Mast cell migration from the skin to the draining lymph nodes upon UV-irradiation represents a key step in the induction of immune suppression

Abstract

The UV radiation in sunlight is the primary cause of skin cancer. UV is also immunosuppressive and numerous studies have shown that UV-induced immune suppression is a major risk factor for skin cancer induction. Previous studies demonstrated that dermal mast cells play a critical role in the induction of immune suppression. Mast cell deficient mice are resistant to the immunosuppressive effects of UV radiation, and UV-induced immune suppression can be restored by injecting bone marrow derived mast cells into the skin of mast cell deficient mice. The exact process however, by which mast cells contribute to immune suppression is not known. Here we show that one of the first steps in the induction of immune suppression is mast cell migration from the skin to the draining lymph nodes. UV-exposure, in a dose dependent manner, causes a significant increase in lymph nodes mast cell numbers. When GFP+ skin was grafted onto mast cell deficient mice, we found that GFP+ mast cells preferentially migrated into the lymph nodes draining the skin. The mast cells migrated primarily to the B cell areas of the draining nodes. Mast cell express CXCR4+ and UV exposure up regulated the expression of its ligand CXCL12, by lymph node B cells. Treating UV-irradiated mice with a CXCR4 antagonist, blocked mast cell migration and abrogated UV-induced immune suppression. Our findings indicate that UV-induced mast cell migration to draining lymph nodes, mediated by CXCR4 interacting with CXCL12, represents a key early step in UV-induced immune suppression.

Introduction

Due to their abundant expression of Fcε receptors and their ability to secrete histamine following IgE binding, mast cells have been traditionally associated with allergic-type immune reactions. However, newer findings indicate that mast cells influence a wide variety of non-allergic immune responses (1) and participate in inducing immune tolerance (2). Immunosuppression and tolerance are necessary counter balances for hyperactive inflammatory-mediated immune responses, in that they inhibit the severity of allergy, and prevent the onset of autoimmune disease. In contrast, un-warranted or ill-timed immune suppression can have significant consequences on the ability of the immune system to combat infections and destroy tumors. The UV wavelengths in sunlight are a prime example of an environmentally acquired immunosuppressant, and suberythemal UV doses are known to cause significant systemic immune suppression and induce tolerance (3). While the DNA damaging properties of sunlight are well known, the mechanisms of how UV suppresses Th1-immune responses and induces tolerance are not as well understood.

Following UV exposure, a cytokine cascade that bias the immune response toward a Th2 reaction is initiated, which ultimately leads to the formation of CD4⁺CTLA-4⁺ regulatory T cells (4, 5). However, the cells and inflammatory mediators involved in the initial steps towards suppression and tolerance (i.e. those within the first hours following UV exposure) are still unknown. Hart et al. and later Alard et al. demonstrated that mast cells are required for both systemic (6) and local (7) UV-induced immune suppression. In these studies, mast cell deficient mice were resistant to the immunosuppressive effects of UV radiation, and suppression was restored in knock out mice reconstituted with wild type bone marrow derived mast cells (BMMC)³.In addition, mast cell density in human skin correlates with susceptibility to both melanoma (8) and non-melanoma skin cancers (9) suggesting that the immunomodulatory function of mast cells is likely to be important for the development of skin tumors. This is perhaps not surprising when one considers the wide range of inflammatory mediators and cytokines that mast cells have been shown to produce (10). Indeed, many of the inflammatory mediators released by mast cells including, histamine (10, 11), prostaglandin E₂ (12), serotonin (13), platelet activating factor (PAF) (14, 15) TNF, IL-4 and IL-10 (16) are critical mediators of UV-induced immunosuppression. Grimbaldeston and colleagues recently demonstrated that mast cell derived IL-10 limits the skin pathology associated with contact dermatitis and chronic inflammation induced by UV exposure, again reinforcing the growing appreciation for the ability of mast cells to regulate inflammation and the immune response (17).

