TY - JOUR
AU - Le,, Shery
AB - Abstract The induction of epidermal immunity depends on activation of local dendritic cells (DC), Langerhans cells (LC), to migrate from the skin to local lymph nodes and mature into potent immunostimulatory cells. We have previously shown that progressor skin tumors, which evade immunological destruction, prevent contact sensitizer-induced LC migration from the skin to draining lymph nodes. In contrast, regressor tumors, which evoke protective immunity, did not inhibit DC mobilization. In this study we utilized the skin explant model to determine the factors produced by skin tumors which regulate LC migration from the skin. Supernatants from two progressor squamous cell carcinoma lines both inhibited LC migration, whereas supernatants from two regressor squamous cell carcinoma lines both enhanced LC mobilization. Transforming growth factor (TGF)-β1 inhibited, while IL-10 enhanced, LC migration from cultured skin. Both reduced the ability of LC to mature into potent allostimulators. Antibody neutralization identified that TGF-β1 produced by the progressor tumor was responsible for inhibition of LC migration, while IL-10 produced by the regressor tumor enhanced LC mobilization. Thus these studies show that skin tumors influence DC mobilization from tumors by production of cytokines, and that TGF-β1 is one factor produced by tumors which can immobilize LC and keep them in an immature form. This is likely to be an important mechanism of tumor escape from the immune system as progressor tumors inhibited, while regressor tumors enhanced DC mobilization. cell trafficking, cytokines, dendritic cells, tumor immunity DC dendritic cells, EC epidermal cells, GM-CSF granulocyte macrophage colony stimulating factor, LC Langerhans cells, MECLR mixed epidermal cell lymphocyte reaction, NCS newborn calf serum, SCC squamous cell carcinoma, TGF transforming growth factor, TNF tumor necrosis factor Introduction Dendritic cells (DC) are bone marrow-derived antigen-presenting cells that are found in most tissues. In the epidermal layer of the skin they are called Langerhans cells (LC). DC are the most potent type of antigen-presenting cell and appear to be particularly important for the induction of primary immunity (1). In the skin, epidermal LC are relatively immature and are unable to provide the accessory signals required for activation of naive T cells. They need to mature and migrate to local lymph nodes in order to initiate immune responses (2). LC mature in response to contact allergens (3) and viruses (4) prior to development of an immune response. It is unknown whether maturation and migration are coordinated biological responses during activation or whether they can be induced independently. A number of cytokines produced in the skin have been shown to induce maturation and migration of LC. Tumor necrosis factor (TNF)-α and IL-1β are both able to activate LC to migrate from the skin to local lymph nodes (5), but TNF-α does not cause their functional maturation (6). Granulocyte macrophage colony stimulating factor (GM-CSF) is an important inducer of LC maturation (7,8), but does not appear to be a major mediator of migration from the skin (9). It is probable that multiple signals are required from a range of cytokines for the coordinated migration and maturation of LC. Other cytokines can also influence LC. IL-10 treatment of LC inhibits their ability to activate T cells (10). Enhanced migration of LC from the skin of IL-10 knockout mice has lead to the suggestion that this cytokine is involved, at least indirectly, in LC migration (11). Transforming growth factor (TGF)-β1 knockout mice lack epidermal LC (12) and TGF-β1 causes a preference for LC production from DC precursors (13). LC infiltrate skin tumors (14), and some studies have shown a relationship between the presence of LC and tumor development or progression (15). The demonstration that immunotherapy of human cancer patients with DC is possible (16) has indicated the importance of determining how tumors influence DC. We have recently examined the cellular mechanism responsible for infiltration of skin tumors with large numbers of LC (17,18). In these studies we compared progressor skin tumor lines, which evade immunological destruction and therefore grow progressively, with regressor tumors, which grow for the first 11–14 days after transplantation and then are immunologically rejected so that they regress. By studying the effects of skin tumor-derived factors on migration of LC from parental skin grafted onto F1 hybrid mice and contact sensitizer-induced LC migration, we showed that progressor skin tumors produce a factor(s) which inhibits LC migration from the skin to draining lymph nodes. The progressor tumor did not interfere with migration of DC precursors into the tissue. Comparison of progressor with regressor tumor lines in athymic mice showed that the progressor tumors, which are infiltrated with large numbers of LC, inhibit LC migration, whereas regressor tumors, which are infiltrated with lower numbers, do not inhibit LC mobilization from the tissue. This suggests that skin tumors produce factor(s) which inhibit LC migration from the tumor to lymph nodes and that this leads to an accumulation of DC in the tissue. This may be an important tumor escape mechanism, as regressor tumors did not inhibit LC migration. LC migration from cultured skin (19) can be used to study mediators of LC migration (20). We have used this system to show that