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Cell Growth & Differentiation Vol. 11, 111-121, February 2000
© 2000 American Association for Cancer Research

Articles

Epidermal Overexpression of Granulocyte-Macrophage Colony-Stimulating Factor Induces Both Keratinocyte Proliferation and Apoptosis1

Kai Breuhahn2, Amrit Mann2, Gabriele Müller, Arnd Wilhelmi, Peter Schirmacher, Alexander Enk and Manfred Blessing3

SFB-432, I. Medical Department [K. B., A. M., M. B.], Department of Dermatology [G. M., A. E.], Boehringer Ingelheim Research Group, SFB-311, I. Medical Department [A. W., M. B.], and Institute of Pathology [P. S.], Johannes Gutenberg University, D-55131 Mainz, Germany

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is released by keratinocytes in sizeable amounts only under pathological conditions, e.g., after topical application of a tumor promoter, in atopic dermatitis (AD), and after wounding. To study the biological function of this cytokine release, we generated transgenic mice that constitutively overexpress GM-CSF in the epidermis. An increase in the numbers of mast cells and Langerhans cells (LCs) in transgenics versus nontransgenic controls was observed but no severe inflammation. This is consistent with a central role of this cytokine in the development and maturation of LCs. Mitotic activity in the epidermis of transgenic mice was elevated, but epidermal thickness and differentiation were normal. Homeostasis is maintained by an increase of apoptosis in the epidermis. We describe the differential expression of regulators of apoptosis and discuss a potential mechanism for this novel proapoptotic activity of GM-CSF on keratinocytes. Both stimulation of proliferation and promotion of apoptosis are of great relevance to tumorigenesis. The latter may be a means of removing damaged cells after genotoxic stress or injury.

Introduction

GM-CSF4 is a cytokine regulating proliferation, differentiation, and survival of hematopoetic cells (1, 2, 3) . Besides hematopoetic cells, other cell types including keratinocytes have been identified, both as sources and as targets for GM-CSF (4, 5, 6, 7) . The release of this cytokine by keratinocytes is triggered by disturbances in skin homeostasis such as tumor promotion or wounding (4, 5, 6 , 8) . Because GM-CSF has been shown to stimulate keratinocyte proliferation as well as immune cell proliferation and maturation, it had been proposed to mediate both the proliferative burst in keratinocytes as well as inflammation after disturbances of the homeostasis in the skin. In previous studies, keratinocyte hyperplasia and hyperproliferation were induced by GM-CSF and accompanied by the induction of the stress keratin pair 6/16, indicative of the alternative or regenerative pathway of keratinocyte differentiation (5 , 9 , 10) . In the dermis, neutrophil and macrophage infiltration and fibrosis were observed (10) . However, because these studies involved the application of GM-CSF by injections or the infection with a replication-deficient adenoviral vector, it is not clear whether these effects resulted from GM-CSF alone or from synergistic effects with these secondary stimuli. Also, the significance of keratinocyte-derived GM-CSF could not be addressed in these studies because they involved either systemic levels of GM-CSF or intradermal expression of this cytokine (5 , 10) .

In regard to the skin, GM-CSF is also particularly important for the generation and maturation of epidermal LCs as well as the survival of macrophages (11 , 12) . In this context, a causal role of GM-CSF overproduction in the establishment and chronicity of lesions in AD patients has been discussed (7 , 12) . In addition to increased tissue levels of IL-3, IL-4, IL-5, IL-10, and IL-13, AD lesions display highly elevated levels of GM-CSF and an increase in dendritic cells as well as infiltration of T cells, macrophages, and monocytes (13) .

