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[Cancer Research 61, 2311-2319, March 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Up- and Down-Regulation of Granulocyte/Macrophage-Colony Stimulating Factor Activity in Murine Skin Increase Susceptibility to Skin Carcinogenesis by Independent Mechanisms1

Amrit Mann2, Kai Breuhahn3, Peter Schirmacher, Arnd Wilhelmi, Carsten Beyer, Andrea Rosenau, Suat Özbek, Stefan Rose-John3 and Manfred Blessing4

I. Medical Department, Section of Pathophysiology, Johannes Gutenberg-University, D-55131 Mainz [A. M., K. B., A. W., C. B., A. R., S. O., S. R-J., M. B.], and Institute of Pathology, University of Cologne, 50931 Cologne [P. S.], Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumorigenesis is complex. On the one hand, GM-CSF can promote tumor cell growth, survival, and even metastasis. On the other hand, it can stimulate tumor cell rejection. In skin, it is early expressed after topic application of tumor-promoting agents and therefore may be responsible for changes that correlate with skin tumor promotion (e.g., epidermal hyperproliferation and inflammation). To analyze GM-CSF function in skin tumorigenesis, we generated transgenic mice epidermally overexpressing either GM-CSF or a GM-CSF antagonist. Both types of transgenic mice exhibited significantly increased numbers of benign tumors in a two-step skin carcinogenesis experiment using 7',12'-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate as tumor promoter. However, only animals expressing GM-CSF displayed a significantly elevated carcinoma burden following a single-step carcinogenesis protocol consisting of tumor initiation only. Therefore, endogenous promotion is responsible for elevated tumor development in GM-CSF-overexpressing mice. In antagonist transgenic animals, an increased tumorigenicity of modified B16 tumor cells after cutaneous transplantation as compared with nontransgenic or GM-CSF transgenic mice was observed. Thus, the antitumor activity leading to the repression of tumor cell growth in control mice is GM-CSF dependent and is compromised in mice expressing the antagonist. We suggest that both, up-regulation and down-regulation of GM-CSF activity in skin, increase the incidence and growth of tumors via two independent mechanisms: endogenous tumor promotion in the case of increased GM-CSF activity and compromised tumor cell rejection in the case of decreased GM-CSF activity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytokine GM-CSF4 is a glycoprotein characterized by its ability to induce proliferation, differentiation, and survival of bone marrow-derived hematopoietic cells (1, 2, 3) . GM-CSF has been shown to be secreted and perceived by a variety of normal and transformed cell types, including endothelial cells, keratinocytes, and melanoma cells (4, 5, 6, 7, 8) . In skin for example, GM-CSF is readily secreted by keratinocytes shortly after irritation and mediates keratinocyte hyperproliferation in an autocrine manner (9, 10, 11) . Because of its role in regulating both nonimmune and immune cell proliferation, differentiation, and apoptosis, GM-CSF is likely to affect different aspects of skin tumor initiation and promotion, such as epidermal hyperproliferation and immune responses.

However, the role of GM-CSF during skin tumorigenesis is complex. On the one hand, squamous cell carcinomas as well as cultured carcinoma cells frequently produce GM-CSF and profit from its stimulatory effect on cell proliferation and cell migration promoting both tumor growth and metastasis (12, 13, 14, 15, 16) . Similarly, melanoma cells secrete this cytokine (7) . On the other hand, GM-CSF as well as other cytokines such as IFN-{gamma} or hyper-IL-6 have been reported to enhance antitumor cell responses against melanoma cells or squamous cell carcinoma-derived cell lines, which had been engineered to produce these cytokines (17, 18, 19, 20, 21, 22, 23) . For example, experiments with murine B16 melanoma cells exemplified a GM-CSF-mediated antitumor response dependent on CD4+ lymphocytes (20 , 24) and enhanced by CD8+ cells (25) . Because GM-CSF is a potent stimulator of dendritic cell development, it has been suggested that the induction of immunity against B16 melanoma cells may be attributable to the ability of GM-CSF to induce these APCs to process and present tumor antigens to both CD4+ and CD8+ T cells (26, 27, 28) . As a consequence of these antitumor activities in murine model systems, GM-CSF has already been tested in the treatment of melanoma patients (29, 30, 31, 32) , despite the fact that both tumor-promoting as well as immunosuppressive activities of GM-CSF have been described (7 , 13, 14, 15, 16 , 33 , 34) .

To evaluate the tumorigenic and antitumorigenic effects of GM-CSF in both induction of squamous cell carcinomas as well as melanoma cell growth in vivo, we modulated the activity of GM-CSF in the skin of transgenic animals. We performed chemically induced skin carcinogenesis experiments (35) as well as cutaneous transplantation experiments with B16 melanoma cells expressing an IL-6 analogue with enhanced biological activity (36) . Transgenics expressing GM-CSF in the basal layer of the epidermis under the control of a keratin 5 gene promoter-based expression vector have been described previously (8) . Major alterations seen in these animals were epidermal hyperproliferation, accumulation of mast cells and LCs, as well as an up-regulation of the antiapoptotic factor A1, a bcl-2 family member (8) .