TGF-β1 inhibits LC migration from skin and is a factor produced by progressor tumors which inhibits LC migration. In contrast, regressor tumors produce IL-10, which enhances LC migration from tumors. Methods Animals Female mice (6–12 weeks old) were used for all experiments. C3H/HeN mice were obtained from the University of Sydney. BALB/c and C57BL/6 mice were obtained from the Animal Resources Centre (Perth, Australia). Mice were supplied with food and water ad libitum, and were used with approval from the University of Sydney animal ethics committee. Tumor cell lines and supernatants The tumor lines, growth in vitro, and production of supernatant from cultured tumor cells has been described previously (17,21). In brief, all tumor lines were established from squamous cell carcinomas (SCC) that arose in mice exposed chronically to UV radiation. The T7 and T79 cell lines were kind gifts from Dr V. Reeve (Department of Veterinary Pathology, University of Sydney) and were derived in Skh:HR-1 mice. The 13.1 (kind gift from Dr M. Kripke, University of Texas) and LK-2 (derived in this Department) cell lines were derived from tumors which arose in C3H/HeN mice. All tumor cell lines were maintained by culture in DMEM [Commonwealth Serum Laboratories (CSL), Melbourne, Australia], supplemented with 10% newborn calf serum (NCS; CSL). To prepare supernatant, tumor cells were grown to confluence on microcarrier beads (Pharmacia, Uppsala, Sweden), washed in serum-free DMEM and cultured in the absence of serum for a further 48 h. The culture supernatant was collected. Cellular debris was removed by centrifugation at 1250 g and the supernatant was concentrated 5-fold under nitrogen pressure using an Amicon concentrator with a 10-kDa cut-off membrane (Amicon, Beverly, MA) at 4°C. The supernatant was filter-sterilized and stored at –80°C. Preparation of epidermal sheets for LC staining and enumeration Epidermal sheets were prepared from skin, and LC were stained and enumerated as described previously (17). Skin was incubated in trizma-buffered isotonic saline containing 20 mM EDTA (pH 7.3) for 3 h at 37°C and the epidermis was then mechanically peeled from the underlying dermis. Epidermal sheets were fixed in acetone for 20 min at room temperature, and stained sequentially with hybridoma supernatant containing mouse anti-murine MHC class II antibody (TIB 93; ATCC, Rockville, MD), biotin-conjugated goat anti-mouse IgG + IgM antibody (Caltag, San Francisco, CA), streptavidin-conjugated alkaline phosphatase (Amersham, Little Chalfont, UK) and New Fuchsin-based alkaline phosphatase substrate. Immunostained cells were blinded and counted with respect to area of epidermis using a true color fully automated image analysis system (Chromatic color image analysis system; L. R. Jarvis, Wild-Leitz, Sydney, Australia). For each epidermal sheet, randomly selected fields were counted until the total area evaluated approximated 1 mm2. Isotype controls were included to ensure specificity of staining. Skin explant culture Skin from C3H/HeN mice was cultured to induce LC migration from the epidermis as described previously (19). Each skin sample was divided into two halves, epidermal sheets were freshly prepared from one of these and stained for LC, and the remaining half was cultured for 3 h prior to preparation of epidermal sheets and LC determination. The difference in LC numbers between fresh and cultured skin is the number of LC that migrated out of the skin explants. Rafts were prepared from gelatine sponge (Spongostan Special; Johnson & Johnson Medical, Reading, UK) which was cut into 1-cm2 pieces and floated at the air/liquid interface on DMEM supplemented with 10% heat-inactivated FCS (CSL) and 0.095 mg/ml pyruvate (Sigma, St Louis, MO). The sponges were pre-incubated for 1 h at 37°C in 5% CO2 in air, then overlayed with Whatman 1 Qualitative filter paper (Whatman, Maidstone, UK). Skin pieces (~1 cm2) were then placed on the rafts and incubated at the air/liquid interface as described above for 3 h. Mixed epidermal cell lymphocyte reaction (MECLR) Epidermal cell (EC) suspensions prepared from the skin of BALB/c mice were used to stimulate the proliferation of C57BL/6 spleen cells as we have described previously (22). The EC were washed and then matured by culture at 8×105 viable cells/ml for 3 days in DMEM supplemented with 10% FCS, 1 mM non-essential amino acids (Cytosystems), 0.025 mM 2-mercaptoethanol (Sigma) and 1% penicillin/ streptomycin (Cytosystems). Cytokines and antibodies (described below) were included during this culture period. EC were washed 3 times in DMEM at the end of this culture and resuspended in the culture medium described above. C57BL/6 spleen leukocytes were depleted of monocytes/macrophages by adherence onto plastic for 1 h at 37°C and non-adherent cells were collected. These monocyte-depleted spleen leukocytes were suspended in the same culture medium as the EC (described above). MECLR were performed by co-culture of matured EC with spleen leukocytes in 200 μl volume 96-well round-bottomed plates; 1 μg/ml indomethacin (Sigma) was included in all cultures. Each experiment contained six or 12 replicate wells with 2×105 spleen leucocytes and 0.5, 1 or 2×104 EC/well. After 78 h incubation at 37°C in an atmosphere of 5% CO2 in air, [3H]thymidine (1 μCi/well; sp. act. 2 Ci/mM; Amersham) was added and culture was continued for a further 18 h. Incorporated [3H]thymidine was quantitated by liquid scintillation spectroscopy. Cytokines and