerance.

Keywords: dendritic cell, quantitative analysis, self-antigen, skin, trafficking


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
Dendritic cells (DCs) are known to play a major role in the regulation of immune responses (1–3). DCs capture foreign antigens, become mature, migrate to secondary lymphoid organs such as lymph nodes (LNs) and present the antigens to naive T cells. Mature DCs up-regulate co-stimulatory molecules such as CD40, CD80 and CD86, produce pro-inflammatory cytokines and activate immune responses (1, 4, 5). Recently, it has been proposed that DCs can capture self-antigens as well as foreign antigens and transport them to LNs (6–8). Constitutive transport of natural self-antigens, which are derived mainly from apoptotic self-cells (9, 10), by DCs has been demonstrated in various organs (9–12). Such DCs show an immature phenotype with low levels of co-stimulatory molecules and migrate to regional LNs without inflammatory stimuli, that is, in the steady state (9). Phenotypic differences among DC populations correlate with the direction of immune responses (2, 10, 13). On the other hand, several reports have pointed out a relationship between the amount of antigen and the magnitude of immune responses, especially, the maintenance of peripheral tolerance in the steady state (7, 14–17). Phenotypic characterization of the DCs has been investigated in detail; however, the amount of antigens transported by the DCs is still obscure.

We previously established a transgenic (Tg) stock of hyperpigmented mice. In these mice, Kit ligand (Kitl) gene is driven by a human keratin 14 promoter (KRT14), and the epidermis is pigmented by melanin granules (MGs) generated by aberrantly proliferating melanocytes, which deliver MGs to keratinocytes (18). In these Tg mice, a large number of MG-laden cells accumulate in regional LNs. Since the MGs are not dissolved unlike other peptide antigens, they function as a tracer of self-antigens in the epidermis. We employed this Tg mouse system to visualize antigen trafficking of skin resident DCs to regional LNs. Consequently, we found constitutive steady-state MG trafficking and observed that the MGs were transported by transforming growth factor (TGF)-ß1-dependent DCs such as Langerhans cells (LCs) (12, 19).

Because gradual increases of MG-laden cells in regional LNs in the steady state were observed in our Tg system (12), we sought to estimate self-antigen trafficking by measuring the amount of MGs in regional LNs in the steady state. Using an MG-solubilization method and spectrophotometry, we determined both the total amount and the temporal increase of MG trafficking. Furthermore, we compared MG trafficking in the steady state and in active states. The Tg mouse system facilitates clarification of the relationship between trafficked self-antigens and levels of immune response.

Here we show that self-antigens were found to continuously traffic from the skin at a constant rate throughout life in the steady state. Recently, presentation of self-antigens by immature DCs in the steady state was considered to be important in the induction of self-tolerance (8, 10, 13, 20, 21). The level of self-antigens may also be related to the maintenance of tolerance (6, 7, 15, 22). We will discuss the relationship between self-antigen trafficking and the maintenance of tolerance.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice
C57BL/6Jjcl (B6Jcl) mice were obtained from Clea (Shizuoka, Japan). B6.129S2-Cd40lgtm1Imx/J homozygous female and hemizygous males (herein called CD40L null) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Tg(KRT14-Kitl)1Takk is a transgene expressing mouse Kitl driven by the KRT14 (herein called Kitl-Tg). These Tg mice show heightened proliferation of melanocytes within the basal layer of the epidermis (18). We used F1 progeny of the following crosses: B6Jcl x Kitl-Tg hemizygous male, CD40L-null homozygous female x Kitl-Tg hemizygous male and Kitl-Tg hemizygous female x CD40L-null hemizygous male for measurement of the melanin content at various ages. All animals were maintained under specific pathogen-free condition at The Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University. Experiments were approved and performed in accordance with the guidelines of the Animal Care and Use Committee of Tottori University.