Besides proliferation and differentiation, apoptosis is another critical parameter affecting homeostasis and tumorigenesis. Apoptosis occurs at high frequencies during certain phases of the hair cycle in follicular cells as well as in interfollicular keratinocytes in response to genotoxic stress (14 , 15) . The latter may be a mechanism to remove genetically damaged cells and therefore prevent the formation of malignancies (14) . GM-CSF has been shown to prevent apoptosis in many immune cells. This activity is mediated through the induction of antiapoptotic factors like Bcl-2 via the ß-chain of the GM-CSF receptor (3 , 12 , 16) . However, there are no reports on the effects of GM-CSF on keratinocyte apoptosis in vivo.

Apoptosis is a complex event and is tightly regulated. Members of the Bcl-2 family of proteins are crucial factors regulating programmed cell death. Among those are the antiapoptotic factors Bcl-2, Bcl-xL, and A1 and the proapoptotic factors Bcl-xs, Bad, Bak, and Bax (17, 18, 19, 20) . Apoptosis is promoted or inhibited by the formation of specific homodimers or heterodimers between family members (17, 18, 19) . Thus, the balance between different members of the Bcl-2 family controls cell survival and cell death. Expression of Bcl-2 in the epidermis is restricted to the basal layer and hair follicles (21 , 22) . Conversely, Bcl-xL is predominantly seen in suprabasal layers, whereas Bax is weakly expressed in all layers (23 , 24) . Strong epidermal expression has been reported recently for Bak (25) . In addition to these intracellular regulators of programmed cell death, keratinocytes have also been shown to express Fas, a major receptor for apoptotic signals. Moreover, expression of Fas is strongly up-regulated in keratinocytes upon treatment with IFN-{gamma}. Thus, besides differential expression of members of the Bcl-2 family, regulation of Fas expression via IFN-{gamma} may be a major mechanism for controlling apoptosis in keratinocytes (26 , 27) .

To analyze the effect of constitutive release of GM-CSF by keratinocytes without additional stimuli on skin architecture as well as keratinocyte proliferation, differentiation, and apoptosis, we generated transgenic mice expressing GM-CSF in the skin under the control of a keratin 5-based expression vector. We observed an increase in the number of LCs and mast cells in the skin of these transgenics that is compatible with the proposed role of GM-CSF in the perpetuation and chronicity of AD lesions. However, no significant infiltration by other inflammatory cells, such as T cells or macrophages, was seen. Both mitotic and apoptotic frequencies were doubled for basal interfollicular keratinocytes in these mice, resulting in the maintenance of regular epidermal thickness and the standard pathway of keratinocyte differentiation. This novel proapoptotic activity of GM-CSF may be mediated through an induction of Fas by IFN-{gamma} and changes in the expression of Bcl-2 family members. Apoptosis induced by GM-CSF release from keratinocytes may be a means to eliminate damaged keratinocytes after genotoxic stress.

Results

Generation of Transgenic Mice.
Transgenic mice were generated by pronuclear injection of the keratin 5-based expression vector for GM-CSF (Fig. 1)Citation . Three independent transgenic founder animals with autosomal integration of the transgene were obtained. All were fertile and produced transgenic offspring at the expected Mendelian frequency. The resulting transgenic lines were designated according to the guidelines set by the Institute for Laboratory Animal Research (Washington, DC) as TgN(K5mGMCSF)1 Mbl, TgN(K5mGMCSF)2 Mbl, and TgN(K5mGMCSF)3 Mbl. For simplicity, these lines are being abbreviated hereafter as Tg1, Tg2, and Tg3, respectively. Animals of all three lines displayed no obvious gross abnormalities. The density of hair follicles and the outer appearance of skin and fur were normal. A deficit in total body weight of ~10% in comparison to controls was noted during the first 2–3 months of the life span. However, this difference vanished at later time points, and neither life expectancy nor fertility was compromised.



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  		Fig. 1. Expression vector for GM-CSF. A cDNA coding for murine GM-CSF was brought under the control of the bovine keratin 5 promoter. Downstream of the cDNA the splice and polyadenylation signals derived from the bovine keratin 5 gene were included. B, BamHI; E, EcoRI; S, SalI; X, XhoI.