anti-cytokine antibodies In some experiments cytokines or anti-cytokine antibodies were included in the skin explants or the MECLR. The cytokines used were hTGF-β1 (R & D Systems, Minneapolis, MN) and rmIL-10 (R & D Systems). Polyclonal rabbit pan-specific anti-TGF-β IgG (R & D Systems; 5 μg/ml neutralizes 50% of bioactivity) was used at 10 μg/ml in the skin explant cultures and MECLR. The control rabbit IgG (Sigma) was used at the same concentration. Polyclonal goat anti-mouse IL-10 IgG (R & D Systems; 2–4 μg/ml neutralizes 50% of the bioactivity due to 2.5 ng/ml) was used at 10 μg/ml in the skin explant cultures and MECLR. Control goat IgG (R & D Systems) was used at the same concentration. Statistical analysis Results are presented as means ± SEM and test groups are statistically compared to the control group using a two-tailed unpaired Student's t-test. P < 0.05 was regarded as significant. N values in figures refer to the number of skin explants. Results Progressor tumors inhibit, and regressor tumors enhance, LC migration from the epidermis Supernatants were prepared from cultured progressor and regressor tumor cell lines. The tumor supernatants were concentrated 5-fold and included during the culture of skin explants. Preliminary dose–response experiments (not presented) showed that the maximum effects on LC migration were produced with 4% 13.1, 80% T7, 40% LK-2 and 40% T79 concentrated supernatant, and therefore these concentrations were used throughout the remainder of the study. Similar results were produced with higher or lower concentrations, but were less pronounced or reproducible. Culture of skin in the absence of tumor supernatant (cultured control) reduced the LC density from 648 ± 9 to 454 ± 10 cells/mm2 (Fig. 1). This level of LC migration was regarded as the control and therefore the effects of tumor supernatants on LC migration were determined by comparison to cultured control epidermis. Both progressor tumor supernatants significantly prevented LC migration from occurring to the extent that was observed from cultured control epidermis, increasing the number of LC retained in cultured epidermis. In contrast, both regressor tumor supernatants significantly enhanced the reduction in LC compared to control cultured skin. Thus the progressor tumors inhibited, and the regressor tumors enhanced, LC migration from the skin during explant culture. TGF-β1 inhibits and IL-10 enhances LC migration from cultured skin Compared to the level of LC migration from cultured control epidermis, TGF-β1 retained LC numbers at significantly higher levels (Fig. 2). This contrasts with IL-10, which significantly reduced the number of epidermal LC compared to control cultured skin (Fig. 3). Thus TGF-β1 inhibits, while IL-10 enhances, LC migration out of the epidermis. Both TGF-β1 and IL-10 inhibit LC maturation into potent allostimulatory cells LC freshly prepared from the epidermis are relatively immature compared to DC found in secondary lymphoid organs, but can be induced to mature by culture. A widely used assay for functional maturation of LC is their ability to stimulate proliferation of allogeneic lymphocytes, which increases several fold upon maturation by culture (1,6,7,8,19,22). Thus to examine functional LC maturation, EC suspensions were cultured for 3 days in the presence or absence of cytokines or neutralizing antibodies to induce LC maturation. The ability of the LC to stimulate proliferation of allogeneic spleen cells was then examined to determine the effect of the cytokines or antibodies on maturation. In our experiments this maturation of LC caused a 4-fold increase in ability to induce proliferation of allogeneic spleen cells. Culture with TGF-β1 at 1–50 ng/ml caused a significant inhibition of LC maturation as it reduced the ability of the LC to stimulate proliferation of allogeneic spleen cells; 50 ng/ml had a greater effect than 1 or 10 ng/ml (Fig. 4). Furthermore, neutralizing anti-TGF-β antibody significantly enhanced maturation (Fig. 5). This indicates that TGF-β produced during the maturation-inducing culture of the EC, limits LC maturation and confirms that TGF-β inhibits LC maturation. IL-10 also caused a significant inhibition of LC maturation (Fig. 6) as pre-culture of the EC with IL-10 reduced their ability to stimulate proliferation of the allogeneic spleen cells; 50 ng/ml had a greater effect than 1 or 5 ng/ml. Anti-IL-10 antibody included during LC maturation significantly enhanced the ability of these cells to stimulate allogeneic spleen cell proliferation (Fig. 7), confirming that IL-10 inhibits LC maturation. As for TGF-β, this also suggests that IL-10 produced during EC culture limits the ability of LC to fully mature. TGF-β produced by progressor tumors inhibits LC migration Since TGF-β inhibits LC migration from the skin, it was examined whether this was the factor produced by the progressor tumors which inhibits LC mobilization. Skin explants were cultured with the T7 tumor supernatant in the presence and absence of anti-TGF-β or control antibody (Fig. 8). Skin culture reduced the LC from 689 ± 9.5 to 509 ± 10.2/mm2, indicating that 180 LC/mm2 migrated from the skin during this time. Neither the control IgG nor anti-TGF-β on their own affected LC migration. In the presence of T7 supernatant and control IgG there were significantly larger numbers of LC retained in cultured epidermis, indicating that the T7 supernatant reduced LC migration to 116/mm2. Neutralization of TGF-β in the T7 supernatant