Spectrophotometric quantitative measurements of melanin
The amounts of melanin in lymph node (LN–melanin) were measured by spectrophotometry according to Ozeki et al. (23) with minor alterations. A skin regional LN (axillary, brachial and inguinal LNs) or all skin regional LNs from a half of the body (sub-mandibular, axillary, brachial, inguinal and poplitial LNs) were removed and placed into 1-ml test tubes, respectively. The volumes were adjusted to 100 µl by adding distilled water. These samples were frozen and thawed three times with alternating liquid nitrogen and hot water. The refrozen samples were put into heat-resistant screw-capped glass test tubes (10-ml ST-SPITZ tube, NEG, Kobe, Japan). Tissue solubilizer Soluene®-350 (900 µl) (Perkin-Elmer, Boston, MA, USA) was added to each sample tube. The tubes were mixed by a Vortex mixer, capped firmly and incubated at 100°C for 30 min in a hot water bath. An additional 15-min incubation was done after cooling and mixing the boiled samples. The samples were then transferred to plastic test tubes, and then centrifuged at 10 000 r.p.m. for 10 min. The concentration of solubilized melanin was analyzed at 500 nm using a quartz cell and spectrophotometer (Hitachi U-1000, Tokyo, Japan). The amounts of melanin were calculated by a regression curve. Standard melanin samples (0.05, 0.1, 0.25 and 0.5 mg ml–1) were prepared using synthesized melanin (M-8631, Sigma, St Louis, MO, USA) with distilled water, processed in the same way as LN samples and a regression curve was prepared with every experiment. We subtracted the calculated amount of melanin in wild-type (WT) littermate samples from that of Kitl-Tg samples, and obtained the amount of LN–melanin. Samples were taken every week from 3 to 10 weeks of age, and at 15, 20, 30 and 50 weeks of age. Microsoft Excel 2004TM (Microsoft, Redmond, WA, USA) was used to analyze data, calculate regression and approximate curves. Data were expressed as the mean ± SD. Statistical significance was assessed by Student's t-test.

Flow cytometry of DCs in regional LN
To obtain DCs in regional LN (LNDCs), LN cells were dissociated as described (24) with minor alterations. In brief, skin regional LNs from at least three Kitl-Tg and littermate control mice were minced with scissors, suspended in PBS/5% fetal bovine serum (FBS) and then dissociated with 1 mg ml–1 Collagenase D (Roche, Penzberg, Germany) and 0.02 mg ml–1 DNAse I (Amersham Bioscience, Piscataway, NJ, USA) at 37°C for 45 min. Cells were collected in 15-ml tubes through nylon mesh, washed and re-suspended in 10 ml of RPMI1640 (GIBCO, Auckland, New Zealand)/10% FBS/2-mercaptoethanol/penicillin and streptomycin. Granulocyte macrophage colony-stimulating factor-induced bone marrow-derived DCs (BMDCs) (25) were used as positive and negative controls for surface expression of activation markers. Half of the BMDCs were stimulated with 10 µg ml–1 LPS (Escherichia coli 055:B5W, Sigma) for 24 h and used as a positive control. The unstimulated cells were used as a negative control. CD11c-positive DCs were positively sorted using MACS beads [CD11c (N418), Miltenyi Biotec, Bergisch Gladbach, Germany] according to manufacturer's instructions. The purity of sorted cells was ~85% for LNDCs and >99% for BMDCs. Anti-mouse antibodies used for staining were as follows: biotinylated antibodies against CD40 (3/23, rat IgG2a, BD Bioscience, Franklin Lakes, NJ, USA), CD54 intercellular adhesion molecule-1 (ICAM-1): YN1/1.7.4, eBioscience, San Diego, CA, USA), CD86 (GL1, rat IgG2a, BD Bioscience), CD45R (B220) (RA3-6B2, rat IgG2a, BD Bioscience), CD90.2 (Thy1.2) (30-H12, rat IgG2b) and FITC-conjugated anti-CD11c (HL3, BD Bioscience). The cells were then incubated in ExtrAvidin–PE (Sigma). Anti-CD90.2 antibody was also used for control IgG staining in Fig. 3. Flow cytometry was done by using EPICSTMXL (Beckman Coulter, Palo Alto, CA, USA). We also collected CD11b-positive macrophages in LNs by using MACS beads (anti-PE beads, Miltenyi Biotec) and anti-CD11b–PE antibody (M1/70, eBioscience) and analyzed their surface markers as was done for LNDCs.