In this report, we describe the generation and characterization of transgenic mice expressing a GM-CSF double mutant antagonist (K14E/E21K) under the control of a keratin 10 gene-based expression vector in the suprabasal layers of the skin. Briefly, the receptor for GM-CSF consists of a cytokine-specific {alpha} chain (GM-{alpha}R) and a signal transducing ß-chain (GM-ßR), which is shared with IL-3 and IL-5 (37) . A double mutant ligand (GM-CSF) with charge reversals at amino acid positions 14 and 21 created a potent GM-CSF antagonist (K14E/E21K). In vitro, this double mutant protein had lost the ability to activate the GM-CSF receptor in the GM-CSF-dependent myeloid leukemia cell line NFS60 (38) . Furthermore, a 1000-fold excess of the double mutant protein over GM-CSF inhibited the proliferative response of NFS60 cells to GM-CSF (38) . Thus, the double mutant is an effective competitor, binding, and titrating the receptor {alpha} chain without inducing signal transduction by the receptor ß-chain.

A restriction of the respective GM-CSF activity changes predominantly to the skin in both types of transgenics was desired to avoid the development of pathological changes as seen in generalized GM-CSF-overexpressing mice or in GM-CSF or GM-CSF receptor ß-chain knock-outs (18 , 39 , 40) . Our results from the chemical-induced skin carcinogenesis experiments as well as from tumor cell transplantation models suggest that both activities of GM-CSF, stimulation of tumor cell proliferation as well as stimulation of immune cells, independently modulate tumor development in these models. Increased tumor frequencies in GM-CSF transgenics warrant a closer evaluation of antitumorigenic treatment protocols using GM-CSF.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation and Maintenance of Transgenic Mice.
A cDNA coding for the K14E/E21K double mutant antagonist to GM-CSF (38) was derived from a murine cDNA for GM-CSF by site-directed mutagenesis (41) . Using oligonucleotides ant-1 (5'-C CGG CCT TGG GAA CAT GTA GAG GCC ATC AAA AAG GCC CTG-3'; corresponding to position 12–52; GenBank accession no. X05906; Ref. 42 ), ant-2 (5'-CCT GCT CGA ATA TCT TCA GGC G-3'; position 167–145; GenBank accession no. X05906; Ref. 42 ), and the murine cDNA for GM-CSF as template, a fragment carrying the K14E/E21K mutations (underlined codons in primer ant-1) was generated by PCR. The PCR fragment was used to replace the corresponding fragment of the cDNA via StyI and BsgI restriction enzyme digestion and ligation.

The resulting cDNA was verified by sequence analysis and inserted into a keratin 10 gene promoter-based expression vector using the restriction endonuclease SmaI (43) . The expression cassette was excised out of pBluescript by KpnI digestion, gel purified, and used for pronuclear microinjection of fertilized eggs of strain FVB/N, essentially as described (44) . Offspring were biopsied at ears or tails for transgene analysis by PCR using a keratin 10 gene promoter-specific primer (5'-TAA CAC ATG TGG GAT ACA CCC-3'; position 1021–1042, GenBank accession no. Z32746; Ref. 45 ) and a murine GM-CSF cDNA-specific oligonucleotide (5'-CTG GCT GTC ATG TTC AAG GCG-3'; position 218–198; GenBank accession no. X05906; Ref. 42 ).

Transgenic mice overexpressing GM-CSF under the control of the bovine keratin 5 promoter have been described elsewhere (8) . All transgenic lines were maintained as heterozygotes on a FVB/N background. For B16 melanoma cell transplantation experiments, F1 crosses to C57Bl6 mice were used as recipients. For all experimental procedures, age matched nontransgenic littermates were used as controls.

Northern Blot Analysis.
Northern blot analysis was carried out essentially as described (8) . Twenty-five µg of total RNA per lane were gel separated and blotted onto nylon membranes (Hybond N; Amersham, Braunschweig, Germany). The hybridization probes were generated by reverse transcription-PCR with spleen cDNA serving as template, essentially as described previously (8) : for A1, A1-1 was 5'-GCC TCC AGA TAT GAT TAG GG-3' (position 28–48; GenBank accession no. L16462) and A1-2 was (5'-CTG ATA ACC ATT CTC GTG GG-3'; position 698–678; GenBank accession no. L16462; Ref. 46 ); for GM-CSF, GM-1 was 5'-TGG GGA AGC CCA GGC CAG CT-3' (position 3185–320; GenBank accession no. X03020) and GM-2 was 5'-CAA AAA ACG TAA GTT TCC CC-3' (position 3541–3521; GenBank accession no. X03020; Ref. 47 ); and for GAPDH, GAPDH-1 was 5'-CAA CTA CAT GGT CTA CAT GTT C-3' (position 159–181; GenBank accession no. M32599) and GAPDH-2 was 5'-ACC AGT AGA CTC CAC GAC-3' (position 340–322; GenBank accession no. M32599; Ref. 48 ). Each PCR product was verified by sequence analysis.

Histology and Immunostaining.
For histopathological studies, skin samples were fixed in neutral buffered 4% formaldehyde overnight, processed for paraffin embedding, and stained routinely with H&E or with May-Grünwald/Giemsa stain for detection of mast cells (8) . Preparation of samples for immunohistological procedures and antibodies used in this study have been described previously (8) .