 
Expression of the Transgene.
Tissue specificity of the expression of the transgene was determined by RT-PCR analysis using RNAs extracted from various tissues as templates. Expression of the GM-CSF transgene was demonstrated in the skin of both lines Tg1 and Tg2 (Fig. 2)Citation . Additionally, a weak signal was obtained in heart of line Tg2 (Fig. 2B)Citation . Expression of the transgene was undetectable by this method in kidney, lung including trachea, liver, brain, and spleen in both transgenic lines.



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  		Fig. 2. Expression of the transgene-derived RNA. RT-PCR analysis with total RNAs from kidney (Lanes 1), lung (Lanes 2), heart (Lanes 3), liver (Lanes 4), skin (Lanes 5), brain (Lanes 6), and spleen (Lanes 7) was performed. Lane 8 (A) shows genomic DNA from transgenic mice containing an intron sequence. Mice of line Tg1 (A) expressed the transgene only in the skin (A, Lane 5), whereas Tg2 transgenics (B) showed a signal in skin (B, Lane 5) and also in the heart (B, Lane 3). Nontransgenic mice exhibit no expression of transgene-specific transcripts (data not shown). BMP-6 was used as a control for the RT-PCR reaction (A and B).

 
Cell type-specific expression of the GM-CSF transgene in skin was verified by in situ hybridization experiments and immunohistochemistry on skin sections (Fig. 3)Citation . In situ hybridization experiments using a transgene-specific antisense cRNA probe revealed an accumulation of transgene-derived transcripts in interfollicular keratinocytes as well as follicular cells in the skin of transgenic neonates, whereas no specific signal was seen in skin sections from nontransgenic control neonates (Fig. 3, A and B)Citation . The corresponding sense cRNA probe produced no specific signals in skin sections of control mice or of transgenic mice (data not shown). By immunohistochemistry using a GM-CSF-specific antibody, no significant staining was observed in normal adult skin (Fig. 3C)Citation . By contrast, strong cytoplasmic staining was observed in interfollicular keratinocytes as well as follicular cells in the skin of transgenic adults (Fig. 3D)Citation . Control experiments with secondary antibody alone resulted in no staining in skin sections of control mice or of transgenic mice (data not shown).



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  		Fig. 3. In situ localization of transgene derived RNA (A and B) and protein (C and D). In situ hybridization experiments using a transgene-specific probe derived from the 3' untranslated region of the keratin 5 gene on skin sections from newborn wt (A) and transgenic mice (B; Tg1). Immunohistochemical staining of adult wt (C) and transgenic skin sections (D; Tg1) using a rat antimouse GM-CSF monoclonal antibody. Both GM-CSF mRNA and protein are localized in the cytoplasm of keratinocytes in the transgenic epidermis (B and D). No staining is observed in wt littermates (A and C). Bars, 100 µm.

 
Tissue concentrations of murine GM-CSF were compared between transgenic lines and nontransgenic controls by ELISA on extracts prepared from dorsal skin. Whereas GM-CSF concentrations in skin extracts from nontransgenic controls were below the level of detection, extracts prepared from transgenic skin revealed tissue concentrations for GM-CSF ranging from 42 pg/g of skin in line Tg1 to 182 pg/g skin in line Tg2 (Table 1)Citation . In both transgenic lines Tg1 and Tg2 as well as nontransgenic controls, serum and plasma levels for GM-CSF were below the threshold of detection for this method (Table 1)Citation .


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  		Table 1 Concentrations of GM-CSF in skin, serum, plasma, and supernatants of cultured keratinocytes

GM-CSF concentrations in skin, serum, and plasma of transgenic (Tg1, Tg2, and Tg3) and nontransgenic mice were determined by ELISA. Furthermore the concentrations of GM-CSF in the supernatants of cultured keratinocytes were determined.