Figure 3
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  		Fig. 3 Levels of surface markers expressed on LNDCs in Kitl-Tg mice were not elevated when compared with that in WT littermate. (A) Surface expression of activating markers [CD40, CD54 (ICAM-1) and CD86] on CD11c positively sorted LNDCs of Kitl-Tg (black line) and WT littermate (filled gray peak). (B) Comparisons between LNDCs and BMDCs. BMDCs were stimulated with 10 µg ml–1 of LPS for 24 h, and used as positive controls for surface expression of activating markers. Non-stimulated immature BMDCs were also used as negative controls. Upper panels: levels on Kitl-Tg LNDCs (black line) and immature BMDCs (filled gray peak). Lower panels: levels on Kitl-Tg LNDCs (black line) and activated BMDCs (filled gray peak). Gray line: control IgG staining. The experiment was repeated twice, and representative data are shown.

 
Immunohistochemistry and histology
For immunohistochemistry, cryostat sections were cut at 7-µm thickness and stained with rat anti-mouse monoclonal antibodies against CD68 [FA11 (26), Serotec, Oxford, UK], CD205 (NLDC145, a kind gift of K. Inaba, Kyoto University) and MOMA-2 (Serotec). Rabbit anti-rat IgG (H + L) (BA-4001, Vector, Burlingame, CA, USA) was used as a secondary antibody, and ExtrAvidin–FITC or tetramethylrhodamine isothiocyanate (TRITC) (Sigma) was used for tertiary staining. Organs (spleen, liver, lung, kidney and thymus) of 30- and 50-week old Kitl-Tg mice and serially LPS-injected 7- and 11-week old Kitl-Tg mice were fixed in 4% PFA and embedded in polyester wax (BDH, Poole, UK), cut at 7-µm thickness. Hematoxylin-eosin-stained specimens were prepared by standard protocols. Some specimens were observed without any staining for detection of MGs. All specimens were observed by microscope (BX-60, Olympus, Tokyo, Japan) and images were taken using a digital camera (Polaroid PDMC II i, Nihon Polaroid, Tokyo, Japan).

LPS injection
Twenty micrograms of LPS (E. coli 055:B5W, Sigma) in 0.2 ml of PBS was intravenously injected three times every 3 days to 6- and 10-week old Kitl-Tg and littermate control mice. At 1 day after the last injection (i.e. 1 week after the first injection), regional LNs were taken at 7 and 11 weeks of age, and the amounts of melanin were measured as described above. Some groups of mice were treated with one injection of 20 or 40 µg of LPS. Four days after injection (at 7 or 11 weeks of age), the mice were prepared for analysis. Mice injected with 0.2 ml of PBS were simultaneously prepared as controls.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
Levels of MGs increased linearly in skin regional LNs of Kitl-Tg mice in the steady state
Macroscopic observation showed that the LNs of Kitl-Tg mice gradually become pigmented from 3 to 30 weeks of age (Fig. 1). In the LNs, there were CD205-positive DCs which develop from LCs (27) containing few MGs, and CD68-positive and MOMA-2 [a marker of tissue resident macrophages (28)]-positive macrophages carried large amounts of MGs (Supplementary Fig. 1, available at International Immunology Online). We also found no specific accumulation of MG-laden cells in any organs except for regional LNs even in 30- and 50-week old Kitl-Tg mice similar to 7- to 9-week old Kitl-Tg mice described previously (12, 19) (data not shown). These results indicate that even in the older mice, MG-bearing DCs migrated only to regional LNs and accumulated MGs were only present in regional LNs.