Chemically Induced Skin Carcinogenesis.
DMBA and TPA were purchased from Sigma-Aldrich Chemie (Deisendorf, Germany) and solubilized in acetone. Each experimental group consisted of 25–40 mice. Initiation and promotion were performed essentially as described (35 , 49) . For a one-step protocol, initiation was achieved by topic application of TPA (2.5 µg) on the shaved back (mid-dorsum) of mice of 8 weeks of age, followed by a single topical application of 100 µg of DMBA 18 h later. Thereafter, the experimental groups were kept for 60 weeks without any further treatment. For a two-step protocol, mice were initiated as described above. Tumor promotion was carried out by treating the mice with TPA (2.5 µg) twice weekly for 50 weeks after initiation. In both experiments, the mice were monitored weekly, and the tumor incidence and mortality were recorded. Mice bearing carcinomas were sacrificed as soon as the well being of the animals was significantly compromised. All tumors were histologically classified by a senior pathologist (P. S.).

LPS Treatment.
Mice were injected i.p. with 100 µg of LPS (Serotype, 026:B6; Sigma Chemical Co., St. Louis, MO) in 200 µl of PBS. Blood was collected from the lateral tail vein at different time points after LPS injection. For serum preparation, the blood was left for clotting at room temperature for 2 h. After centrifugation at 6000 rpm (Sorvall; FA-Micro) for 10 min, the supernatants were used for ELISA.

Cytokine Assays.
Serum and skin extracts were prepared essentially as described previously (8) . Levels of IFN-{gamma}, TNF-{alpha}, and GM-CSF were evaluated using cytokine-specific ELISA kits (R & D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The K14E/E21K double mutant antagonist for GM-CSF could also be quantified using the GM-CSF-specific ELISA.

Quantification of Apoptosis and Cell Proliferation.
BrdUrd labeling experiments were carried out using the In Situ Cell Proliferation kit, FLUOS (Boehringer Mannheim, Mannheim, Germany), as well as detection and quantification of programmed cell death in the epidermis. Both experiments were carried out as described previously (8) . For each experiment, values were obtained from at least three age- and sex-matched animals of each genotype.

s.c. Challenge Experiment.
Mice were shaved on the back and challenged s.c. with 105 B16 melanoma cells or transfected B16 melanoma cells secreting hyper-IL-6 (22 , 23) in PBS. Mice were sacrificed after 2 weeks, when the tumors displayed severe necrosis or reached a size of 300 mm2. All tumors were excised, and tumor growth was monitored by measuring perpendicular diameters and by weighing.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation and Phenotype of Antagonist Transgenic Mice
Transgenic mice were generated by pronuclear injection of the keratin 10 gene promoter-based expression vector for the GM-CSF antagonist (Fig. 1A)Citation . Three independent transgenic founder animals were identified. All were fertile, but only two founders transmitted the transgene and established lines. In both lines, the transgene was transmitted to offspring animals at the expected Mendelian frequency without any bias in regard to gender, suggesting autosomal integration sites. The transgenic lines were designated according to the guidelines set by the Institute for Laboratory Animal Research (Washington, DC) as TgN(K10AGMCSF)1 Mbl and TgN(K10AGMCSF)2 Mbl. For simplicity, these lines are being abbreviated hereafter as TgAnt1 and TgAnt2.



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  		Fig. 1. Generation and characterization of keratin 10/GM-CSF antagonist transgenic animals. A, schematic representation of the keratin 10 gene promoter-based GM-CSF antagonist expression vector. Black rectangle, the antagonist cDNA under the control of the bovine keratin 10 promoter. The cDNA is preceded by a ß-globin gene-derived intron and followed by the polyadenylation signal of the gene encoding human growth hormone. B, Northern blot analysis with total RNAs (25 µg) extracted from skin (Lane 1), brain (Lane 2), lung and trachea (Lane 3), spleen (Lane 4), heart (Lane 5), liver (Lane 6), and kidney (Lane 7) of transgenic animals using GM-CSF cDNA as a probe. Note the restriction of transgene expression to skin. GAPDH was used as a loading control (C–E). Immunohistochemical staining of skin sections derived from nontransgenic (C) and transgenic (D, TgAnt1; E, TgAnt2) mice using a rat antimouse GM-CSF-specific monoclonal antibody (bars, 200 µm; F and G) is shown. High-power magnification of skin sections derived from transgenic animals basally overexpressing GM-CSF under the control of the keratin 5 promoter (F, Tg1) and mice suprabasally expressing the GM-CSF antagonist under the control of the keratin 10 promoter (G, TgAnt1) is shown. Arrowheads, dermal-epidermal junction. Bars, 40 µm.

 
Animals of both lines TgAnt1 and TgAnt2 showed no obvious abnormalities. No difference in the proliferation rate of epidermal cells was seen between antagonist transgenics and controls. Similarly, no significant changes in the rate of apoptotic cells as determined by terminal deoxynucleotidyltransferase-mediated nick end labeling assay were observed in the basal epidermal layer between the antagonist transgenics (TgAnt1, 0.331% ± 0.059%; TgAnt2, 0.306% ± 0.062%) and nontransgenic animals (0.366% ± 0.017%).

