Department of Medicine (Dermatology), Melanoma and Skin Cancer Research Institute, University of Sydney at Royal Prince Alfred Hospital, NSW 2006, Sydney, Australia
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Abstract |
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Abbreviations: CHS, contact hypersensitivity; DETC, dendritic epidermal T cell; DMEM, Dulbecco's modification of Eagle's medium; EC, epidermal cell; LC, Langerhans cell; mAb, monoclonal antibody; MHC, major histocompatibility complex; TCR, T-cell receptor.
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Introduction |
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Alternatively, UV radiation may promote skin cancer by increasing the production of growth-promoting factors which can support tumor growth (14). Studies have shown that the growth of UV-induced skin tumors in mice is stimulated by the production of paracrine growth factors from infiltrating granulocytes (1517). Others have shown the presence of cytokine-producing resident and inflammatory leukocytes in the skin 13 days after exposure to UV radiation (1821). Although the profile of cytokines produced by these cells have not been fully elucidated, UV radiation is known to up-regulate a large number of growth factors and cytokines from a variety of cutaneous cell populations, for example keratinocytes (22).
Here, we present evidence that enhancement of growth of a squamous carcinoma cell line is associated with the presence of inflammatory cells in the epidermis of UV-irradiated hosts but not with immunosuppression. Furthermore, we demonstrate that unresponsiveness to hapten can be induced when oxazolone is painted on to UV-irradiated skin which contains a variety of changes within the cellular milieu of the epidermis.
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Materials and methods |
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Antibodies
Anti-F4/80 (rat IgG2b, HB-198), anti-DEC-205 (rat IgG2a, NLDC-145), anti-CD16/32 (rat IgG2b, 2.4G2), anti-CD11c (hamster IgG, N418) and anti-CD3 (hamster IgG, 145-2C11) monoclonal antibodies (mAb) (all American Type Culture Collection, Rockville, MD, USA) were used as hybridoma culture supernatants. Anti- T-cell receptor (TCR) (hamster IgG, GL3), anti-V
3 TCR (hamster IgG, F536), anti-Gr-1 (rat IgG2b, RB68C5), biotinylated anti-I-Ak (murine IgG2b, 11-5.2) and phycoerythrin (PE)-conjugated anti-CD45 (rat IgG2b, 30-F11) mAbs were purchased from PharMingen (San Diego, CA, USA). Anti-CD11b (rat IgG2b, M1/70) mAb was purchased from Boehringer-Mannheim (Mannheim, Germany). Rat IgG2b, hamster IgG and biotinylated murine IgG2b were all obtained from PharMingen, and PE-conjugated rat IgG2b was obtained from Serotec (Oxford, UK).
UV-irradiation
UV radiation was provided by a single UVB-emitting tube (FS72 T12-UVB-HO; Philips, Amsterdam, The Netherlands). The spectrum emitted by the UVB-emitting tube was determined by Dr Frank Wilkinson (CSIRO Division of Applied Physics, National Measurement Laboratory, West Lindfield, NSW, Australia). There was no detectable emission below 280 nm (UVC), and the UVB:UVA ratio was 17:5. Irradiance was monitored using an IL1350 radiometer/photometer fitted with a SED240 UVB sensor and SED038 UVA sensor (International Light, Newburyport, MA, USA). The average irradiance of the UVB-emitting tube was 0.471 mW/cm2 UVB (280320 nm) and 0.125 mW/cm2 UVA (320400 nm).
The dorsal trunks of mice were shaved with an electric shaver. After 24 h mice were placed unrestricted in lidless plastic boxes and exposed to a single inflammatory dose of UV radiation, consisting of 410 mJ/cm2 UVB and 100 mJ/cm2 UVA, at a distance of 35 cm from the UVB-emitting tube. Unirradiated control mice were shaved at the same time as the UV-irradiated mice. For CHS experiments, all the ear surfaces of mice were protected from UV with zinc oxide cream (Zinc White (32% (w/w) zinc oxide); FH Faulding & Co., Salisbury, SA, Australia) applied 10 min before UV-irradiation. Control mice were treated in an identical fashion but were not irradiated. The minimum edemal dose of UV radiation for C3H/HeN mice was determined to be 60 mJ/cm2 UVB (results not shown).
Tumor inoculation into mice
UV-irradiated and unirradiated control mice received 2x106 viable LK2 tumor cells in 50 µL phosphate-buffered saline (Trace Biosciences) intradermally into each flank within the area of treatment. UV-irradiated mice received tumor cells 2, 3 or 4 days after irradiation. Unirradiated control mice received tumor cells at the same time as UV-irradiated mice. Tumor growth was monitored by measuring two perpendicular diameters of each tumor using Vernier callipers (Mitutoyo, Tokyo, Japan). A mouse was defined as tumor bearing if it had at least one tumor with an average diameter of 1 mm. Tumor growth is expressed as the mean tumor diameter per total number of inoculated mice per group.