Figure 1
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  		Fig. 1 Skin regional LNs of Kitl-Tg mice become pigmented gradually with age even in the steady state. Skin regional LNs (inguinal LNs of Kitl-Tg mice) are shown. Other skin regional LNs showed similar pigmentation. WT: wild type littermate. Bars = 1 mm.

 
To estimate the accumulation of MGs quantitatively, we measured the total amount of melanin in individual LNs (LN–melanin) of Kitl-Tg mice using spectrophotometry following MG solubilization (23). We took every skin regional LN from a half of the body (see Methods; hereafter designated as ‘half of LNs’). Each skin regional LN (axillary, brachial and inguinal LNs) was also taken from 3- to 50-week old Kitl-Tg mice. The amount of LN–melanin increased linearly (Fig. 2A and B). The data showed increase in levels of LN–melanin almost at the same rate from 3 to 50 weeks; the increasing rate was 64 µg per week in half of LNs (y = 0.064x – 0.228, R2 = 0.998) (Fig. 2A), and each LN also showed an increase in the amount of LN–melanin almost at a steady rate (axillary LN: y = 0.010x – 0.050, R2 = 0.990; brachial LN: y = 0.021x – 0.092, R2 = 0.995; inguinal LN: y = 0.026x – 0.116, R2 = 0.992) (Fig. 2B). We also estimated the ratio of the amount of melanin in each LN compared with the total amount of melanin in half of LNs. Brachial and inguinal LNs showed ~30% of total accumulation, and axillary LNs showed ~12% of total accumulation. These rates were maintained almost at the same level throughout the observation period (Fig. 2C). These results suggest that 128 µg of melanin was continuously trafficking to regional LNs per week, and that the rate of trafficking was constant both periodically and regionally in the steady state.


Figure 2
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  		Fig. 2 The amount of LN–melanin increased linearly with age in regional LNs in the steady state. (A–C) The amount of melanin in half of LNs (all skin regional LNs from a half of the body (A)]. Each LN [axillary, brachial, inguinal LN, respectively (B)] and levels of melanin in each LN against that of half of LNs (C). Samples were taken every week from 3 to 10 weeks, and at 15, 20, 30 and 50 weeks. In each experiment, at least two Kitl-Tg mice and two WT littermates were studied. The experiment was repeated at least three times per age, except the data of 50-week-old Kitl-Tg mice in Fig. 2(A) (n = 4, two experiments). All data obtained for each age group are integrated. Total numbers of samples are 97 (WT) and 128 (Kitl-Tg), respectively. y: the function of regression curve. R2: R square.

 
Activation markers on DCs are not elevated in regional LNs of Kitl-Tg mice
To assess whether the heightened pigmentation in the skin of the Kitl-Tg mice resulted in activation of the immune system, we determined expression of surface markers on CD11c-positive LNDCs. CD11c-positive LNDCs were enriched by magnetic cell sorting, and surface expression of activation markers were observed. The levels of CD40, CD54 (ICAM-1) and CD86 on LNDCs in Kitl-Tg and WT control mice were similar (Fig. 3A). CD11b-positive macrophages in the LNs were also observed and we found no specific up-regulations of these markers in Kitl-Tg (data not shown). The results suggested a lack of spontaneous inflammation in the Kitl-Tg mice. Furthermore, we compared the expression of these molecules on Kitl-Tg LNDCs with those on immature and LPS-activated BMDCs. Although the expression levels of CD40 and CD54 on Kitl-Tg LNDCs were slightly higher, the level of CD86 was lower than immature BMDCs (Fig. 3B, upper panels). Furthermore, the expression levels of all these markers on the Kitl-Tg LNDCs were lower than that on activated BMDCs (Fig. 3B, lower panels). These results indicated that the accumulation of MGs did not likely result from inflammatory responses.