No gross abnormalities in hair follicle morphology, dermal cellularity, or epidermal thickness were seen in H&E-stained skin sections of antagonist transgenic animals as compared with nontransgenic controls (data not shown). Likewise, Giemsa staining and immunostaining with anti-CD117 (c-kit) revealed no differences in mast cell numbers in the dermis of antagonist transgenics versus controls (data not shown). Also, histopathological studies of lung samples revealed no differences in transgenics compared with nontransgenic animals.

Expression of the Transgene in Antagonist Transgenics
Tissue-specific expression of the keratin 10 gene promoter-driven transgene in lines TgAnt1 and TgAnt2 was confirmed using Northern blot analysis. Using GM-CSF cDNA as a probe, signals were obtained only in RNA from skin of both transgenic lines (Fig. 1B)Citation . Expression of the transgene was undetectable by this method in heart, brain, liver, lung and trachea, spleen, and kidney in both transgenic lines. The size of the transgene-derived RNA was determined to be ~0.86 kb, whereas endogenous GM-CSF mRNA with a size of 1.2 kb was not detectable by this method. In addition, no signal was obtained in RNAs from nontransgenic mice (data not shown).

Cell type specificity of transgene expression was confirmed by immunohistochemistry using a GM-CSF-specific monoclonal antibody. No staining was observed in normal adult epidermis (Fig. 1C)Citation , whereas significant staining was detectable in the suprabasal layers of the interfollicular epidermis in animals of both transgenic lines (Fig. 1, D and E)Citation . High power magnification revealed that keratin 5 promoter-directed expression of GM-CSF is restricted to the basal layer of the epidermis (Fig. 1F)Citation as compared with keratin 10 promoter-directed expression in the suprabasal layers of the epidermis in antagonist transgenics (Fig. 1G)Citation . Control experiments with secondary antibodies alone were negative in skin sections of control or transgenic mice (data not shown).

Tissue concentrations of the GM-CSF antagonist in both transgenic lines and nontransgenic controls were determined by ELISA. Because we did not observe induction of the endogenous gene for GM-CSF in lines TgAnt1 and TgAnt2 by Northern blot experiments, the gross majority of the specific protein measured by ELISA in extracts from these transgenics must be a transgene-derived K14E/E21K double mutant antagonist for GM-CSF. Whereas GM-CSF concentrations in skin extracts and sera from nontransgenic controls were below the threshold of detection, extracts prepared from transgenic skin revealed well-detectable protein concentrations ranging from 107.02 ng/g of skin in line TgAnt1 to 417.14 ng/g of skin in line TgAnt2 (Table 1)Citation . In both transgenic lines, serum levels were ranging from 1.77 ng/ml serum in line TgAnt1 to 14.65 ng/ml serum in line TgAnt2 (Table 1)Citation .


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  		Table 1 Concentrations of GM-CSF antagonist in skin and serum

 
Antagonist Function in Vivo
To investigate the bioactivity of the GM-CSF antagonist in vivo, animals expressing the GM-CSF agonist (Tg1) were crossed with animals expressing the GM-CSF antagonist (TgAnt1). Double transgenic offspring was analyzed for the changes described in the skin of agonist transgenics, such as the induction of the antiapoptotic factor A1, LC accumulation, and epidermal hyperproliferation. In addition, GM-CSF-dependent systemic effects, such as the rise in IFN-{gamma} serum levels after acute phase induction by LPS, were analyzed in antagonist transgenics:

(a) Expression of the antiapoptotic factor A1 in the skin was determined using Northern blot analysis (Fig. 2A)Citation . Although transgenic animals overexpressing GM-CSF in the skin showed a strong induction of A1 mRNA, mice expressing GM-CSF antagonist exhibited no induction of A1 RNA levels (Fig. 2A)Citation . Also, mice expressing both agonist and antagonist show the same induction in A1 mRNA as was observed in agonist transgenic animals. Thus, in the double transgenics the antagonist was not able to eliminate the A1 induction caused by GM-CSF.



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  		Fig. 2. Bioactivity of the GM-CSF antagonist. A, RNAs extracted from the skin of nontransgenic animals (Lane 1), animals of line Tg1 expressing GM-CSF (Lane 2), animals of line TgAnt1 expressing the GM-CSF antagonist (Lane 3), and double transgenic animals Tg1 x TgAnt1 (Lane 4) were gel separated, blotted, and hybridized with a probe for A1 and rehybridized with a probe for GAPDH. Levels of A1 expression were equally induced in Tg1 and Tg1 x TgAnt1 mice. B, immunofluorescence staining of epidermal ear sheets using a monoclonal LC-specific antibody (NLDC145). The frequency of LCs is increased in transgenics overexpressing GM-CSF (Tg1) in comparison with nontransgenics and animals expressing the antagonist (TgAnt1) but not in double transgenics (Tg1 x TgAnt1). Bars, 200 µm. C, the rate of S-phase keratinocytes in the basal layer of the epidermis of nontransgenic controls, agonist transgenics (Tg1), and antagonist transgenics (TgAnt1), as well as double transgenics (Tg1 x TgAnt1) was determined by BrdUrd labeling experiments. Note the significant reduction in the rate of S-phase nuclei in double transgenic animals versus agonist transgenics. Bars, SD. D, serum levels for IFN-{gamma} were determined 7 h after injection (i.p.) of 100 µg of LPS by ELISA. A significant reduction of IFN-{gamma} was observed in both transgenic lines expressing the GM-CSF antagonist 7 h after acute phase induction (P < 0.05). A minimum of nine animals/group were used for this experiment. Nontransgenic littermates served as controls.