reduced the number of LC in cultured skin to control values, indicating that this enhanced LC migration to 207/mm2. IL-10 produced by regressor tumors enhances LC migration It was next determined whether IL-10 was the factor produced by regressor tumors which enhances DC mobilization. Skin explants were cultured with the regressor tumor supernatant T79 in the presence of control IgG or anti-IL-10 neutralizing antibody (Fig. 9). Compared to control cultured skin, the addition of neither control IgG or anti-IL-10 affected LC migration. T79 supernatant with control IgG significantly decreased the number of epidermal LC, indicating that this supernatant enhanced LC migration. The inclusion of anti-IL-10 antibody inhibited the T79 supernatant from enhancing LC migration as these epidermal sheets contained similar numbers of LC to control skin. Thus, IL-10 produced by the regressor tumor enhances LC migration from the skin. Discussion Previous studies of LC migration from skin grafted onto recipient mice and contact sensitizer-induced LC migration have shown that progressor skin tumors inhibit LC from migrating out of the skin, leading to a local increase in their numbers (18). Our present study confirms this using a skin explant culture system and shows that regressor tumors, which are infiltrated with lower numbers of LC, enhance DC mobilization from the skin. TGF-β1 was found to inhibit LC migration and functional maturation, and to be a factor produced by progressor tumors which inhibits LC migration. In contrast, IL-10 was found to enhance LC migration from the skin while also inhibiting functional maturation. IL-10 produced by the regressor tumors was responsible for enhancing LC migration. These experiments cannot exclude the possibility that skin tumors also produce other factors that regulate LC mobilization or maturation. These studies suggest that TGF-β1 produced by progressor tumors inhibits DC mobilization from the tissue, leading to DC accumulation, while IL-10 produced by regressor tumors enhances LC migration from the tissue. As the progressor tumors fail to evoke immunological destruction, whereas the regressor tumors are immunologically destroyed, the ability of LC to migrate from tumor tissue is likely to be important for host defense against skin tumors. The reduction in epidermal LC which occurs in response to culture of whole skin has been shown to be due to migration of LC from the epidermis and has been used to examine LC migration from the skin in other studies (19,20). Using this model, we have shown that TGF-β1 inhibits LC migration from the skin. TGF-β1 also inhibited LC maturation in response to culture. Furthermore, TGF-β1 produced during EC culture limits LC maturation as the inclusion of anti-TGF-β1 antibody during the maturation-inducing culture of EC enhanced the ability of the LC to stimulate allogeneic spleen cells. In these experiments the EC were cultured with the antibody for 3 days prior to assessment of allostimulatory capacity which would have provided sufficient time for production of biologically relevant amounts of TGF-β1. The migration assay was only for 3 h and therefore the inability of anti-TGF-β to influence LC migration in the absence of tumor supernatant was probably due to insufficient TGF-β1 production by the skin during this 3-h period. TGF-β1 has previously been implicated as a regulator of LC. TGF-β1 knockout mice lack epidermal LC (12), although these mice have been shown to contain the LC precursor in their bone marrow, which is able to populate TGF-β1 null skin grafted onto BALB/c athymic mice (23). This suggestion of a role for TGF-β1 in LC development is supported by the observation that the in vitro development of LC-like DC from DC precursors in serum-free conditions is dependent upon the presence of TGF-β1 (24). TGF-β1 enhances the production from DC precursors of LC-like cells expressing high levels of E-cadherin (13). E-cadherin on LC is thought to be important in maintaining these cells within the epidermis (25) and cytokines which induce DC mobilization from the skin reduces the levels of cellular E-cadherin (26). Thus, the increase in E-cadherin on LC in response to TGF-β1 is consistent with our data that this cytokine inhibits LC migration from the skin. Expression of the chemokine receptor CCR7 is important for LC migration from the skin (27) and therefore the observation that TGF-β1 suppresses expression of CCR7 mRNA in DC (13) is consistent with our observation that this cytokine inhibits DC mobilization. TGF-β1 has been reported to inhibit the up-regulation of MHC class II (28), which is one of the steps occurring during LC maturation. The ability of matured LC to stimulate proliferation of allogeneic lymphocytes is reduced by 2-h exposure to TGF-β1 (29). Whereas this is a different experimental design to that which we used, our studies are in agreement that this cytokine has a suppressive effect on the ability of LC to induce T cell stimulation. Thus TGF-β1 inhibits LC from leaving the skin, as well as maintaining them in an immature state, where they would have limited capacity to activate T cells. Antibody neutralization experiments showed that TGF-β1 is the factor produced by the progressor UV-induced tumor T7 that inhibits LC migration from the skin. We found IL-10 to enhance LC migration from the skin and inhibit the ability of LC to mature into potent activators of allogeneic lymphocytes during culture. This appears to be at odds with the recent