One hallmark of UV-immunosuppression is the generation of suppressor lymphocyte populations. During the early phase (i.e. within hours of UV exposure) an IL-10 producing suppressor B cell is activated (18, 19), followed a few days to weeks later by CD4⁺ regulatory T cells (4, 20, 21). Mast cells are not only potent B cell activators (22, 23), they are capable of producing Th2-polarizing cytokines (24) that preferentially activate CD4⁺ Th2 cells (25). Most of these earlier studies used in vitro cultured BMMC and so it is still not clear exactly how a mast cell in the periphery influences lymphocyte activation, although their ability to reach draining lymph nodes (DLN) where lymphocyte activation occurs would seem to be a necessary pre-requisite. In this study we show that UV exposure triggers mast cell migration to the draining lymph node through CXCR4 expressed on mast cells and CXCL12 expressed on lymph node cells. Blocking mast cell migration into the DLN by a CXCR4 antagonist abrogates UV-induced immune suppression.

Materials and Methods

Mice

C57BL/6 wild type mice, mast cell deficient mice on a C57BL/6 background (Kit^W-sh/W-sh), and GFP+ mice (C57BL/6-Tg(UBC-GFP) 30Scha/J) were obtained from the Jackson Laboratories. The mice were housed in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International. The University of Texas M.D. Anderson Cancer Center Institutional Animal Care and Use Committee approved all the animal procedures described here.

UV exposure and Contact Hypersensitivity Reactions

On day 0, the mice were exposed to an immunosuppressive dose of UV radiation (80 kJ/m² of solar simulated radiation; 290 to 400 nm; containing approximately 8 kJ/m² of UVB; 290 to 320 nm) supplied by a 1000W xenon arc solar simulator (Oriel), as described previously (26). Four days later the mice were sensitized by applying 50μl of 0.3% DNFB (2,4-dinitro-1-fluorobenzene; Sigma Chemical Co. diluted in 4:1 acetone: olive oil) to the un-irradiated, shaved abdominal skin. Six days later, the ears of each mouse was measured with a micrometer and the animals were challenged by applying 5μl of 0.2% DNFB in the same diluent to the ventral and dorsal surface of each ear. Twenty-four h later, the change in ear thickness (post challenge – pre challenge ear thickness) was determined (18, 19).

In some experiments the effect of UV exposure on mast cell deficient mice reconstituted with wild-type bone marrow derived mast cells was examined. Bone marrow stem cells were isolated from the femurs and tibias of 6-week-old C57BL/6 mice and then cultured at a concentration of 10⁶ cells/ml in complete RPMI 1640 supplemented with murine recombinant IL-3 (10 ng/mL; Peprotech) and SCF (10 ng/mL; Peprotech). Non-adherent cells were transferred to fresh culture medium twice a week for 4 to 5 weeks at which point more than 98% of viable cells were mast cells as verified by flow cytometry (CD45⁺CD117⁺FcεR1α⁺CD3⁻B220⁻) and positive staining for toluidine blue. A total of 1 × 10⁶ BMMC were injected into multiple sites underlying the dorsal skin of mast cell-deficient mice (6). Six weeks later, the mice were exposed to UV radiation as described above.

To activate BMMC in vitro, 10⁶ cells were incubated for 6 hr with 5 μg/ml purified mouse IgE (Sigma) to cross-link Fcε receptors. BMMC were then washed and incubated in RPMI containing 0.5% BSA and 100 ng/ml DNP-KLH (Sigma) for 18h. 72h post activation, BMMC were labeled with antibodies against CD117, FcεR1α, CXCR4 (BD Pharmingen), and CCR7 (eBioscience).

