Enhanced tumor growth in UV-irradiated skin is associated with an influx of inflammatory cells into the epidermis

Ronald Sluyter and Gary M. Halliday1

Department of Medicine (Dermatology), Melanoma and Skin Cancer Research Institute, University of Sydney at Royal Prince Alfred Hospital, NSW 2006, Sydney, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
UV radiation causes a number of cellular changes within the skin which play a role in tumor outgrowth, including immunosuppression and production of growth-enhancing cytokines. Both of these enable tumors to grow but their relative importance in carcinogenesis is poorly defined. In this study, C3H/HeN mice were exposed to a single inflammatory dose of 410 mJ/cm2 UVB radiation (plus 100 mJ/cm2 UVA radiation) followed by the inoculation of a regressor squamous cell carcinoma into or the painting of oxazolone onto the treated skin. Tumors transplanted 2 or 3 but not 4 days after irradiation had a significantly higher growth rate than tumors inoculated into unirradiated control mice. In contrast, mice failed to respond to hapten when it was applied 2, 3 or 4 days after irradiation. Cytofluorimetric analysis demonstrated that the number of F4/80+ Langerhans cells was not significantly reduced until 4 days after irradiation, while the number of dendritic epidermal T cells was significantly lower at all time points observed after UV-irradiation. Furthermore, a large cellular infiltration of CD11b+, Gr-1+, CD45+ MHC class II+ and CD45+ MHC class II cells into the epidermis was observed 2 and 3 days after irradiation, which corresponded with the enhanced tumor growth. To a lesser extent tumor growth was also associated with CD45+ MHC class IIhi cells, possibly the previously described UV-induced macrophage. In contrast, suppression of contact hypersensitivity corresponded with the reduction in dendritic epidermal T cells but not with other cell changes. The results suggest that, in this model, where immunosuppression did not appear to be responsible for enhanced tumor growth, inflammatory infiltrates may contribute to the promotion of skin tumor growth within UV-irradiated skin.

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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exposure to UV radiation remains the largest risk factor associated with the development of skin cancer (1). In particular, the UVB wavelengths (280–320 nm) of UV radiation initiate and promote tumors within the epidermis (2). One mechanism by which UV radiation promotes skin tumors is by inducing immunosuppression (35), which shares certain steps with the suppression of contact hypersensitivity (CHS) to haptens (6). Exposure to UV radiation causes a number of changes within the epidermis which have been linked to immunosuppression. UV radiation can reduce the density of epidermal Langerhans cells (LC) and dendritic epidermal T cells (DETC) (79). It can cause the infiltration of macrophages, granulocytes and T cells into the epidermis (9,10). Furthermore, LC, DETC and macrophages from UV-irradiated epidermis can induce hapten-specific immunosuppression and tolerance (1113) suggesting that UV radiation causes a functional alteration in these cells. However, it is unknown how these changes in epidermal cell (EC) populations relate to the suppression of anti-tumor immunity.

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 1–3 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice and squamous carcinoma cell line
Inbred female C3H/HeN mice were 8–12 weeks of age at the beginning of the experiments. Mice were used with approval from the University of Sydney and Central Sydney Area Health Service Animal Ethics Committees. The regressor UV-induced squamous carcinoma cell line, LK2, was originally derived from a skin tumor which arose in a chronically UV-irradiated C3H/HeN mouse (23). The cell line was maintained in Dulbecco's modification of Eagle's medium (DMEM) containing 20 mM HEPES (Trace Biosciences, Castle Hill, NSW, Australia), and supplemented with 2.5% fetal calf serum and 7.5% newborn bovine serum (both from CSL Laboratories, Parkville, Victoria, Australia).

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-{gamma}{delta} T-cell receptor (TCR) (hamster IgG, GL3), anti-V{gamma}3 TCR (hamster IgG, F536), anti-Gr-1 (rat IgG2b, RB6–8C5), 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 (280–320 nm) and 0.125 mW/cm2 UVA (320–400 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 16–18 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 streptavidin–FITC (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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enhancement of tumor growth in mice exposed to UV radiation
The regressor squamous carcinoma cell line, LK2, when inoculated intradermally into mice, grew significantly more quickly in mice that had been UV-irradiated 2 or 3 days previously than in unirradiated control mice (Figure 1Go). However, no significant increase in tumor growth was observed when LK2 cells were inoculated into the dermis 4 days after irradiation.