 
(b) Mice epidermally overexpressing GM-CSF exhibit accumulation of LCs as determined by NLDC145 staining in comparison with the nontransgenic controls and antagonist transgenics (Fig. 2BCitation ; nontransgenic 1286 LCs/mm2, Tg1–3871 LCs/mm2, and TgAnt1–1586 LCs/mm2). However, in double transgenics, the antagonist was able to reduce the GM-CSF-dependent LC accumulation (Fig. 2BCitation ; double transgenics, 1265 LCs/mm2).

(c) The GM-CSF antagonist was able to suppress GM-CSF-mediated epidermal hyperproliferation in the double transgenic animals (Fig. 2C)Citation , whereas both nontransgenic mice and animals expressing GM-CSF antagonist exhibit ~2 labeled nuclei per 100 total cells. This frequency is significantly higher in GM-CSF-overexpressing mice (8) . However, in double transgenic animals, the proliferative index is reduced significantly and is comparable with that of nontransgenic controls and antagonist transgenics (Fig. 2C)Citation .

(d) The rise of IFN-{gamma} serum levels after acute phase induction, which is reduced in GM-CSF knock-out mice, was analyzed. There were no significant differences in the induction of TNF-{alpha} between antagonist transgenic and nontransgenic animals 1 h after LPS injection (data not shown). However, there was a significant reduction in the IFN-{gamma} production 7 h after induction of acute phase in antagonist transgenics (Fig. 2D)Citation . This decrease in serum IFN-{gamma} levels was shown in both transgenic lines TgAnt1 and TgAnt2 and demonstrates systemic effects of the GM-CSF antagonist.

Skin Carcinogenesis
Single Treatment with a Tumor Promoter.
The kinetics of GM-CSF levels in the skin of agonist transgenic and nontransgenic animals for up to 7 days were investigated after a single topical application of TPA. The protein levels remained elevated in wild-type animals until 24–48 h (<20 pg/g skin) after TPA treatment and declined thereafter below the threshold of detection (Fig. 3A)Citation . In the agonist transgenic line Tg1, GM-CSF levels rose from the basal level of 37.79 pg/g skin to 101.19 pg/g skin after 24 h, followed by a continuous decrease back to basal level at 96 h after treatment (Fig. 3A)Citation . Similar kinetics were observed in the second agonist transgenic line Tg2 (data not shown). Transgenic animals expressing the GM-CSF antagonist could not be analyzed in this setting because the high levels of antagonist in the skin (>100 ng/g skin in line TgAnt1 and >400 ng/g skin in line TgAnt2) were constitutively maintained, thereby masking the 3-orders-of-magnitude lower levels of endogenous GM-CSF (data not shown).



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  		Fig. 3. Tissue levels (skin) of GM-CSF and BrdUrd labeling indices in interfollicular epidermis of transgenic and nontransgenic animals at different time points after a single topical TPA application. A, GM-CSF levels in the skin as determined by ELISA in animals overexpressing GM-CSF agonist (Tg1, {triangleup}) and control mice ({circ}) rise within 24 h after TPA application and return to normal within 96 h after application. Bars, SD. B, rates of S-phase nuclei in the basal layer of interfollicular epidermis after TPA treatment were determined by BrdUrd labeling for mice overexpressing the antagonist (TgAnt1, {square}) and for transgenics expressing GM-CSF (Tg1, {triangleup}), as well as for the appropriate nontransgenic groups ({circ}). Note the pronounced hyperproliferation in GM-CSF transgenics. Bars, SD.

 
The rate of S-phase nuclei in the basal layer of interfollicular epidermis was determined using BrdUrd labeling at different time points after TPA treatment. In nontransgenic animals, the number of mitotic cells peaked 24 h after TPA application ({approx}15% labeled nuclei) and declined back toward the basal level ({approx}2% labeled nuclei) by 7 days (Fig. 3B)Citation . Transgenic mice expressing the GM-CSF antagonist showed no significant differences in the frequencies of S-phase nuclei in comparison with nontransgenic animals (Fig. 3B)Citation . However, animals overexpressing the GM-CSF agonist exhibited significantly higher numbers of proliferating cells at all time points after TPA treatment (Fig. 3B)Citation . In these animals 24 h after TPA treatment, 21% (Tg1) and 26% (Tg2) of S-phase nuclei were counted in the basal epidermal layer (Fig. 3B)Citation . The rate of mitotic cells declined back toward the basal level during the course of the experiment but always remained higher in comparison with nontransgenic animals. These results demonstrate that TPA treatment of transgenic mice expressing GM-CSF (Tg1 and Tg2) induces a more pronounced epidermal hyperproliferation compared with lines expressing the GM-CSF antagonist (TgAnt1 and TgAnt2), which perform comparably with nontransgenics.