observation of increased LC migration from the epidermis to lymph nodes in IL-10 knockout mice (11). However, keratinocyte production of TNF-α and IL-1 was increased in these mice, and neutralization of either of these cytokines reduced LC migration. Based on these observations in IL-10 knockout mice, the authors suggested that IL-10 might inhibit LC migration by down-regulation of TNF-α and IL-1. The ability of IL-10 to inhibit LC maturation was confirmed by showing that neutralization of IL-10 during the 3-day maturation period enhanced LC maturation. Keratinocytes have been shown to produce IL-10 (30) and our results therefore suggest that IL-10 produced by keratinocytes during the maturation process limits LC maturation. IL-10 inhibition of LC maturation is consistent with previous studies that IL-10 inhibits the ability of LC and splenic DC to present antigen to Th1 cells (10,31). Antibody neutralization identification of IL-10 as a cytokine produced by the regressor tumor that enhances DC mobilization is consistent with our observation that IL-10 is able to enhance LC migration from the skin. It therefore seems probable that the reason why these tumors do not accumulate such large numbers of DC is because they have a cytokine microenvironment that enables DC to leave the tissue. The prevention of DC accumulation in tumors by antisense neutralization of IL-10 (32) is consistent with our suggestion that this cytokine induces DC mobilization from the tumor. Because IL-10-treated DC induce anergy rather than immunity (33), it appears paradoxical that IL-10 produced by a regressor tumor, which is destroyed by the immune response, enhances DC mobilization. It is possible that IL-10 enhances LC migration from the tumor and, once in the lymph node, the local cytokine microenvironment may enable the LC to mature into a cell capable of inducing tumor regression. This is consistent with the observation that the deficiencies in co-stimulatory molecule expression and T cell-stimulating function of DC isolated from human basal cell carcinomas can be overcome by culture of the DC with GM-CSF for 3 days (34). It is also possible that the regressor tumor produces other cytokines that counteract the suppressive effect of IL-10, enabling the activation of an effective immune response. We have previously shown that the regressor tumors produce GM-CSF in vivo (35) and it is possible that this may prevent IL-10 from converting the DC into inducers of tolerance. A reduced number of DC reaching the lymph node with tumor antigen may be a general step in tumor evasion of the induction of tumor immunity, although the mechanisms by which this occurs may differ between tumors. It has recently been shown that the D459 tumor, which was constructed by transfection of 3T3 cells with EJ ras and mutant human p53, produces vascular endothelial growth factor which interferes with DC production, leading to a reduction in the number of epidermal LC (36). A consequence of this reduced LC density was a lower number of DC migrating from the skin to the local lymph node. In conclusion, we have shown that TGF-β1 can inhibit, while IL-10 enhances, DC mobilization from the skin and that the UV-induced progressor SCC T7 interferes with DC mobilization by production of TGF-β1, while the UV-induced regressor SCC enhances DC mobilization by production of IL-10. These are all new observations that extend the understanding of the role of these cytokines in regulation of skin immunity. These studies also indicate that DC mobilization from the tumor is likely to be a critical event in the induction of anti-tumor immunity. However, the T cell-activating properties of DC within the tumor microenvironment may also be important as down-regulation of this function would fail to support the anti-tumor activity of activated T cells which have migrated into the tumor. Fig. 1. Open in new tabDownload slide Progressor tumors inhibit, and regressor tumors enhance, LC migration from the epidermis. Skin was cultured for 3 h in the absence (cultured control) or presence of supernatant from progressor (13.1 and T7) or regressor (LK-2 and T79) tumor cells. Uncultured fresh skin is also shown. Epidermal sheets were then prepared and stained for LC which were enumerated. Results are a pool of three separate experiments and are presented as mean ± SEM LC which remained in the cultured epidermis. P values compared with cultured control; two-tailed unpaired Student's t-test. Fig. 1. Open in new tabDownload slide Progressor tumors inhibit, and regressor tumors enhance, LC migration from the epidermis. Skin was cultured for 3 h in the absence (cultured control) or presence of supernatant from progressor (13.1 and T7) or regressor (LK-2 and T79) tumor cells. Uncultured fresh skin is also shown. Epidermal sheets were then prepared and stained for LC which were enumerated. Results are a pool of three separate experiments and are presented as mean ± SEM LC which remained in the cultured epidermis. P values compared with cultured control; two-tailed unpaired Student's t-test. Fig. 2. Open in new tabDownload slide TGF-β1 inhibits LC migration from cultured skin. Skin was removed from mice and the epidermis was either prepared immediately and stained for LC (fresh), or cultured for 3 h in the absence (cultured control) or presence (cultured TGF-β) of 50 ng/ml TGF-β1. Epidermal sheets were then prepared and stained for LC. Results are a pool of four separate experiments and are presented as mean ± SEM LC in the