 
Secretion of GM-CSF by keratinocytes was demonstrated in keratinocyte cultures from neonates. After 4 days of culture, supernatant from wt keratinocytes exhibited 9.36 pg of protein/ml, whereas supernatant from transgenic keratinocytes of line Tg1 showed 156.19 pg of protein/ml (Table 1)Citation .

Effects of GM-CSF Overexpression on Skin Architecture and Inflammation.
No gross abnormalities of hair follicle morphology and in numbers of hair follicles were seen in H&E-stained skin sections of transgenic adults. The thickness of the epidermis was unchanged (Fig. 4, A–D)Citation . However, there was an increased cellularity in the dermis of these transgenics (Fig. 4, A–D)Citation . Giemsa staining revealed an increase in mast cell numbers in the dermis of transgenics by a factor of approximately 2–3 (data not shown). These results were confirmed by immunostaining with an anti-CD117 (c-kit) antibody (Fig. 4, E and F)Citation . Mast cells had been described to be the only cell type expressing c-kit in the dermis (28) . Immunostaining with anti-Mac1, anti-CD4, and anti-CD8 antibodies revealed no prominent infiltrates of macrophages or T cells (data not shown). Thus, the increased cellularity in the dermis of transgenic adults is most likely attributable to an increased number of resident dermal cells, such as fibroblasts, and increased numbers of mast cells.



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  		Fig. 4. Skin architecture and morphology in wt and GM-CSF transgenic animals. By using H&E staining (A and B), an increase in dermal cellularity can be observed in transgenics (B; Tg1) as compared with the wt littermates (A), whereas the thickness of the epidermis is not affected (C and D). Immunostaining with anti-CD117 (c-kit) antibody revealed a 2–3-fold increase in mast cell numbers in transgenic mice (F; Tg2) in contrast to wt mice (E). Arrowheads, individual mast cells. Immunofluorescence staining of epidermal ear sheets using a monoclonal LC-specific antibody (NLDC145) showed a 3-fold increase in transgenic mice (H) as compared with wt animals (G). Bars: A and B, 200 µm; C and D, 50 µm; E and F, 200 µm; G and H, 200 µm.

 
Because GM-CSF had been described as a factor promoting generation and maturation of epidermal LCs, we prepared epidermal sheets from the ears of normal controls and transgenic mice of lines Tg1 and Tg2. Immunostaining with anti-NLDC145 antibody, a dendritic cell-specific marker (29 , 30) , revealed an increase in LCs in the epidermal sheets prepared from the two transgenic lines in comparison to nontransgenic controls (Fig. 4, G and H)Citation . Although it had been described previously that injection of GM-CSF in the mouse skin evokes inflammation, a robust inflammatory infiltrate was not seen in transgenic skin. However, a difference in the cytokine milieu was noted. Extracts from transgenic dorsal skin revealed an increase in the tissue concentration of IFN-{gamma} as compared with nontransgenic controls (Table 2)Citation . By contrast, IL-12 and IL-1ß levels were unchanged, pointing toward a nonactivated state of the epidermal LCs (Table 2)Citation .


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  		Table 2 Cytokine concentrations in skin

IFN-{gamma}, IL-12, and IL-1ß concentrations in skin extracts of wt and transgenic mice were determined by ELISA. An increase of IFN-{gamma} level was observed in transgenic mice as compared with wt animals. IL-1ß concentrations were high in skin extracts but revealed no significant differences between wt and transgenic animals. The mean values and SD from three to six animals are figured.