Real time RT-PCR for CXCL12 expression

Twenty-four h after UV exposure the inguinal lymph nodes were removed, snap frozen in liquid nitrogen and pulverized with a mortal and pestle. Control groups were shaved but un-irradiated. Total RNA was extracted with Trizol (Invitrogen) and further purified by treating with RNeasy RNA cleanup protocol (Qiagen). The concentration of isolated RNA was measured and 0.5μg converted to cDNA using the Retroscript RT kit (Ambion). 25ng of cDNA was subjected to real-time RT-PCR using a sequence detector (Model ABI Prism 7500) and target mixes for CXCL12 and GAPDH (Taqman Gene Expression Assay, Applied Biosystems). Cycle threshold (C_T) values for CXCL12 were normalized to GAPDH using the following equation: (1.8 ^{(GAPDH-CXCL12)} × 1,000), where GAPDH is the C_T of each GAPDH control, CXCL12 is the C_T of CXCL12, and 1,000 is an arbitrary factor to bring all values above one. There were four mice in each group; RNA was isolated from each individual mouse.

Histological analysis of mast cells

Skin samples from control or UV irradiated mice were embedded in paraffin and 7μm serial sections cut. One section was labeled for CXCR4 using a rat anti-mouse monoclonal antibody (clone 2B11; BD Pharmingen) while the other section was stained for mast cells using toluidine blue. DLN from control or UV-irradiated mice were frozen in liquid nitrogen, 7μm sections were cut, fixed, and then stained with toluidine blue. Care was taken to ensure that sectioning occurred in the same area of each individual lymph node. Lymph node mast cell density was determined by counting the total numbers of mast cells per lymph node section and dividing this count by the area of the lymph node section calculated using NIH Image J software (http://rsb.info.nih.gov/nih-image/).

Flow cytometric analysis to identify lymph node mast cells

Mice were exposed to different doses of UV radiation (0–80 kJ/m²) and 24h later the inguinal lymph nodes removed. Single lymph node cell suspensions were enzymatically digested with collagenase (400U/ml) and DNAse (300U/ml) (Sigma) before labeling for mast cells by flow cytometry. The following antibodies were used: CD45, CD3, CD4, CD8, CD11c, CD49b, NK1.1, Gr-1, CD117, and FcεR1α.

Blocking the CXCR4-CXCL12 pathway using AMD3100

Mice were supplied with AMD3100 (Sigma) in their drinking water (60μg/ml) beginning 2 days prior to UV exposure. For analysis of mast cell densities, AMD3100 was provided 2 days prior to UV and maintained throughout the experiment. In experiments were CHS was measured, AMD3100 supplemented water was provided 2 days prior to UV radiation. Four days after UV irradiation, and one day prior to hapten sensitization (5 days post UV), the mice were put on normal drinking water.

Statistics

In the CHS experiments the mean change in ear thickness (left ear + right ear ÷ 2) was calculated for each animal in each group. There were at least 5 mice/group. The change in thickness ± the SEM was then calculated for the group. Statistical differences between the control and experimental groups were determined using an un-paired two-tailed Student’s t-test. (GraphPad Prism Software V4, GraphPad Inc). In experiments measuring changes in lymph node mast cell numbers, there were at least 3 mice/group. The number of mast cells per mm² for each individual animal was calculated. The mean ± the SEM was then calculated for the group. Similarly, when RT-PCR was used to determine fold-increases in chemokine mRNA levels, values were calculated from back skin samples isolated from 4 individual mice. The means and the SEM for each treatment group were calculated and statistical differences between the experimental groups were determined using an un-paired two-tailed Student’s t-test. Representative experiments are shown; each experiment was repeated at least 3 times.

Results

Mast cells accumulate in skin DLN following exposure to UV radiation

Exposing mice to 80 kJ/m² of solar simulated ultraviolet radiation (290 to 400 nm) significantly (p = 0.006) suppressed contact hypersensitivity (CHS) (Fig. 1a). This was accompanied by increase in the size and cellularity of skin DLN 24h after UV-irradiation of back skin (Fig. 1b). We verified that mast cells are critical mediators of UV-immunosuppression by exposing various groups of mice to UV radiation. In contrast to their wild type littermates, UV-irradiated mast cell deficient mice (Kit^W-sh/W-sh) were resistant to the immunosuppressive effects of UV (Fig. 1c). Injecting 10⁶ wild type bone marrow derived mast cells into the backs of Kit^W-sh/W-sh mice restored immune suppression (p = 0.0003 vs. No UV control), thus confirming that mast cells were required for UV-induced immunosuppression.