cultured or fresh epidermis. P values compared with cultured control; two-tailed unpaired Student's t-test. Fig. 2. Open in new tabDownload slide TGF-β1 inhibits LC migration from cultured skin. Skin was removed from mice and the epidermis was either prepared immediately and stained for LC (fresh), or cultured for 3 h in the absence (cultured control) or presence (cultured TGF-β) of 50 ng/ml TGF-β1. Epidermal sheets were then prepared and stained for LC. Results are a pool of four separate experiments and are presented as mean ± SEM LC in the cultured or fresh epidermis. P values compared with cultured control; two-tailed unpaired Student's t-test. Fig. 3. Open in new tabDownload slide IL-10 enhances LC migration from cultured skin. Skin was removed from mice and the epidermis was either prepared immediately and stained for LC (fresh), or cultured for 3 h in the absence (cultured control) or presence (cultured IL-10) of 5 ng/ml IL-10. Epidermal sheets were then prepared and stained for LC. Results are a pool of three separate experiments and are presented as mean ± SEM LC in the cultured or fresh epidermis. P values compared with cultured control; two-tailed unpaired Student's t-test. Fig. 3. Open in new tabDownload slide IL-10 enhances LC migration from cultured skin. Skin was removed from mice and the epidermis was either prepared immediately and stained for LC (fresh), or cultured for 3 h in the absence (cultured control) or presence (cultured IL-10) of 5 ng/ml IL-10. Epidermal sheets were then prepared and stained for LC. Results are a pool of three separate experiments and are presented as mean ± SEM LC in the cultured or fresh epidermis. P values compared with cultured control; two-tailed unpaired Student's t-test. Fig. 4. Open in new tabDownload slide TGF-β1 inhibits LC maturation into potent allostimulatory cells. Epidermal cell (EC) suspensions were matured by culture for 3 days without supplementation (control, ▪), with 1 (⧫), 10 (•) or 50 (▴) ng/ml TGF-β1. The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of 12 replicate cultures shown; where SEM is not obvious it is too small to be seen. Three out of three experiments gave similar results. All TGF-β-treated EC were significantly lower than the control (P < 0.0001) at each EC concentration; two-tailed unpaired Student's t-test. Fig. 4. Open in new tabDownload slide TGF-β1 inhibits LC maturation into potent allostimulatory cells. Epidermal cell (EC) suspensions were matured by culture for 3 days without supplementation (control, ▪), with 1 (⧫), 10 (•) or 50 (▴) ng/ml TGF-β1. The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of 12 replicate cultures shown; where SEM is not obvious it is too small to be seen. Three out of three experiments gave similar results. All TGF-β-treated EC were significantly lower than the control (P < 0.0001) at each EC concentration; two-tailed unpaired Student's t-test. Fig. 5. Open in new tabDownload slide Anti-TGF-β antibody enhances LC maturation into potent allostimulatory cells. EC suspensions were matured by culture for 3 days without supplementation (control, ▪), with control IgG (•) or with anti-TGF-β IgG (▴). The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of six replicate cultures shown, where SEM is not obvious it is too small to be seen. Five out of five experiments gave similar results. Statistical comparison with control; two-tailed unpaired Student's t-test. Fig. 5. Open in new tabDownload slide Anti-TGF-β antibody enhances LC maturation into potent allostimulatory cells. EC suspensions were matured by culture for 3 days without supplementation (control, ▪), with control IgG (•) or with anti-TGF-β IgG (▴). The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of six replicate cultures shown, where SEM is not obvious it is too small to be seen. Five out of five experiments gave similar results. Statistical comparison with control; two-tailed unpaired Student's t-test. Fig. 6. Open in new tabDownload slide IL-10 inhibits LC maturation into potent allostimulatory cells. EC suspensions were matured by culture for 3 days without supplementation (control, ▪), with 1 (⧫), 5 (•) or 50 (▴) ng/ml IL-10. The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of 12 replicate cultures shown; where SEM is not obvious it is too small to be seen. Three out of three experiments gave similar results. All IL-10-treated EC were significantly lower than the control (P < 0.005) at each EC concentration; two-tailed unpaired Student's t-test. Fig. 6. Open in new tabDownload slide IL-10 inhibits LC maturation into potent allostimulatory cells. EC suspensions were matured by culture for 3 days without supplementation (control, ▪), with 1 (⧫), 5 (•) or 50 (▴) ng/ml IL-10. The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of 12 replicate cultures shown; where SEM is not obvious it is too small to be seen. Three out of three experiments gave similar results. All IL-10-treated EC were significantly lower than the control (P < 0.005) at each EC concentration; two-tailed unpaired Student's t-test. Fig. 7. Open in new tabDownload slide Anti-IL-10 antibody enhances LC maturation into potent allostimulatory cells. EC suspensions were matured by culture for 3 days without supplementation (control, ▪), with control IgG (•) or with anti-IL-10 IgG (▴). The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of 12 replicate cultures shown; where SEM is not obvious it is too small to be seen. Two out of two experiments gave similar