Contact hypersensitivity
CHS to a minimum sensitizing dose of oxazolone (4-ethoxymethylene-2-phenyloxazol-5-one; Sigma Chemical Co., St Louis, MO, USA) was used to assess immunosuppression and tolerance. UV-irradiated and unirradiated control mice were sensitized on the treated dorsal skin with 50 µg oxazolone in 50 µl acetone. UV-irradiated mice were sensitized 2, 3 or 4 days after irradiation. Seven days after sensitization, mice received 25 µg of oxazolone in 5 µl acetone on to each surface (dorsal and ventral) of one ear. After 24 h, the thickness of the challenged and unchallenged ears was measured using a spring-loaded engineer's micrometer (Mercer, St Albans, UK). Naive mice (irritant control mice), which had not been previously sensitized to oxazolone, were used to determine the level of non-specific ear swelling. The CHS response for each mouse was calculated as the difference in ear thickness between challenged and unchallenged ears.
To determine the induction of tolerance, mice were rested for 3 weeks after the initial sensitization with oxazolone and anaesthetized with 2,2,2-tribromoethanol (Aldrich Chemical Co., Milwaukee, WI, USA); the ventral surface of their trunk was shaved. The anaesthetized mice were resensitized with 50 µg oxazolone in 50 µl acetone on the shaved ventral surface. The CHS response was elicited 7 days later and determined as above except that mice were challenged on the ear that previously did not receive hapten. Ear swelling was measured in a blinded fashion and experiments were performed twice.
Cytofluorimetric analysis of EC suspensions
EC were prepared from killed mice 2, 3 or 4 days after UV-irradiation, or from killed unirradiated control mice using a modification of a method previously described (24). Excised skin was cut into 1 cm2 pieces and placed into Hank's balanced salt solution (without Ca2+ or Mg2+; Trace Biosciences) containing 0.3% trypsin (Boehringer-Mannheim) for 1618 h at 4°C. The epidermis was removed from the dermis using forceps, and the resulting epidermis incubated in Hank's balanced salt solution containing 0.03% trypsin and 300 U/ml deoxyribonuclease I (Amersham International, Amersham, UK) for 20 min at 37°C/5% CO2. An equal volume of DMEM containing 10% fetal calf serum was then added and the mixture was agitated by hand for 5 min at room temperature. The suspension was filtered through 160 µm nylon gauze (Swiss Screens, Australian Filter Specialists, Huntingwood, NSW, Australia) and washed twice with DMEM.
For one-color cytofluorimetric analysis, blocked EC (20% normal goat serum) were incubated in specific primary mAb or isotype control mAb, followed by staining with PE-conjugated goat anti-rat IgG or fluorescein isothiocyanate (FITC)-conjugated goat anti-hamster IgG (both from Caltag Laboratories, San Francisco, CA, USA). For two-color cytofluorimetric analysis, blocked EC (2.4G2 supernatant) were incubated with biotinylated mAb, followed by streptavidinFITC (Caltag Laboratories), and finally with PE-conjugated mAb. All antibodies and conjugates not acquired as hybridoma culture supernatants were diluted in DMEM containing 10% fetal calf serum. EC were analyzed by collecting data from 5x104 gated events using a FACScaliber flow cytometer and CellQuest® software (both from Becton Dickinson, Sunnyvale, CA, USA).
Statistics
Differences in tumor growth between groups over all time points throughout the experiment were assessed by multivariate analysis of variance. Differences in mean ear swelling for CHS experiments, and differences in cell percentages for cytofluorimetric studies, were compared using the two-tailed Student's unpaired t-test. The differences were considered statistically different when P was <0.05.
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Results |
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Specific staining with anti-CD11b and anti-Gr-1 mAb was used to further examine cellular changes within the epidermis of irradiated hosts. Shaved unirradiated epidermis contained 1.8% CD11b+ cells and 0.9% Gr-1+ cells (Figure 3c). There was a significant increase in CD11b+ and Gr-1+ cells within the epidermis 2 and 3 days after irradiation. Irradiated epidermis 2 and 3 days after UV-irradiation contained 18.1% and 10.9% CD11b+ cells, respectively. Similar increases were observed with the Gr-1+ population on days 2 (17.1%) and 3 (13.2%). However, by 4 days after UV exposure, the percentages of CD11b+ and Gr-1+ cells were declining (5.6% and 4.2%, respectively), although remaining significantly higher than control EC.