Figure 1. Mast cells accumulate in skin DLN following exposure to immunosuppressive doses of UV radiation.

(a) CHS immune response to DNFB with and without UV (negative irritant control (3.7 ± 0.8 mm⁻²) subtracted. * p = 0.006 vs. No UV control. (b) Whole lymph node cell counts from control un-irradiated and 24h post UV mice, * p = 0.0003 vs. No UV control. (c) CHS immune response to DNFB with and without UV in wild type (WT), mast cell knockout (KIT^W-Sh/W-Sh) and mast cell knockout mice engrafted with 10⁶ WT bone marrow derived mast cells. Negative irritant controls (WT = 3.4 ± 0.5 mm⁻²; KIT^W-Sh/W-Sh = 2.2 ± 0.4 mm⁻²) subtracted. * p = 0.0003 UV-irradiated Mast cell KO + BMMC vs. No UV; p = 0.0001 UV-irradiated WT vs. No UV WT. (d) Lymph node mast cell density * p = 0.0001 vs. No UV control (No UV, n = 21; UV, n = 16 pooled from 3 separate experiments). (e) Gating strategy for identifying mast cells. (f) UV dose-response curve. * p = 0.02 vs. No UV. (g) Skin mast cell density. * p= 0.004 vs. 0 time.

Next we examined the effect of UV radiation on lymph node mast cell density. Twenty-four h post UV exposure a substantial (75%) and significant (p = 0.0001 vs. No UV control) increase in mast cell density was observed in the DLN of irradiated mice (Fig. 1d). This mast cell increase was confirmed by flow cytometry. Mast cells were identified by double staining with anti-CD117 and FcεR1 (Fig. 1e). There was a doubling in mast cell numbers in the skin DLN 24h after exposure to 80 kJ/m² of UV radiation (p = 0.02 vs. No UV control) (Fig. 1f). We did not observe a significant increase in mast cell densities or numbers in non-DLN (data not shown).

Next we examined changes in dermal mast cell numbers following UV exposure. We observed a significant increase mast cell density 6h post UV (Fig. 1g, p = 0.004 vs. 0 time;), which returned to normal at the 24 h time point. This rise and fall in skin mast cell densities was not due to UV-induced changes to dermal thickness as there was no significant difference in skin area between un-irradiated (0.4 ± 0.03 mm²) and UV exposed groups (6h = 0.3 ± 0.02 mm²; 24h = 0.3 ± 0.01 mm²). Exposure to UV radiation, therefore, results in an initial increase in dermal mast cell density, with a return to baseline levels at 24h. At the same time we noted a concordant increase in the density and number of lymph node mast cells.

Mast cells infiltrating lymph nodes of UV irradiated mice are skin derived

A limitation of the experiment described above is the difficulty in distinguishing between skin-derived and blood-derived lymph node mast cells. To differentiate between these two populations, we grafted skin from GFP+ mice onto the backs of congenic mast cell deficient (Kit^W-sh/W-sh) mice. After allowing 5 weeks for the skin grafts to take, the mice were exposed to UV radiation and 24h later the draining (inguinal, brachial, axillary) as well as non-draining (popliteal) lymph nodes were excised and analyzed by flow cytometry. GFP⁺ cells were only found in lymph nodes draining the back skin (Fig. 2a-R1). The DLN from mast cell−/− mice grafted with GFP⁺ skin, but not exposed to UV, were infiltrated by a small number of GFP⁺ cells (0.63%), which was not much greater than background (0.45%, data not shown). We attribute this small increase of GFP+ cells in un-irradiated animals to the migration of dendritic cells from the graft to recipient DLN (27), which is supported by the fact that the majority of these cells (> 97%) were positive for CD11c, CD4, CD8 or CD19 (Fig. 2b). Gating on CD11c⁻CD3⁻CD4⁻CD8⁻CD19⁻ cells (Fig. 2b-R2) revealed a population of GFP⁺ cells found exclusively in the DLN of UV-irradiated mice (Fig. 2b, R2). This subset of GFP⁺ cells also had high forward and side scatter profiles and consistent with a mast cell phenotype were CD117⁺ FcεR1α⁺ (Fig. 2c). As the transplanted skin was the only source of GFP⁺ cells, we conclude that UV exposure triggers the migration of mast cells from the skin to DLN.