Accumulation of melanin in regional LNs from CD40L-null Kitl-Tg mice
To measure the amount of LN–melanin trafficking strictly in the steady state, we employed Kitl-Tg mice lacking CD40L. Cd40lgtm1Imx/J [abbreviated as female CD40L(–/–) and male CD40L(–/Y)] mice have been reported to be defective in T cell-dependent antigen responses, germinal center formation, and migration of LCs in response to skin irritation (29, 30). We reasoned that if the accumulation of MGs observed in Kitl-Tg mice resulted from inflammatory responses, Kitl-Tg mice lacking CD40L should show lower levels of LN–melanin accumulation. Since the Cd40lg gene is X-linked, we prepared CD40L(–/Y)-+/Kitl-Tg male and CD40L(–/+)-+/Kitl-Tg female mice as a control from female CD40L(–/–) mice crossed with male +/Kitl-Tg mice. CD40L(+/Y)-Kitl-Tg/+ male and CD40L(+/–)-Kitl-Tg/+ female mice from the opposite crossing pair were also prepared as additional controls to compare influences of the Cd40lg gene more strictly (see Methods). Hereafter, +/Kitl-Tg and Kitl-Tg/+ are abbreviated as Kitl-Tg.

We measured the amount of LN–melanin in these F1 mice at 5 and 10 weeks of age. The accumulation of LN–melanin in CD40L(–/Y)-Kitl-Tg male mice was not abolished (Fig. 4A). At 5 weeks, the amount of LN–melanin in CD40L(–/Y)-Kitl-Tg male mice was lower than that in their female littermates [CD40L(–/+)-Kitl-Tg, P = 0.015, Fig. 4A, left panel]. However, the amount of LN–melanin in CD40L(–/Y)-Kitl-Tg mice was higher than that in CD40L(+/Y)-Kitl-Tg mice (P = 0.044, Fig. 4A, left panel). In all strains of mice examined at 10 weeks of age, similar amounts of LN–melanin accumulated (Fig. 4A, right panel). Although the reason for the difference between 5-week-old CD40L(–/Y)-Kitl-Tg male and CD40L(–/+)-Kitl-Tg female littermates is not obvious, it is noteworthy that no significant decrease was observed in CD40L(–/Y)-Kitl-Tg compared with CD40L(+/Y)-Kitl-Tg male mice. Furthermore, the increased rate of LN–melanin in each mouse from 5 to 10 weeks of age was comparable, although levels of LN-melanin in CD40L(+/Y)-Kitl-Tg mice were slightly higher than others (Fig. 4B). The amount of LN–melanin in CD40L(–/Y)-Kitl-Tg mice was higher than that of Kitl-Tg mice which was observed in Fig. 2(A) (Fig 4A); this difference might be caused by sub-strain differences between C57BL/6J and C57BL/6J-Jcl. The result that the trafficking of MG-laden DCs to regional LNs was not impaired even if CD40 signaling is abrogated suggests that the observed accumulation of MGs does not result from an inflammatory response.


Figure 4
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  		Fig. 4 The amount of LN–melanin is not markedly decreased in CD40L-null-Kitl-Tg mice. All mice are Kitl-Tg positive, and the CD40L of each mouse is indicated in this figure. The amounts of LN–melanin were measured. (A) The amount of LN–melanin in F1 progeny of CD40L-null mice and Kitl-Tg mice at 5 and 10 weeks of age. Open bars: CD40L(+/–) female mice and CD40L(+/Y) male mice from female Kitl-Tg x male CD40L-null crossing pair. Filled bars: CD40L(–/+) female mice and CD40L(–/Y) male mice from female CD40L-null x male Kitl-Tg crossing pair. Gray bars: single Kitl-Tg mice (indicated as ‘WT’). *P = 0.015, {dagger}P = 0.044. Numbers in each column indicate the numbers of mice. (B) Increased amounts of LN–melanin from 5 to 10 weeks in the mice displayed in the right panels. Symbols indicate single Kitl-Tg mice (gray triangles, indicated as ‘WT’), CD40L(+/–)-Kitl-Tg mice (open circles) and its littermate CD40L(+/Y)-Kitl-Tg mice (open squares), CD40L(–/+)-Kitl-Tg mice (filled circles) and its littermate CD40L(–/Y)-Kitl-Tg mice (filled squares), respectively.