The major histocompatibility complex (MHC) is a group of cell surface molecules, consisting of MHC class II and MHC class I, which present antigenic peptides to CD4+ and CD8+ T cells, respectively. MHC class II expression is generally confined to antigen-presenting cells such as LC and macrophages and is useful in their identification, while MHC class I is expressed on most cell types. Therefore, two-color cytofluorimetric analysis of EC, using anti-CD45 and anti-MHC class II (I-Ak) mAb, was used to characterize further the cellular changes within the UV-irradiated epidermis. Shaved unirradiated epidermis contained 2.0% CD45+ MHC class II+ cells (LC) and 7.6% CD45+ MHC class II cells (DETC) (Figure 3d). The percentage of CD45+ MHC class II+ EC and CD45+ MHC class II EC was significantly higher in each of the irradiated groups (Figure 3d
).
Shaved unirradiated epidermis also contained 0.23 ± 0.05% (mean ± SEM; n = 6 mice) CD45 MHC class II+ cells (presumably MHC class II+ keratinocytes). UV-irradiation caused a small but significant increase in MHC class II expression on CD45 EC 3 and 4 days after irradiation (0.62 ± 0.13% (P < 0.02) and 0.71 ± 0.16% (P < 0.02), respectively) but not on day 2 (0.45 ± 0.10%, P > 0.05).
The increase in CD45+ MHC class II+ EC after UV-irradiation was presumably due to the infiltration of macrophages and/or other antigen-presenting cells. Others have previously identified different cell populations infiltrating the epidermis after UV-irradiation based on differences in MHC class II expression (9). Analysis of the flow cytometric profiles indicated populations of CD45+ cells in control and UV-irradiated epidermis differing in levels of expression of MHC class II. Therefore, CD45+ MHC class II+ EC were subdivided into three different populations based on their level of MHC class II expressionlow (MHC class IIlo), medium (MHC class IImed) or high (MHC class IIhi)as illustrated in Figure 4. The marker regions were based on CD45+ MHC class II+ EC, presumably LC, within unirradiated control skin falling predominantly within the MHC class IImed population. The number of MHC class IIlo cells was significantly increased 24 days after irradiation (Figure 5a
). In contrast, MHC class IImed cells were maintained 2 and 3 days after irradiation at control levels with no significant changes, with a small significant increase on day 4 (Figure 5b
). Two days after irradiation there was a small population of CD45+ MHC class IIhi EC present, which began to decline on day 3 (Figure 5c
).
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Discussion |
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UV-induced suppression of CHS responses and UV-induced suppression of anti-tumor immune responses in mice may share certain steps in their pathways (6), and suppression of CHS is often used as a surrogate for suppression of anti-tumor immunity. However, mice exposed to 410 mJ/cm2 UVB radiation were unresponsive to hapten applied to the irradiated site 2, 3 or 4 days after irradiation. Therefore, this suppression did not correlate with suppression of anti-tumor immunity as it did not resolve by day 4. In support of the findings presented here, it has been shown that the enhanced local growth of melanoma in chronic, low-dose UV-irradiated mice is unrelated to local suppression of the CHS response (25). Furthermore, it has been suggested recently that the mechanisms by which UV radiation induces immunosuppression differ depending on the antigen (26,27).
The enhanced tumor growth and suppression of CHS may be partly due to changes in EC populations in response to UV-irradiation. The enhanced tumor growth was associated with an infiltration of CD11b+ and Gr-1+ cells and, to a lesser extent, CD45+ MHC class IIhi cells. In contrast, neither LC nor DETC densities correlated with enhanced tumor growth. The quantification of LC, however, was confounded by the lack of a more specific marker; attempts using dendritic cell markers, DEC-205 or CD11c, were unsuccessful (results not shown) possibly due to trypsin sensitivities of these epitopes (28). Therefore, anti-F4/80 mAb, which specifically recognizes LC in normal mouse skin (29), was used to quantify LC. Surprisingly, LC were not significantly reduced until 4 days after irradiation, in contrast with findings from previous studies in mice (9,12). These differences are difficult to reconcile but may reflect differences in the dose or spectrum of UV radiation. Studies in humans have found similar levels of LC 23 days after UV-irradiation compared with control skin (30,31). Alternatively, since F4/80 may also be expressed on macrophages (29), it may be that LC are reduced as soon as 2 days after UV-irradiation and replaced by a population of F4/80+ macrophages which then leave the epidermis after day 3. This latter point is consistent with the influx of inflammatory cells.