 
Effects of GM-CSF Overexpression on Proliferation, Differentiation, and Apoptosis of Keratinocytes.
GM-CSF had been reported to stimulate keratinocyte proliferation in culture (4 , 31) . To investigate whether GM-CSF promotes keratinocyte proliferation in vivo, we performed BrdUrd labeling experiments and determined the epidermal mitotic indices in two transgenic lines, Tg1 and Tg2, as well as in nontransgenic control littermates. To identify basal keratinocytes, we counterstained the specimens with an anti-keratin 5 antibody. BrdUrd labeling experiments revealed an increase in numbers of S-phase nuclei in interfollicular keratinocytes of GM-CSF overexpressing transgenic mice (Fig. 5, A and B)Citation . Whereas two S-phase nuclei per 100 total cells were found in the basal layer of control epidermis, four and six labeled nuclei per 100 total cells were found in the basal layers of mice of lines Tg1 and Tg2, respectively (Table 3)Citation .



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  		Fig. 5. Epidermal proliferation and apoptosis in the skin of adult transgenic mice (Tg1) as determined by BrdUrd labeling experiments (A and B) and TUNEL staining (C and D). S-phase nuclei in the basal layer of the epidermis of wt (A) and transgenic littermates (B) were analyzed and counted using fluorescence microscopy. Arrowheads, S-phase nuclei. Apoptosis in wt and transgenic animals was assessed in both control (C) and transgenic (D; Tg1) mice using the FITC-tagged TUNEL method. Arrowheads, apoptotic cells in the basal layer. A murine anti-keratin 14 antibody and a CyTM3-labeled secondary antibody were used for counterstaining the basal layer of the epidermis in both cases. Bars, 200 µm.

 

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  		Table 3 Proliferative and apoptotic frequencies

Proliferative and apoptotic frequencies in interfollicular keratinocytes of wt and transgenic littermates are shown. For the quantification of mitotic indices, at least 3000 cells in the basal layer of the epidermis were counted with a minimum of three animals from each line. For the determination of apoptotic frequencies, a minimum of 1600 basal cells with specimens from at least three animals were counted after TUNEL reaction and anti-keratin 14 staining (P < 0.05).

 
Apoptosis in the epidermis was analyzed by double labeling experiments using the TUNEL assay for the detection of apoptotic cells and an anti-keratin 5 staining for the identification of keratinocytes. An increase of stained apoptotic keratinocytes in the epidermis of transgenic mice versus nontransgenic controls was noticed (Fig. 5, C and D)Citation . For quantification, apoptotic keratinocytes only in the basal layer of interfollicular epidermis were counted because the normal process of keratinocyte differentiation that takes place in suprabasal epidermal layers bears some resemblance to certain apoptotic changes. We found that the frequency of apoptotic basal keratinocytes in normal interfollicular epidermis was 0.31 per 100 keratinocytes (Table 3)Citation . By contrast, this frequency is doubled in transgenic mice to 0.66 apoptotic keratinocytes per 100 keratinocytes (Table 3)Citation . The increase of apoptosis in keratinocytes was corroborated by double-label FACS analysis on epidermal cells stained with annexin-V and a LC-specific antibody (NLDC145) as well as an antibody to MHC class II (KH118) molecules (Fig. 6)Citation . There is an increase in transgenics versus nontransgenics of MHC class II-positive, nondendritic epidermal cells undergoing apoptosis (Fig. 6, B and C)Citation . These cells are most likely keratinocytes, because up-regulation of MHC class II molecules had been shown on keratinocytes from these transgenic mice.5



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  		Fig. 6. FACS analysis of epidermal cell suspensions. Isotype control is shown in the upper panels (A). Single-cell suspensions were treated with AnnexinV-FITC and KH118/MHC class II (B) as well as NLDC145/DEC205 (dendritic cell-related marker; C). The numbers of vital MHC class II+ (B, upper left) and vital DEC205+ (C, upper left) cells are increased in transgenics. Also, MHC class II+ (B, upper right) and DEC205- (C, lower right) cells undergoing apoptosis are more abundant in transgenics.