 
Amount of LN–melanin in Kitl-Tg mice is not markedly increased following LPS stimulation
Next, to assess active-state trafficking, we determined the accumulation of LN–melanin in Kitl-Tg mice following stimulation with LPS. Because administration of LPS induces LC migration from the epidermis to regional LNs (31), we injected LPS into Kitl-Tg mice and the amount of LN–melanin was measured. LPS (20 µg) was administered to 6- and 10-week old Kitl-Tg mice three times, every 3 days, and 1 day after the last injection, LN–melanin was quantitated. In LPS-treated mice, we observed enlargement of spleens, higher expression of MHC class II antigen on LCs and partial disappearance of LCs from ear epidermal sheets by serial LPS injections (data not shown). Therefore, the LPS injections resulted in expected immune responses. However, we could not detect significant differences between the amount of LN–melanin in PBS-injected Kitl-Tg mice and LPS-injected Kitl-Tg mice at 7 weeks (Fig. 5). In LPS-injected 11-week-old Kitl-Tg mice, the amounts of LN–melanin were decreased significantly rather than increased (Fig. 5, P = 0.019). Single LPS injection into Kitl-Tg mice also did not result in an increase in the amount of LN–melanin (data not shown). These results indicate that active-state accumulation of self-antigens may be comparable to or smaller than the steady-state accumulation. Moreover, ectopic accumulation of MGs was not observed in any organs other than skin regional LNs in the LPS-injected Kitl-Tg mice (data not shown), suggesting that MG-laden DCs in the epidermis maintained trafficking to only regional LNs in the active state.


Figure 5
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  		Fig. 5 Active-state accumulation of LN–melanin did not significantly increase above steady-state levels. Kitl-Tg mice (6- and 10-weeks old) were injected with 20 µg LPS three times per 3 days, and 1 day after the last injection, LN–melanin was measured at 7- and 11-weeks old, respectively. PBS-injected littermates were prepared as steady-state controls. The mean amount of LN–melanin of left- and right-side LNs was used as the amount of LN–melanin per mouse. Numbers in each column indicate the numbers of mice. The experiments were repeated three times, and all data were integrated. *P = 0.019.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
In this study, we observed that MGs, as an endogenous tracer for self-antigens, were trafficking to regional LNs continuously in the steady state, and that the amount of steady-state trafficking of skin antigens was maintained at a nearly constant rate throughout life.

Several reports showed steady-state migration of DCs carrying self-antigens to regional LNs (8–11). However, these reports were not concerned with the amount of transported self-antigens. First, we have attempted to estimate the total numbers of MG-containing LNDCs and macrophages as self-antigen-bearing cells by flow cytometry, and found that it was difficult to determine the total numbers in regional LNs, because preparation of DCs and macrophages without loss was not possible. Then, we employed the system shown here. MGs are not catabolized in DCs and macrophages, and accumulate in these cells unlike peptide antigens (Supplementary Fig. 1, available at International Immunology Online). The MG-solubilization system enables us to estimate the total amount of antigen accumulation in LNs without any loss.