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Fig. 1. UV radiation enhances growth of tumors inoculated 2 or 3 but not 4 days after irradiation. The dorsal trunks of mice were shaved and 24 h later exposed to 410 mJ/cm2 UVB. Two (•), 3 ({circ}) or 4 ({blacksquare}) days after UV-irradiation mice were inoculated intradermally with 2x106 LK2 tumor cells in each treated flank. Control mice ({triangleup}) were shaved 3 days before tumor inoculation but not irradiated. Tumor growth was monitored over 5.5 weeks. *P < 0.05 compared with control group (multivariate analysis of variance). Results are pooled from two separate experiments using 15–17 mice per group.

 
Induction of local suppression and tolerance to contact sensitization by UV-irradiation
The CHS response in mice exposed to 410 mJ/cm2 UVB radiation was significantly smaller than the CHS response in control mice when oxazolone was painted on to the UV-exposed skin 2, 3 or 4 days after irradiation (Figure 2Go). In contrast, mice that were originally sensitized through irradiated skin 3 or 4 days, but not 2 days, after irradiation developed tolerance to oxazolone.



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Fig. 2. UV radiation induces local suppression and tolerance of CHS. Mice were exposed to a single dose of 410 mJ/cm2 UVB 2, 3 or 4 days before sensitization by application of oxazolone on to the irradiated surface. Unirradiated control mice were sensitized at the same time (day 0). Seven days after sensitization (i.e. 9, 10 or 11 days after UV), mice were challenged with oxazolone on the right ear and the CHS response (filled bars) was determined 24 h later (mean ear swelling ± SEM). Three weeks after initial sensitization, oxazolone was painted on to the shaved abdominal skin of previously treated mice. Seven days later, mice were challenged with oxazolone on the left ear and the CHS response was determined 24 h later (mean ear swelling ± SEM) to assess the induction of tolerance (open bars). ***P < 0.01 compared with unirradiated control mice (unpaired Student's t-test). There were six to eight mice in each group. Results are representative of two replicate experiments.

 
Changes in EC populations after UV-irradiation
F4/80 expression was used to determine the percentage of LC within control and irradiated epidermis by cytofluorimetric analysis. F4/80+ LC comprised 3.1% of the shaved unirradiated epidermis (Figure 3aGo). Similar levels of F4/80+ LC (3.2%) were observed 2 days after irradiation. In contrast, F4/80+ LC began to decline by 3 days after exposure to UV radiation (2.7%), and were significantly reduced by day 4 (1.3%).



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Fig. 3. UV radiation causes changes in EC populations 2–4 days after irradiation. Cytofluorimetric analysis was performed as described in Materials and methods. The mean percentage of positive cells ± SEM at each time point is shown for EC expressing (a) F4/80 (•); (b) {gamma}{delta} TCR (•) and CD3 ({circ}); (c) CD11b (•) and Gr-1 ({circ}); (d) CD45+ MHC class II+ (•) and CD45+ MHC class II ({circ}). *P < 0.05; **P < 0.02; ***P < 0.01 compared with unirradiated control mice (unpaired Student's t-test). Results are pooled from two separate experiments using a total of six mice per group. Where the SEM is not obvious, it is too small to be seen.

 
To determine the percentage of DETC in control and irradiated epidermis by cytofluorimetric analysis, two markers were used, {gamma}{delta} TCR and CD3. {gamma}{delta} TCR+ DETC comprised 4.5% of the cells within control epidermis (Figure 3bGo). The level of {gamma}{delta} TCR+ DETC significantly decreased 2 days after UV-irradiation (2.0%) and remained significantly depressed on days 3 (1.2%) and 4 (0.8%). Staining with anti-CD3 mAb revealed similar percentages of positive cells as the anti-{gamma}{delta} TCR mAb at each time point (Figure 3bGo).