Figure 2. Infiltrating Mast cells are skin derived.

(a) Kit^W-Sh/W-Sh mice received skin grafts from GFP+ donors, exposed to UV, and the DLN were analyzed for the presence of GFP⁺ mast cells (R1). Percentage of total lymph node cells shown in the upper right hand corner. b) R1 = GFP⁺, CD3⁺,4⁺,8⁺,11c⁺,19⁺ cells. R2 = cells negative for these markers. Top number is percentage of GFP⁺ cells in the R2 gate; bottom number is the percentage of GFP+, CD117+, FcεR1α+, CD3⁻,4⁻,8⁻,11c⁻,19⁻ cells per lymph node. (c) R2 = GFP+, CD117+, FcεR1α+, CD3⁻,4⁻,8⁻,11c⁻,19⁻ cells; Numbers represent percent of R2

Mast cells express CXCR4 and UV radiation increases CXCL12 expression in DLN

The chemokine receptor CXCR4 is abundantly expressed on cultured mast cells and these cells migrate towards the CXCR4-specific ligand CXCL12 (28). Although many cells in skin express CXCR4⁺ (Fig. 3b and 3d), toluidine blue positive mast cells were CXCR4⁺ in serial skin sections (Fig. 3c-f). Similarly, in vitro activated BMMC express CXCR4⁺ (Fig. 3g).

Figure 3. Mast cells express CXCR4.

(a) Isotype and (b) CXCR4 staining of serial un-irradiated skin sections. (c and e) Toluidine Blue and (d and f) CXCR4 staining of serial skin sections. Arrows indicate toluidine blue⁺ CXCR4⁺ cells. (g) Activated CD117+ FcER1α⁺ CD45⁺BMMC were stained with CXCR4.

UV exposure significantly increased the expression of the CXCR4 specific ligand CXCL12 (SDF-1α) mRNA in DLN (Fig. 4a). On the other hand, UV exposure had no effect on the expression of CXCL12 in the skin (Fig. 4b). Thus, UV radiation establishes a CXCL12 chemokine gradient potentially directing CXCR4⁺ cells towards CXCL12⁺ DLN.

Figure 4. UV radiation increases CXCL12 production in draining lymph nodes.

(a) RNA from whole inguinal lymph nodes was assessed for CXCL12 expression by real time PCR (n = 4 individual mice; representative of 3 separate experiments showing the same results). (b) Expression of CXCL12 by real time PCR in normal and UV-irradiated skin (n = 4). (c) CD4⁺ and CD8⁺ T cells, and CD19⁺ B cells were isolated from DLN of UV-irradiated and non-irradiated mice, using a BD FACSAria to greater than 98% purity before RNA was isolated from each lymphocyte subset and analyzed for CXCL12. * p = 0.0001 comparing CXCL12 levels in CD19+ cells from UV-irradiated mice vs. CXCL12 levels in CD19+ cells from non-irradiated mice; † p = 0.0001 comparing B cell CXCL12 levels in unirradiated lymph nodes with CD4⁺ and CD8⁺ T cells from non-irradiated mice; n = 5; representative of 3 separate experiments showing the same results. (d) CD45⁻ cells in inguinal lymph nodes were purified and RNA was isolated before analysis of CXCL12 expression (UV, n = 5; No UV, n = 4, representative of 2 separate experiments showing the same results). mRNA amounts are shown as arbitrary units relative to the amount of GAPDH mRNA and normalized to the unirradiated controls.