Since melanin trafficking appeared constant from the skin to regional LNs in the steady state (Fig. 2A), we carefully confirmed that these Kitl-Tg mice were in the steady state. Surface activation markers for DCs were not up-regulated on CD11c-positive LNDCs in Kitl-Tg mice compared with that in WT littermates (Fig. 3). Moreover, the results of studies with CD40L-deficient Kitl-Tg mice also confirmed the absence of spontaneous inflammatory responses on DC migration in Kitl-Tg (Fig. 4). These results indicated that the constant rate of trafficking did not result from DC activation. It should be noted that the periodic increase of LN–melanin was constant throughout the observation period (Fig. 2A, y = 0.064x – 0.228). Age-related changes may affect various immune responses. LC density and the migration capacity decrease with age (32). Five-week-old mice are smaller than 50-week-old mice. The differences of body mass and hormonal regulation with aging might affect this steady-rate trafficking. Nevertheless, similar amounts of self-antigens are constantly transported to LNs. These results indicate the presence of strict homeostatic regulation of the amount of self-antigen trafficking to regional LNs.

Surprisingly, accumulation of melanin in the active state was not increased compared with that in the steady state, and such accumulation decreased in 11-week-old mice (Fig. 5). First, we considered the possible influence of a mechanism similar to LPS tolerance, unresponsiveness following LPS stimulations after an initial LPS stimulation (33–35). However, it may be unlikely that LPS tolerance plays a major role in our model, because the accumulation of LN–melanin following multiple LPS injections we used in this study was comparable to levels following a single LPS injection (data not shown). We speculate the following possibilities. First, LCs migrating in the active state might be different from that in the steady state. We previously supposed that the migration of LCs was dependent on CCR7–CCL21 serine signaling in the active state while independent in the steady state (19). It is possible that there are distinct subsets of LCs, or LCs themselves change their ability to capture self-antigens and migrate to LNs by stimulation. Alternatively, because of the difference of turnover of LCs between the steady state and the active state, the amount of MGs captured by LCs might be different. Turnover of LCs is considered to be relatively slow in the steady state [about 18 days (36) to nearly over a month (37–39)] compared with active-state trafficking of LCs (31, 38–42). It may provide sufficient time for steady-state LCs to capture self-antigens, because self-antigens should be abundant. In each situation described above, LCs in the active state would capture less self-antigen than in the steady state. Smaller amounts of MGs in active-state LCs might reflect no increase of the amount of LN–melanin. It is also possible that the active-state migration might inhibit the steady-state migration, though it is still unclear whether or not steady- and active-state migrations occur simultaneously. Significant increase of the number of MG-laden DCs following LPS injection (19) seems inconsistent with this hypothesis; however, it might be caused by far larger numbers of active-state migration of LCs. Actually, LPS stimulation causes extensive emigration of LCs from the epidermis (19, 31). More investigation will be required to clarify this point.

It is accepted that the state of maturity and activation of DCs may regulate immune response, that is, activation or tolerance (1–3). Especially, immature DCs, which express low levels of co-stimulatory molecules such as CD40, CD80 and CD86, transport self-antigens in the steady state to induce peripheral immune tolerance (2, 10, 13). On the other hand, several reports underscored the importance of the amount of self-antigens in the induction of peripheral tolerance (7, 14–17). Findings that low levels of antigen expression in peripheral organs were not sufficient to maintain tolerance (7, 8, 43) imply the requirement that higher amounts of self-antigens are needed to maintain tolerance. Although it is not clear how much self-antigen is actually required, self-antigens traffic constantly. We accept that trafficking of MGs may not accurately reflect trafficking of other self-antigens. Moreover, our system might not precisely reflect the amount of self-antigens especially in the active state. Although further study is required to determine the precise relationship between MGs and self-antigens, our system should have an advantage over other Tgs expressing peptide antigens such as green fluorescence protein or ovalbumin expressed in the skin (44, 45) for determining the total accumulation and the temporal change of trafficking of self-antigens (Fig. 2A).