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 3cGo). 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 3dGo). The percentage of CD45+ MHC class II+ EC and CD45+ MHC class II EC was significantly higher in each of the irradiated groups (Figure 3dGo).

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 expression—low (MHC class IIlo), medium (MHC class IImed) or high (MHC class IIhi)—as illustrated in Figure 4Go. 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 2–4 days after irradiation (Figure 5aGo). 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 5bGo). Two days after irradiation there was a small population of CD45+ MHC class IIhi EC present, which began to decline on day 3 (Figure 5cGo).



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Fig. 4. Representative flow cytometric profiles illustrating MHC class II expression on irradiated and unirradiated CD45+ EC. Two-color cytofluorimetric analysis of EC 2, 3, and 4 days after UV-irradiation (solid line), and of unirradiated control EC (dotted line) was performed as described in Materials and methods. The marker regions shown were used to define MHC class II expression on CD45+ EC: MHC class IIlo (M1), MHC class IImed (M2) and MHC class IIhi (M3). For clarity, histograms show MHC class II staining minus non-specific binding by the murine IgG2b isotype control mAb from the corresponding quadrant on CD45+ cells.

 


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Fig. 5. UV radiation causes alterations in the CD45+ MHC class II+ cell populations within the epidermis 2–4 days after irradiation. Cytofluorimetric analysis was performed as described in Materials and methods. The mean percentage of positive cells ± SEM at each time point is shown for (a) CD45+ MHC class IIlo cells, (b) CD45+ MHC class IImed cells and (c) CD45+ MHC class IIhi cells. *P < 0.05; **P < 0.02; ***P < 0.01 compared with unirradiated control mice (unpaired Student's t-test). Results are pooled from two separate experiments using a total of six mice per group. Where the SEM is not obvious it is too small to be seen.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Changes in the epidermis in response to UV radiation are thought to contribute to immunosuppression to haptens, but less is known about the effect of such changes on the outgrowth of tumor cells. Here, we show that enhanced tumor growth occurred when a regressor UV-induced squamous carcinoma cell line was inoculated intradermally into the irradiated skin of mice 2 or 3 but not 4 days after exposure to a single dose of UV radiation (approximately 6.8 times the minimal edemal dose). The dose of 410 mJ/cm2 UVB radiation (with accompanying 100 mJ/cm2 UVA radiation) was selected to allow significant enhancement of LK2 tumor growth in UV-irradiated skin and was administered as a single dose to prevent UV radiation affects on recently immigrated cells in the skin which may have arisen if repeated, consecutive doses were used. The increased tumor growth was modest and UV radiation did not prevent rejection of the majority of tumors in the irradiated groups. Other studies have shown enhanced growth of UV-induced tumors transplanted into mice exposed to UV radiation (35), but the cumulative UV doses were much higher and more chronic than those used here. The critical issue for this study is that the UV-induced changes responsible for enhancing tumor growth were present 2 and 3 days after UV radiation, but had resolved by day 4. This differs from UV-induced suppression of CHS and enabled this effect to be differentiated from other effects of UV radiation on tumor growth.

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 2–3 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 2–4 days after a single exposure to four times the minimal erythemal dose of UV radiation (10). The similar percentages of {gamma}{delta} 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{gamma}3 TCR, was unsuccessful (results not shown) again presumably due to trypsin sensitivity (32). Interestingly, although the V{gamma}3 TCR epitope appeared to be trypsin sensitive, the {gamma}{delta} 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-{alpha} 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.


    Notes
 
1 To whom correspondence should be addressed Email: garyh{at}med.usyd.edu.au Back


    Acknowledgments
 
We gratefully acknowledge support by the University of Sydney Cancer Research Fund and the University of Sydney Dermatology Research Foundation. R.S. was the recipient of an Australian Postgraduate Award from the University of Sydney. We also gratefully acknowledge Dr Linda Johnston for advice on flow cytometry.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 29, 2000; revised July 26, 2000; accepted July 31, 2000.