In the experiment described above (Fig. 4a), whole lymph nodes were used to isolate mRNA for CXCL12 analysis. It was unclear which cells within the DLN up regulates CXCL12. To address this question we sorted CD19⁺, CD4⁺ and CD8⁺ lymphocytes by FACS (greater than 98% purity) and isolated mRNA from the purified cells. As can be seen in Fig 4c, CD19⁺ B cells were the major source of CXCL12 in lymph nodes, and UV exposure up regulates the expression of CXCL12 on B cells. Lymph node high endothelial venules also express CXCL12 (29), so non-hematopoietic derived populations (i.e. CD45⁻ cells) were also analyzed. As expected, these cells also expressed CXCL12 although no difference in expression between control and UV-irradiated groups was observed (Fig. 4d). These results demonstrate that the increase in lymph node CXCL12 expression observed after UV radiation was predominantly B cell derived.

Lymph node infiltrating mast cells preferentially home to B cell areas

To determine the significance of the UV-induced CXCL12 production by B cells, we analyzed the localization of the infiltrating mast cells. Using a two-step immunohistochemical staining procedure we were able to visualize both CD19⁺ B cells and toluidine blue stained mast cells (Fig. 5). Most of the resident mast cells in DLN from un-irradiated animals were found in the sub capsular sinus and medulla regions of the node (Fig. 5a). In contrast, mast cells in the DLN of UV-irradiated animals were often found in close association with CD19⁺ B cells (Fig. 5b and 5c). We quantified this association by counting the number of B cells that were in physical contact with mast cells (Fig. 5d). In resting lymph nodes isolated from un-irradiated mice, the majority of mast cells (> 50%) were not associated with B cells. UV-irradiation significantly enhanced mast cell-B cell interactions so that almost half of all mast cells in the DLN were in direct physical contact with B cells. Indeed, mast cells were sometimes found deep within the B cell follicles (Fig. 5b and 5c). Almost 1 in 4 mast cells (24.3 ± 4.7%) in the DLN of UV-exposed mice were in direct contact with 5 or more B cells, and a significant number (almost 5%) were in contact with more than 9 B cells (Fig. 5c & d). In comparison, only 1 in 50 mast cells in resting DLN (2.3 ± 0.7%) were in contact with 5 or more B cells and no lymph node mast cells were observed to be in contact with more than 9 B cells.

Figure 5. Infiltrating mast cells preferentially home to B cell areas.

24 h post UV, DLN were first stained with anti-CD19 (brown), followed by counterstaining with toluidine blue. (a) No UV, (b and c). (d) Mast cell – B cells interactions were quantified. * p = 0.001 vs. No UV control.

Blocking CXCR4/CXCL12 inhibits mast cell migration to skin DLN and abolishes UV-induce immune suppression

A UV-induced CXCL12 gradient towards the B cell areas of DLN, combined with mast cell CXCR4 expression suggested that this chemokine pathway maybe driving the UV-induced mast cells migration. To investigate this possibility, we treated mice with AMD3100, a CXCR4 antagonist (30). Normal mice treated with AMD3100 (Fig. 6a black bars, No UV) generate a CHS reaction that is not statistically different (> 0.05) from that found in mice maintained on normal drinking water (Fig. 6a, open bars, No UV). As expected, UV suppressed CHS in mice maintained on normal drinking water (p = 0.015 vs. No UV control). On the other hand, when mice maintained on AMD3100 supplemented drinking water were exposed to an immunosuppressive dose of UV radiation, no immune suppression was noted (p > 0.05 UV vs. No UV).

Figure 6. Blocking the CXCR4 pathway inhibits UV-induced immune suppression and mast migration.

(a) CHS; * p = 0.001, UV vs. UV + AMD 3100. † p = 0.01 No UV vs. UV (b) Mast cell migration; * p = 0.015 vs. UV only control. Closed bars = AMD3100-treated mice; open bars = mice maintained on normal drinking water.