Two-Step Carcinogenesis Protocol.
Tumor initiation was performed by administration of DMBA on the dorsal skin, followed by promotion carried out with TPA twice weekly. After repeated exposure to TPA, benign tumors were observed in nontransgenic mice as early as 7 weeks after initiation. The number of benign tumors increased steadily and achieved a plateau after 25 weeks (Fig. 4, A and B)Citation . The number of benign tumors in all transgenic mice, agonist as well as antagonist transgenics, was more than doubled when compared with the appropriate nontransgenic control groups after 31 weeks of promotion (Fig. 4C)Citation . In addition, tumors showed an earlier onset in mice overexpressing the GM-CSF agonist at 4 weeks as compared with 7 weeks in nontransgenic controls (Fig. 4A)Citation .



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  		Fig. 4. Tumor induction in transgenic and nontransgenic mice. Animals were initiated by topical treatment with DMBA, followed by application of TPA twice weekly (two-step protocol). Representative kinetics of benign skin tumor formation after initiation are shown for GM-CSF transgenics (A, Tg2, {triangleup}) and GM-CSF antagonist transgenics (B, TgAnt2, {square}) as well as for the respective control group ({circ}). C, ratios of benign skin tumors in all transgenic lines versus respective littermate controls at 31 weeks after initiation. The number of carcinomas in mice overexpressing GM-CSF (D, Tg2, {triangleup}) and in transgenics expressing the GM-CSF antagonist (E, TgAnt1, {square}) were plotted versus time after initiation. F, ratios of carcinomas in all transgenic lines versus respective littermate controls at 40 weeks after initiation.

 
A slightly higher incidence of malignant tumors in the skin of transgenic mice (agonist as well as antagonist) was observed when compared with nontransgenic animals (Fig. 4, D and E)Citation . Nevertheless, in all transgenic lines the number of carcinomas did not increase proportionally to the number of papillomas (Fig. 4, C and F)Citation . As a consequence, the conversion rates in all transgenic lines were decreased in comparison with the appropriate control groups (Table 2)Citation . Taken together, these results demonstrate that an increase of bioactive GM-CSF (Tg1 and Tg2) leads to enhanced tumor development in transgenic mice. Surprisingly, a reduction of GM-CSF activity by overexpression of a competitive antagonist (TgAnt1 and TgAnt2) evoked an increased tumor incidence in comparison with control animals as well.


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  		Table 2 Tumor conversion frequency

 
One-Step Carcinogenesis Protocol.
To evaluate endogenous tumor promotion, transgenic and nontransgenic mice were initiated by a single application of DMBA. Mice expressing GM-CSF (Tg2) showed a very early onset of carcinoma appearance in week 12 in comparison with week 29 in the control group (Fig. 5A)Citation . The GM-CSF transgenic animals developed significantly more carcinomas as compared with the nontransgenic controls. In contrast, animals expressing the GM-CSF antagonist (TgAnt1) performed similarly to control animals, with a late onset and a low total number of carcinomas (Fig. 5B)Citation . The number of carcinomas in agonist transgenics (Tg1 and Tg2) were >4-fold higher as compared with the nontransgenic controls, whereas antagonist transgenics (TgAnt1 and TgAnt2) developed approximately the same number of carcinomas as the nontransgenics (Fig. 5C)Citation . These data suggest an endogenous tumor-promoting process in GM-CSF transgenics but not in antagonist transgenics.



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  		Fig. 5. Carcinoma induction in transgenic and nontransgenic mice after a single DMBA treatment (one-step protocol). Representative kinetics of carcinoma formation after initiation are shown for GM-CSF transgenics (A, Tg2, {triangleup}) and GM-CSF antagonist transgenics (B, TgAnt1, {square}), as well as for the respective control group ({circ}). C, skin carcinoma frequency in all transgenic lines related to the respective littermate controls at 50 weeks after initiation.

 
Transplantation of Tumor Cells
To characterize the mechanisms underlying the different tumor responses in the one-step protocol, we performed transplantation experiments with highly tumorigenic but poorly immunogenic B16 melanoma cells as well as stably transfected B16 melanoma cells secreting the designer cytokine hyper-IL-6. After s.c. injection of untransfected parental B16 cells in GM-CSF agonist transgenic (Tg1 and Tg2) and nontransgenic mice, tumors developed after 2 weeks with a medium weight of 2.26 g in transgenic mice and 2.11 g in control animals (Fig. 6A)Citation . In contrast, injection of hyper-IL-6-transfected B16 cells did not lead to tumor growth in agonist-transgenic or in nontransgenic animals (Fig. 6A)Citation . This observation clearly demonstrates that hyper-IL-6 is a potent inducer of antitumor immunity in this system.



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  		Fig. 6. Growth of hyper-IL-6-secreting B16, as well as parental B16 melanoma cells, in animals expressing GM-CSF agonist ({triangleup}), GM-CSF antagonist ({square}), and nontransgenic littermates ({circ}). A, s.c. injection of 105 hyper-IL-6 or parental B16 cells in transgenic mice expressing GM-CSF agonist (Tg1/2) and wild-type (wt) controls. B, s.c. injection of 105 hyper-IL-6-secreting or parental B16 cells in transgenic mice expressing GM-CSF antagonist (TgAnt1/2) and wild-type (wt) controls. Note that hyper-IL-6-secreting B16 cells can only grow in antagonist transgenics. Controls used in these experiments were age-matched littermates.