The reduction in DETC is consistent with the depletion of intraepithelial T cells in human skin 24 days after a single exposure to four times the minimal erythemal dose of UV radiation (10). The similar percentages of TCR+ and CD3+ cells at each time point suggest that few, if any, CD3+ T cells infiltrated the epidermis within the first 4 days after irradiation. Quantification of DETC using the lineage-specific marker, V
3 TCR, was unsuccessful (results not shown) again presumably due to trypsin sensitivity (32). Interestingly, although the V
3 TCR epitope appeared to be trypsin sensitive, the
TCR epitope could still be recognized. Collectively, the data suggest that DETC are more sensitive than LC to the effects of UV-irradiation. This is consistent with other observations within our laboratory. Exposure of C3H/HeJ mice to low-dose UV radiation, for 5 days/week for 4 weeks, reduced the density of LC and DETC by 78% and 97%, respectively, in irradiated epidermis compared with unirradiated control epidermis (33).
Both CD11b+ and Gr-1+ inflammatory cells infiltrated the epidermis 2 and 3 days after irradiation, and declined to nearly normal levels by day 4. This was supported by similar increases in the CD45+ cells. Others have observed an infiltration of inflammatory cells including CD45+ MHC class II CD11b+ Gr-1+ granulocytes, CD45+ MHC class II CD11b+Gr-1 macrophages and CD45+ MHC class II+ CD11b+ Gr-1+/ macrophages in murine epidermis after exposure to 1.1 times the minimal erythemal dose of UV radiation (9,12,34). Therefore, it is probable that the inflammatory cells detected here are similar to those observed by Cooper et al.9 Two-color cytofluorimetric analysis revealed that CD45+ MHC class IIhi cells were present after UV-irradiation, a phenotype shared by CD11b+ macrophages known to induce hapten-specific tolerance (9,12).
The differences in EC populations in UV-irradiated skin may be associated with the enhanced growth of tumors and suppressed CHS at different time points after UV-irradiation. The increased LK2 growth 2 and 3 days after irradiation has the same time-course as the large infiltration of CD11b+ and Gr-1+ cells. This suggests that cells within the inflammatory infiltrate may be responsible for the enhanced tumor growth. These cells may contribute to tumor growth either by providing paracrine growth factors, suppressing local effector immune responses, or by activating suppressor and/or regulatory T cells. Previously, paracrine stimulation by Gr-1+ cells was associated with the progression of UV-induced tumors (1517). A paracrine role has also been described for macrophages (35). The small increase in CD45+ MHC class IIhi cells present in irradiated epidermis may represent CD11b+ macrophages which are known to produce interleukin-10 (18) and can induce tolerance possibly via a novel form of T-cell activation that is characterized by deficient interleukin-2 receptor- expression (12,36). As far as we are aware, this is the first time that CD11b+ macrophages have been observed with increased tumor growth in UV-irradiated skin. Furthermore, since others have observed a correlation in inflammatory cells between the dermis and epidermis in UV-irradiated skin (10,20,34), it is tempting to suggest that the cells present in the epidermis after UV-irradiation may also reflect changes within the dermis, and therefore contribute to the growth of LK2 tumors transplanted into UV-irradiated dermis.
Alternatively, F4/80+ LC present 2 and 3 days after irradiation may have contributed to the enhanced tumor growth. LC present in the skin after UV-irradiation are known to have a reduced capacity to stimulate T cells (37,38), presumably due to altered expression of co-stimulatory molecules (39). LC exposed to UV radiation fail to induce protective anti-tumor immunity (40). Interestingly, others have shown the growth of UV-induced skin tumors in UV-irradiated skin in the presence of LC but in the absence of DETC (41,42). This latter observation suggests that the absence of DETC may have also contributed to tumor growth; however, since DETC were also absent on day 4, other cells and/or factors must also be required.
CHS was suppressed when hapten was applied to irradiated skin 2, 3 or 4 days after UV radiation. LC, DETC and CD11b+ macrophages from UV-irradiated skin have been shown to induce unresponsiveness and/or tolerance to haptens (1113), so it is possible that each of these cell types may have contributed to the local immunosuppression and/or the induction of tolerance observed after UV-irradiation. Of note, despite marked differences within the immune cells present in irradiated skin, unresponsiveness to hapten was observed at each time point. The only cellular change consistently associated with suppression of the CHS response was the reduction in DETC.
In summary, enhanced tumor growth was observed when tumor cells were inoculated intradermally into irradiated skin 2 or 3 but not 4 days after UV radiation, whereas local immunosuppression (unresponsiveness) to hapten was induced at days 2, 3 and 4. The increase in tumor growth was associated with the infiltration of CD45+ cells, CD11b+ cells, Gr-1+ cells and, to a lesser extent, CD45+ MHC class IIhi cells. In contrast, unresponsiveness to hapten was associated with a reduction in DETC.
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Notes |
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Acknowledgments |
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References |
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