The effect of AMD3100 on UV-induced mast cell migration is found in Figure 6b. AMD3100 was supplied in the drinking water for the entire experiment. Twenty-four hours following UV exposure (80 kJ/m²), the mice were killed and their lymph nodes were removed and stained with toluidine blue. As before, UV exposure causes a significant increase (p = 0.02) in DLN mast cell density (open bars, UV vs. No UV). However, when the AMD3100-treated mice were exposed to UV radiation, no increase in lymph node mast cell density was observed (p > 0.05). These findings indicate that AMD3100, a drug know to interfere with the binding of CXCL12 to its receptor, CXCR4, blocks UV-induced immune suppression and interferes with the ability of mast cells to migrate into the DLN, supporting our hypothesis that mast cell migration to the DLN is a critical step in the pathway leading to immune suppression.

UV exposure also depletes epidermal Langerhans cells (LC) numbers in the skin, which is thought to be responsible for the ability of UV to inhibit local immune responses (31). Because LC express CXCR4 (32) it was possible that AMD3100 might prevent UV-induced immune suppression (Fig. 6b) by interfering with LC migration. To rule out this possibility we prepared epidermal sheets from the backs of AMD3100-treated, UV-irradiated mice. Counting the density of IA-b⁺ LC revealed that AMD3100 had no effect on the ability of UV to alter LC morphology or deplete LC from the skin (Fig. 7). This indicates that LC migration is not involved in AMD3100-induced abrogation of UV-induced immune suppression.

Figure 7. Blocking the CXCR4 pathway does not inhibit UV-induced Langerhans cell migration.

Epidermal sheets were stained with anti-mouse IA-b-biotin followed by Strepavidin FITC. AMD3100 was supplied to one of the UV irradiated groups for 2 days prior to and for the 24h post UV exposure. 8 random fields for each mouse. * p = 0.0012 vs. No UV control; + p = 0.026 vs. No UV control.

Discussion

The UV radiation present in sunlight damages DNA, induces inflammation, and suppresses the immune response, including the rejection of highly antigenic sunlight-induced skin cancers (21, 33). Exposure to sub-erythemal doses of UV radiation is all that is required to damage DNA and induce immune suppression, and humans are frequently exposed to these doses on a regular basis (34). This makes sunlight one of the most significant and potent human environmental carcinogens and human immunosuppressant. The precise mechanism by which UV suppresses anti-tumor immunity is still unknown even though understanding this process is crucial to our ability to design new treatment regimes aimed at reducing the incidence of skin cancer. A number of different cell types are known to be involved including, dendritic cells (31), immunoregulatory T cells (4, 21), suppressor B cells (18, 19), NKT cells (20), macrophages (35) and mast cells (6). What remains to be shown is how inflammatory events in the skin (i.e. UV exposure) affect the induction of regulatory cells in distant lymphoid tissues leading to antigen specific immune suppression and tolerance.

Exposure to UV radiation induces systemic immune suppression. This is illustrated by the fact that UV-exposure at one site will suppress the immune response to hapten or antigens introduced at a distant non-irradiated site (16). Because the skin effectively absorbs UV radiation, and none of the UV wavelengths penetrate to the DLN, it is still not entirely clear how the suppressive signal is transmitted from the skin to the immune system. Here we present data supporting a novel mechanism by which dermal UV exposure induces immune suppression, mast cell migration from the skin to the DLN. Early following UV exposure we noted a modulation of mast cell density in the skin, and 24h post UV exposure, we observed a doubling of lymph node mast cell density. When skin from GFP+ mice was grafted onto mast cell deficient animals, and the donor grafts were exposed to UV radiation, we observed the appearance of GFP+ mast cells in the lymph nodes of the recipient mast cell deficient mice, confirming the hypothesis that UV-irradiation is triggering the migration of mast cells from the skin to the DLN. The significance of UV-induced mast cell migration was highlighted by the fact that mast cell migration was required for the UV-induced immune suppression. When we used the CXCR4 inhibitor AMD3100 (36), also known as Mozobil in phase III clinical trials, to block CXCR4 binding to CXCL12, we blocked UV-induced mast cell migration, and prevented UV-induced immune suppression.