results. Statistical comparison with control; two-tailed unpaired Student's t-test. Fig. 7. Open in new tabDownload slide Anti-IL-10 antibody enhances LC maturation into potent allostimulatory cells. EC suspensions were matured by culture for 3 days without supplementation (control, ▪), with control IgG (•) or with anti-IL-10 IgG (▴). The EC were then washed and co-cultured at a range of EC concentrations with monocyte-depleted allogeneic spleen cells. Spleen cell proliferation was assessed by [3H]thymidine uptake. Spleen cells in the absence of EC are shown in the control as 0 EC. Mean ± SEM of 12 replicate cultures shown; where SEM is not obvious it is too small to be seen. Two out of two experiments gave similar results. Statistical comparison with control; two-tailed unpaired Student's t-test. Fig. 8. Open in new tabDownload slide TGF-β produced by progressor tumors inhibits LC migration. Skin was cultured for 3 h in the absence (control) or presence of supernatant from the T7 progressor tumor cell line. Rabbit control IgG or rabbit anti-TGF-β was included in some cultures with and without T7 supernatant. Epidermal sheets were then prepared and stained for LC which were enumerated. Results are a pool of three separate experiments and are presented as mean ± SEM LC which remained in the cultured epidermis. P values compared with control; two-tailed unpaired Student's t-test. Fig. 8. Open in new tabDownload slide TGF-β produced by progressor tumors inhibits LC migration. Skin was cultured for 3 h in the absence (control) or presence of supernatant from the T7 progressor tumor cell line. Rabbit control IgG or rabbit anti-TGF-β was included in some cultures with and without T7 supernatant. Epidermal sheets were then prepared and stained for LC which were enumerated. Results are a pool of three separate experiments and are presented as mean ± SEM LC which remained in the cultured epidermis. P values compared with control; two-tailed unpaired Student's t-test. Fig. 9. Open in new tabDownload slide IL-10 produced by regressor tumors enhances LC migration. Skin was cultured for 3 h in the absence (control) or presence of supernatant from the T79 regressor tumor cell line. Goat control IgG or goat anti-IL-10 was included in some cultures with and without T79 supernatant. Epidermal sheets were then prepared and stained for LC which were enumerated. Results are a pool of three separate experiments and are presented as mean ± SEM LC which remained in the cultured epidermis. P values compared with control; two-tailed unpaired Student's t-test. Fig. 9. Open in new tabDownload slide IL-10 produced by regressor tumors enhances LC migration. Skin was cultured for 3 h in the absence (control) or presence of supernatant from the T79 regressor tumor cell line. Goat control IgG or goat anti-IL-10 was included in some cultures with and without T79 supernatant. Epidermal sheets were then prepared and stained for LC which were enumerated. Results are a pool of three separate experiments and are presented as mean ± SEM LC which remained in the cultured epidermis. P values compared with control; two-tailed unpaired Student's t-test. Transmitting editor: A. Kelso This work was supported by the National Health and Medical Research Council of Australia, and the University of Sydney Cancer Research Fund. References 1 Steinman, R. M. 1991 . The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9 : 271 . 2 Streilein, J. W., Grammer, S. F., Yoshikawa, T., Demidem, A. and Vermeer, M. 1990 . Functional dichotomy between Langerhans cells that present antigen to naive and to memory/effector T lymphocytes. Immunol. Rev. 117 : 159 . 3 Grabbe, S. and Schwarz, T. 1998 . Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity. Immunol. Today 19 : 37 . 4 Johnston, L. J., Halliday, G. M. and King, N. J. C. 1996 . Phenotypic changes in Langerhans cells after infection with arboviruses—a role in the immune response to epidermally acquired viral infection. J. Virol. 70 : 4761 . 5 Cumberbatch, M., Dearman, R. J. and Kimber, I. 1997 . Langerhans cells require signals from both tumor necrosis factor-alpha and interleukin-1-beta for migration. Immunology 92 : 388 . 6 Koch, F., Heufler, C., Kampgen, E., Schneeweiss, D., Bock, G. and Schuler, G. 1990 . Tumor necrosis factor a maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation. J. Exp. Med. 171 : 159 . 7 Witmer-Pack, M. D., Olivier, W., Valinsky, J., Schuler, G. and Steinman, R. M. 1987 . Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J. Exp. Med. 166 : 1484 . 8 Heufler, C., Koch, F. and Schuler, G. 1988 . Granulocyte/macrophage colony stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells. J. Exp. Med. 167 : 700 . 9 Cumberbatch, M. and Kimber, I. 1992 . Dermal tumor necrosis factor-alpha induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans' cell migration. Immunology 75 : 257 . 10 Enk, A. H., Angeloni, V. L., Udey, M. C. and Katz, S. I. 1993 . Inhibition of Langerhans cell antigen-presenting function by IL-10—a role for IL-10 in induction of tolerance. J. Immunol. 151 : 2390 . 11 Wang, B., Zhuang, L. H., Fujisawa, H., Shinder, G. A., Feliciani, C., Shivji, G. M., Suzuki, H., Amerio, E., Toto, P. and Sauder, D. N. 1999 . Enhanced epidermal Langerhans cell migration in IL-10 knockout mice. J. Immunol. 162 : 277 . 12 Borkowski, T. A., Letterio, J. J., Farr, A. G. and Udey, M. C. 1996 . A role for endogenous transforming growth factor β1 in Langerhans cell biology: the skin of transforming growth factor β1 null mice is devoid of epidermal Langerhans cells. J. Exp. Med. 184 : 2417 . 