We previously reported abnormal accumulation of MG-laden DCs in some strains of mice using our Tg system. In Tgfb1(–/–)-Kitl-Tg mice which lack LCs (46), MG trafficking to regional LNs was completely abrogated, and alymphoplastic Nikaly/Nikaly-Kitl-Tg (aly/aly-Kitl-Tg) mice which lack peripheral LNs and Peyer's patches (47) showed ectopic accumulation of MGs in liver, spleen, kidney and lung in the steady state (12). By contrast, plt/plt-Kitl-Tg mice which have decreased numbers of T cells and DCs in secondary lymphoid organs (48) showed similar level of accumulation of MG-laden DCs in the skin regional LNs compared with that in their WT littermate in the steady state, whereas active-state trafficking of the DCs were impaired (19). The accumulation of MGs indicates the trafficking of self-antigens in the steady state in our Tg system. Considering the importance of self-antigens for induction of peripheral tolerance as discussed above, the abnormal accumulation of MGs might be correlated with the breakdown of peripheral tolerance to some extent. Tgfb1(–/–) mice develop fulminant autoimmune disease (49, 50), and aly/aly mice and plt/plt mice exhibit exocrinopathy such as Sjögren's syndrome (51, 52). The main cause of autoimmunity in Tgfb1(–/–) mice is thought to be aberrant proliferation of leukocytes (50, 51), and plt/plt mice fall ill even though steady-state accumulation of self-antigens seems not to be impaired. However, we could think that such diseases were caused partly by the abnormal trafficking of self-antigens. Actually, Tgfb1(–/–) mice in which trafficking of self-antigens is completely abrogated showed severe symptoms than the other mouse strains. Additionally, impairment of central tolerance is thought to be crucial for onset of the disease in plt/plt mice (52).

Recent studies suggest that regulatory T (Treg) cells play a critical role in the maintenance of immune tolerance, and that DCs support the induction and proliferation of Treg cells (53–56). Recently, it has also been reported that self-antigen presentation in the regional LNs is required for maintenance of Treg cells to suppress autoimmune responses in vivo (57). Constitutive high levels of trafficking of self-antigens, as shown in our study, may also contribute to maintain Treg cell function. Here, we described a novel system to estimate the amount of steady-state self-antigen trafficking from skin. Future studies will clarify the dose of self-antigens required to maintain immune tolerance.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary Fig. 1 is available at International Immunology Online.


   Acknowledgements
 
We are grateful to S. Niida (National Institute of Longevity Science, Japan) and T. Kurosaki (RIKEN, Japan) for their warm encouragement, T. Kunisada (Gifu University Graduate School, Gifu, Japan) for generating hyperpigmented Kitl-Tg mice, K. Inaba (Kyoto University, Kyoto, Japan) for providing anti-CD205 antibody and K. Wakamatsu (Fujita Health University, Japan) for technical instruction in melanin solubilization. We also thank to M. Tsuneto (Tottori University) for helpful discussions and T. Shibahara (Tottori University) for maintenance of mice, T. Shinohara for technical assistance and C. Gagliardi (Jackson Laboratory) for preparing the manuscript. This work was supported by a Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology and a grant from the Research on Demential and Fracture, Health and Labour Sciences Research Grants, the Japanese Government (H.Y. and S.-I.H.), and National Institutes of Health grant CA34196 and grant JDRF-548 from the Juvenile Diabetes Research Foundation (L.D.S), and support from the Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.


   Abbreviations
 
BMDC, bone marrow-derived dendritic cell
DC, dendritic cell
FBS, fetal bovine serum
ICAM-1, intercellular adhesion molecule-1
KRT14, human keratin 14 promoter
Kitl, Kit ligand
LC, Langerhans cell
LN, lymph node
LNDC, DC in regional LN
LN–melanin, melanin in LN
MG, melanin granule
Treg, regulatory T
Tg, transgenic
TGF, transforming growth factor
WT, wild type

   Notes
 
Transmitting editor: T. Hamaoka

Received 12 May 2006, accepted 8 August 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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L. Wang, L. S. Bursch, A. Kissenpfennig, B. Malissen, S. C. Jameson, and K. A. Hogquist
Langerin Expressing Cells Promote Skin Immune Responses under Defined Conditions
J. Immunol., April 1, 2008; 180(7): 4722 - 4727.
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