The use of AMD3100 as an inhibitor of mast cell migration and immunosuppression is novel and to our knowledge has never been reported before. Unfortunately, verifying these results by UV irradiating CXCR4^−/− mice is not possible due to embryonic lethality. Similarly, reconstituting mast cell knockout mice with CXCR4^−/− embryonic liver derived mast cells is not possible because as we found, this results in the re-population of both the skin and the DLN (data not shown). Finally, while the use of neutralizing anti-CXCR4 antibodies might confirm these results, there are questions surrounding antigen specificity, the potential for agonistic effects and problems associated with CXCR4 heterogeneity that could result in the antibody inhibiting one cell population over another (37), thus confusing the interpretation of such an experiment.

The creation of a chemokine gradient is necessary for directing cellular traffic. In vitro studies have established that mast cells express CXCR4 and migrate towards CXCL12 (28), although demonstrating the existence and importance of a CXCL12 gradient in vivo has not been shown. Mast cells in the skin were found to express CXCR4 and UV radiation increased the expression of the CXCR4 specific chemokine CXCL12 in the DLN. It is not clear how UV exposure sets up a CXCL12 chemokine gradient in the DLN, although we suggest that the cytokines and biological response modifiers released by keratinocytes after UV irradiation may play a role. For example, Silva et al. showed that oxidized lipids, including PAF, induced the expression of a wide range of chemokines (38). Because of the critical role PAF plays in UV-induced immune suppression (14, 15) it is tempting to speculate that PAF is driving UV-induced CXCL12 expression. Another possible CXCL12 trigger might be the multitude of cytokines released following UV exposure including IL-4, IL-10 and TNF (39). TNF for example has been shown to increase CXCL12 production in osteoblasts (40).

It has recently become clear that mast cells not only mediate allergic type immune responses but also have the capacity to influence adaptive immune responses (1) and even induce tolerance (2). The physical separation of these cell populations (lymphocytes being activated in lymphoid tissues and mast cells residing predominantly in the periphery) may be the reason that the immunomodulating function of mast cells has received little attention. However, mast cells can migrate to sites other than the periphery. Antigen sensitization induces dermal mast cell migration to DLN (41). Using experimental allergic encephalomyelitis as a model of multiple sclerosis, Tanzola et al. observed that mast cells only migrated into the lymph nodes after the induction of the disease state (42). More recently, using a model of glomerulonephritis it was shown that mast cell accumulation in the lymph nodes (but not the kidneys) was an essential feature of the ability of mast cells to inhibit disease progression (43). We extend these observations by indicating that a ubiquitous environmental carcinogen, UV radiation, activates mast cell migration to lymph nodes. Perhaps then, it would be more accurate to conclude that mast cells migrate towards sites of inflammation and that the draining lymph node can be considered one such site. In our model, UV radiation not only induces inflammation locally in the skin, but also in the DLN. At early time points after exposure (i.e., 6h), mast cells migrate into the skin, but at 24h post UV, CXCL12 is increased in DLN redirecting mast cells from the skin to “inflamed” hypertrophic nodes. Further supporting this hypothesis was the fact that mast cells did not migrate into un-inflamed, non-DLN, or to un-exposed skin (data not shown).

In summary, our findings indicate that UV-induced mast cell migration from the skin into the DLN represents a critical step in the induction of immune suppression. Blocking mast cells migration, by interfering with CXCR4/CXCL12 interactions blocks both mast cell migration and the induction of immune suppression. We note increased migration of dermal mast cells into B cell regions of the lymph node, suggesting this may be the mechanism by which tolerance-inducing, IL-10-secreting immunoregulatory B cells are activated (18, 19). These findings support the growing appreciation for the ability of mast cells, to regulate adaptive immune reactions. We suggest that mast cell migration represent a critical mechanism for transmitting immunoregulatory signals from the periphery to the immune system after exposure to dermal immune modulating environmental toxins.