13 Zhang, Y., Zhang, Y. Y., Ogata, M., Chen, P., Harada, A., Hashimoto, S. and Matsushima, K. 1999 . Transforming growth factor-beta 1 polarizes murine hematopoietic progenitor cells to generate Langerhans cell-like dendritic cells through a monocyte/macrophage differentiation pathway. Blood 93 : 1208 . 14 McArdle, J. P., Knight, B. A., Halliday, G. M., Muller, H. K. and Rowden, G. 1986 . Quantitative assessment of Langerhans cells in actinic keratosis, Bowen's disease, keratoacanthoma, squamous cell carcinoma and basal cell carcinoma. Pathology 18 : 212 . 15 Becker, Y. 1992 . Anticancer role of dendritic cells (DC) in human and experimental cancers—a review. Anticancer Res. 12 : 511 . 16 Nestle, F. O., Alijagic, S., Gilliet, M., Sun, Y. S., Grabbe, S., Dummer, R., Burg, G. and Schadendorf, D. 1998 . Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med. 4 : 328 . 17 Halliday, G. M., Lucas, A. D. and Barnetson, R. St C. 1992 . Control of Langerhans' cell density by a skin tumor-derived cytokine. Immunology 77 : 13 . 18 Lucas, A. D. and Halliday, G. M. 1999 . Progressor but not regressor skin tumors inhibit Langerhans' cell migration from epidermis to local lymph nodes. Immunology 97 : 130 . 19 Larsen, C., Steinman, R., Witmer-Pack, M., Hankins, D., Morris, P. and Austyn, J. 1990 . Migration and maturation of Langerhans cells in skin transplants and explants. J. Exp. Med. 172 : 1483 . 20 Larsen, C. P. and Austyn, J. M. 1991 . Langerhans cells migrate out of skin grafts and cultured skin—a model in which to study the mediators of dendritic leukocyte migration. Transplant. Proc. 23 : 117 . 21 Cavanagh, L. L. and Halliday, G. M. 1996 . Dendritic epidermal T cells in ultraviolet-irradiated skin enhance skin tumor growth by inhibiting CD4(+) T-cell-mediated immunity. Cancer Res. 56 : 2607 . 22 Dunlop, K. J., Halliday, G. M. and Barnetson, R. St C. 1994 . All-trans retinoic acid induces functional maturation of epidermal Langerhans cells and protects their accessory function from ultraviolet radiation. Exp. Dermatol. 3 : 204 . 23 Borkowski, T. A., Letterio, J. J., Mackall, C. L., Saitoh, A., Wang, X. J., Roop, D. R., Gress, R. E. and Udey, M. C. 1997 . A role for TGF-beta-1 in Langerhans cell biology—further characterization of the epidermal Langerhans cell defect in TGF-beta-1 null mice. J. Clin. Invest. 100 : 575 . 24 Strobl, H., Riedl, E., Scheinecker, C., Bellofernandez, C., Pickl, W. F., Rappersberger, K., Majdic, O. and Knapp, W. 1996 . TGF-beta-1 promotes in vitro development of dendritic cells from CD34(+) hemopoietic progenitors. J. Immunol. 157 : 1499 . 25 Tang, A. M., Amagai, M., Granger, L. G., Stanley, J. R. and Udey, M. C. 1993 . Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature 361 : 82 . 26 Jakob, T. and Udey, M. C. 1998 . Regulation of E-cadherin-mediated adhesion in Langerhans cell-like dendritic cells by inflammatory mediators that mobilize Langerhans cells in vivo. J. Immunol. 160 : 4067 . 27 Saeki, H., Moore, A. M., Brown, M. J. and Hwang, S. T. 1999 . Secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J. Immunol. 162 : 2472 . 28 Epstein, S. P., Baer, R. L., Thorbecke, G. J. and Belsito, D. V. 1991 . Immunosuppressive effects of transforming growth factor-beta—inhibition of the induction of Ia antigen on Langerhans cells by cytokines and of the contact hypersensitivity response. J. Invest. Dermatol. 96 : 832 . 29 Demidem, A., Taylor, J. R., Grammer, S. F. and Streilein, J. W. 1991 . Comparison of effects of transforming growth factor-beta and cyclosporin-A on antigen-presenting cells of blood and epidermis. J. Invest. Dermatol. 96 : 401 . 30 Enk, A. H. and Katz, S. I. 1992 . Identification and induction of keratinocyte-derived IL-10. J. Immunol. 149 : 92 . 31 De Smedt, T., Van Mechelen, M., De Becker, G., Urbain, J., Leo, O. and Moser, M. 1997 . Effect of interleukin-10 on dendritic cell maturation and function. Eur. J. Immunol. 27 : 1229 . 32 Qin, Z. H., Noffz, G., Mohaupt, M. and Blankenstein, T. 1997 . Interleukin-10 prevents dendritic cell accumulation and vaccination with granulocyte-macrophage colony-stimulating factor gene-modified tumor cells. J. Immunol. 159 : 770 . 33 Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, J. and Enk, A. H. 1997 . Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 159 : 4772 . 34 Nestle, F. O., Burg, G., Fah, J., Wronesmith, T. and Nickoloff, B. J. 1997 . Human sunlight-induced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells. Am. J. Pathol. 150 : 641 . 35 Rubel, D. M., Barnetson, R. St C. and Halliday, G. M. 1998 . Bioactive tumor necrosis factor alpha but not granulocyte-macrophage colony-stimulating factor correlates inversely with Langerhans cell numbers in skin tumors. Int. J. Cancer 75 : 210 . 36 Ishida, T., Oyama, T., Carbone, D. P. and Gabrilovich, D. I. 1998 . Defective function of Langerhans cells in tumor-bearing animals is the result of defective maturation from hemopoietic progenitors. J. Immunol. 161 : 4842 . © 2001 The Japanese Society for Immunology
TI - Transforming growth factor-β produced by progressor tumors inhibits, while IL-10 produced by regressor tumors enhances, Langerhans cell migration from skin
JF - International Immunology
DO - 10.1093/intimm/13.9.1147
DA - 2001-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/transforming-growth-factor-produced-by-progressor-tumors-inhibits-HCAnfFCw8D
SP - 1147
EP - 1154
VL - 13
IS - 9
DP - DeepDyve
ER -