 
Transplantation of untransfected B16 melanoma cells in GM-CSF antagonist transgenics (TgAnt1 and TgAnt2) and nontransgenic mice also caused tumor development (Fig. 6B)Citation . Furthermore, injection of hyper-IL-6-transfected B16 cells in GM-CSF antagonist-expressing animals also evoked a significant increase in tumor weight (0.84 g) in comparison with nontransgenic mice (0.086 g). These data demonstrate that the rejection of hyper-IL-6-transfected B16 cells is dependent on the activity of GM-CSF and therefore is reduced in GM-CSF antagonist transgenics (Fig. 6B)Citation . Taken together, these results demonstrate that the immune response to tumor cells is severely compromised in GM-CSF antagonist transgenic mice (TgAnt1 and TgAnt2).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Characterization of GM-CSF Antagonist Transgenic Mice.
GM-CSF is released in the skin by keratinocytes under pathological conditions, such as after wounding, tumor promotion, or in inflammatory skin diseases (10 , 11 , 50) . A variety of cells are targets for this cytokine, including infiltrating cells (monocytes, macrophages, neutrophils, eosinophils, and mast cells) as well as resident skin cells (keratinocytes and epidermal LCs; Refs. 6 , 8 , and 51, 52, 53, 54 ). To investigate the role of keratinocyte-derived GM-CSF in vivo, we previously generated transgenic mice overexpressing murine GM-CSF under the control of a constitutively active keratin 5 gene promoter (8) . Here we describe the generation of transgenic mice expressing an antagonist for GM-CSF (38) in the suprabasal layers of the epidermis under the control of a keratin 10 gene promoter-based expression vector (43 , 55) .

These mice showed the expected cell type-specific expression of the antagonist in suprabasal interfollicular keratinocytes and displayed very high levels of GM-CSF antagonist in the skin. In fact, the antagonist levels in skin were at least three orders of magnitude above the GM-CSF levels observed in the keratin 5/GM-CSF transgenic mice or in control mice after TPA treatment (Ref. 8 and this study). Because of these extremely high tissue levels, a spillover into the circulation was observed that was not the case in the keratin 5/GM-CSF transgenics (8) . However, GM-CSF antagonist transgenics did not develop lung disorders analogous to alveolar proteinosis such as in GM-CSF or receptor ß-chain knock-out mice (39 , 40) , indicating that the systemic antagonist level was not sufficient to induce these pathologies in the lung.

Although in keratin 5/GM-CSF transgenic mice an increase in the number of proliferative and apoptotic keratinocytes, an accumulation of mast cells and LCs, and an induction of the bcl-2 family member A1 was observed (8) , no such changes were observed in animals expressing the antagonist. These findings indicate that the double mutant antagonist does not exert any agonistic activity in vivo, which is consistent with previous in vitro studies (38) .

Bioactivity of the GM-CSF Antagonist.
Thus far, bioactivity of the GM-CSF antagonist (K14E/E21K) has been shown only in vitro with GM-CSF-dependent NFS60 myeloid leukemia cells (38) . To assay bioactivity in vivo, we crossed keratin 5/GM-CSF transgenic mice (8) with keratin 10/GM-CSF antagonist transgenic mice. In double transgenics, the antagonist was not able to suppress GM-CSF-dependent A1 induction (8) . In contrast, the frequencies of S-phase nuclei and LCs were significantly reduced in the skin of double-transgenic animals in comparison with GM-CSF agonist-expressing mice. Thus, the antagonist is able to suppress GM-CSF-dependent keratinocyte hyperproliferation and LC accumulation in vivo (8) . We conclude that in GM-CSF agonist/antagonist double transgenics, many but not all activities of GM-CSF are blocked, indicating different pathways of intracellular signal transduction. This conclusion is supported by previous reports that the cytoplasmic membrane-proximal region is important for cells to progress into mitosis, whereas the distal region mediates a signaling pathway essential for antiapoptotic activity of GM-CSF (56 , 57) . Because the induction of endogenous GM-CSF expression during tumor promotion is much weaker when compared with GM-CSF expression in the keratin 5/GM-CSF transgenics, an even more effective blocking of GM-CSF activity in keratin 10/GM-CSF antagonist single transgenics during tumor promotion can be anticipated.

In contrast to the agonist transgenics (8) , transgene-derived protein builds high serum levels in the antagonist transgenics, raising the question of systemic effects. Indeed, we observed a significant reduction of the IFN-{gamma} response in GM-CSF antagonist transgenic mice in comparison with control mice after acute phase induction by LPS. In accordance with these findings, the rise in IFN-{gamma} levels is decreased in GM-CSF-deficient mice after LPS induction (58 , 59) .

Chemically Induced Skin Carcinogenesis and Tumor Cell Transplantation.
The cutaneous response after exposure to a single TPA application includes inflammation (60) and epidermal hyperproliferation (61) . The proliferative response was significantly more pronounced in mice expressing the agonist than in antagonist-expressing mice, which behaved like controls in this assay. This finding extends our previous data for untreated skin, where only in GM-CSF agonist transgenics was an increased epidermal mitotic index observed (8) . Despite these different responses in both agonist transgenics and antagonist transgenics, a significantly enhanced papilloma and dysplasia formation could be seen in a two-step carcinogenesis experiment consisting of initiation and promotion. By contrast, the incidence of carcinomas in both types of transgenics was only marginally higher as compared with controls.

In a single-step initiation protocol (35) , the increased frequency of tumors in GM-CSF transgenics in comparison with antagonist transgenics and nontransgenic animals proves that epidermal overexpression of GM-CSF but not of the GM-CSF antagonist constitutes an endogenous tumor promotion. This type of endogenous tumor promotion in conjunction with hyperproliferation has been described for other growth factors as well. It was shown that overexpression of TGF-{alpha} in the skin of transgenic mice leads to an increased number of papillomas by acting synergistically with TPA to enhance epidermal hyperproliferation (62, 63, 64, 65) .

Regarding the GM-CSF antagonist-expressing transgenics, the increase in tumor formation in the two-step carcinogenesis model is not attributable to an endogenous tumor promotion in the classical sense, because there is no enhanced hyperproliferation after TPA treatment nor an increase in tumor formation in the one-step protocol in these animals versus the controls. A possible explanation for the increased tumor incidence in GM-CSF antagonist transgenics during two-step skin carcinogenesis could be a compromised antitumor immune response in these animals. To test this hypothesis, we compared the ability of these mice to reject transplanted tumor cells to control mice and GM-CSF-expressing mice. Mice implanted with tumor cells engineered to express cytokines, such as IL-2 (66) , IFN-{gamma} (21) , TNF-{alpha} (67) , and GM-CSF (20) , can reject these tumor cells as well as the nontransduced parental tumor cells after a challenge. We injected highly tumorigenic B16 melanoma cells as well as B16 melanoma cells engineered to secret hyper-IL-6 (22 , 23) into transgenics overexpressing GM-CSF and in transgenics expressing the GM-CSF antagonist as well as in control animals: (a) all animals failed to effectively reject the parental B16 melanoma cells. Thus, the mere increase of GM-CSF activity in the skin of GM-CSF transgenics is not sufficient to significantly enhance the antitumor response in this experimental setting. This is consistent with previous reports demonstrating that tumor cell-derived cytokines are much more potent adjuvants in comparison with cytokines not derived from the tumor cells (20 , 68) ; and (b) as expected from the above-mentioned previous studies, hyper-IL-6-secreting B16 melanoma cells were effectively rejected in nontransgenics and GM-CSF agonist mice. However, this antitumor response was greatly diminished in GM-CSF antagonist transgenics. These data are consistent with experiments indicating that GM-CSF is important for the induction of antitumor immunity in B16 melanoma cell vaccination experiments (19 , 20) . Thus, the reduced antitumor immune response of the antagonist transgenics provides an explanation for the increased frequency of tumor formation in these animals during two-step skin carcinogenesis. This finding also raises the question of potential risks in the application of GM-CSF in patients (29, 30, 31, 32) .

The cellular mechanisms of the GM-CSF antagonist-mediated failure of antitumor immunity are speculative. However, it is well known that APCs are major targets of GM-CSF (26 , 27 , 69 , 70) . In addition, GM-CSF-mediated antitumor immunity depends on both CD4+ and CD8+ lymphocytes (19 , 20 , 25 , 71) . Thus, the induction of immunity against transfected B16 melanoma cells may be dependent on the ability of GM-CSF to stimulate APCs during the priming phase to process and present tumor antigens to both CD4+ and CD8+ lymphocytes (26, 27, 28) . Therefore, the blocking of endogenous GM-CSF activity in our antagonist transgenics may be responsible for an impaired LC function, leading to a failure in T-cell activation. We are currently in the process of evaluating LC functions in both GM-CSF and GM-CSF antagonist transgenics by sensibilization experiments in vivo and T-cell stimulation assays in vitro.


   ACKNOWLEDGMENTS
 
We thank Karin Nicol, Martina Protschka, Nicole Voltz, and Katharina Petmecky for excellent technical assistance and Professor Galle for constant encouragement and support.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant SFB432/B1 from the Deutsche Forschungsgemeinschaft and a grant from the Boehringer Ingelheim Foundation. Back

2 These two authors contributed equally to this work. Back

3 Present address: Institute of Biochemistry, University of Kiel, D-24098 Kiel, Germany. Back

4 To whom requests for reprints should be addressed, at Johannes Gutenberg-University, I. Medical Department, Obere Zahlbacher Strasse 63, D-55131 Mainz, Germany. Phone: (49)-6131-3933357; Fax: (49)-6131-3933364; E-mail: Blessing{at}mail.unimainz.de Back

5 The abbreviations used are: GM-CSF, granulocyte/macrophage-colony stimulating factor; APC, antigen-presenting cell; BrdUrd, 5-bromodeoxyuridine; DMBA, 7',12'-dimethylbenz[a]anthracene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; LC, Langerhans cell; TGF, transforming growth factor; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; LPS, lipopolysaccharide. Back

Received 8/31/00. Accepted 1/ 3/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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C. M. Gutschalk, C. C. Herold-Mende, N. E. Fusenig, and M. M. Mueller
Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Promote Malignant Growth of Cells from Head and Neck Squamous Cell Carcinomas In vivo
Cancer Res., August 15, 2006; 66(16): 8026 - 8036.
[Abstract] [Full Text] [PDF]