 
To see how this increase in the frequency of basal keratinocyte apoptosis is conveyed, we analyzed the expression of several genes relevant to apoptosis and cell survival by Northern blotting experiments using RNAs extracted from control skin and skin from mice of lines Tg1 and Tg2 (Fig. 7)Citation . To exclude the influence of the hair cycle on the expression of proapoptotic or antiapoptotic genes, RNA samples were prepared from age-matched animals. No changes were observed for the factors Bcl-2, Bcl-x, Bak, Bax, and Bad at the RNA level. However, a strong increase in transgenics versus nontransgenics was seen for the antiapoptotic factor A1 (Fig. 7)Citation . 18S and 28S RNA as well as ß-actin were used as loading controls. No differences were observed in intensity (data not shown).



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  		Fig. 7. Expression of mRNAs for several Bcl-2-related genes in wt, Tg1, and Tg2 animals. Northern blots were prepared by loading 25 µg/lane of total RNA isolated from the skin. Although no changes in expression of Bak, Bad, Bax, and Bcl-x were seen, an up-regulation of the antiapoptotic factor A1 was observed in transgenic skin. Bcl-2 showed a weak but steady transcriptional product at ~6.5 kb.

 
Analysis of keratinocyte differentiation markers as keratins 1, 10, and 14 and especially of the hyperproliferative marker keratin 6 in interfollicular epidermis by immunohistochemistry and Western blotting revealed no differences in expression of these markers between transgenic epidermis and nontransgenic control epidermis (data not shown).

Discussion

GM-CSF is a cytokine that is normally not released by keratinocytes. However, under pathological conditions such as after wounding, during tumor promotion, or in inflammatory skin diseases such as AD, keratinocytes secrete GM-CSF (4 , 7 , 8 , 31) . GM-CSF has been described to affect a broad spectrum of different cell types including T cells, monocytes, neutrophils, eosinophils, epidermal LCs, as well as melanocytes and keratinocytes (4 , 5 , 11 , 12 , 32 , 33) . Therefore, the contribution of keratinocyte-derived GM-CSF to these pathological conditions appears to be complex. To investigate the effect of GM-CSF release by keratinocytes in an in vivo situation, we generated transgenic mice overexpressing murine GM-CSF in the epidermis under the control of a constitutively active keratin 5 promoter-based expression vector (34) .

Phenotype and Skin Architecture of Keratin 5/GM-CSF Transgenic Mice.
The transgenic mice were healthy and fertile and displayed elevated levels of GM-CSF in the skin. Secretion of this cytokine by keratinocytes was demonstrated in culture experiments. The bioactivity of keratinocyte-derived GM-CSF had been demonstrated previously (4) . These transgenics did not develop accumulations of macrophages, blindness, or a fatal syndrome of tissue damage that had been described previously in transgenic mice carrying multiple copies of the GM-CSF gene (35) . This difference probably results from the local restriction of GM-CSF overproduction in our transgenics, which do not show elevated serum levels for this cytokine. By contrast, the mice carrying multiple copies of the GM-CSF gene do exhibit measurable levels of GM-CSF in the serum, peritoneal cavities, and pleural cavities, thereby evoking systemic effects (36 , 37) . Histological examination as well as immunohistochemistry of skin sections revealed no gross abnormalities in the skin of our transgenics. We did not observe significant epidermal thickening or hyperplasia. There was an increase in epidermal LC numbers and dermal mast cells. The former confirms the bioactivity of keratinocyte-derived GM-CSF because it is consistent with the already described effects of GM-CSF on the generation and maturation of cells belonging to the dendritic cell lineages and may also contribute to the chronicity of lesions in AD (2 , 7 , 11 , 38) . An increase in mast cell numbers has also been described in lesional skin of AD patients (39) . However, because the GM-CSF transgenic mice do not spontaneously develop AD-like lesions, other factors must be crucial to the development of this disease.

Besides the increased number of mast cells, no prominent inflammatory reaction was observed. There was no infiltration of T cells, macrophages, neutrophils, or eosinophils in the dermal compartment of our transgenics. Consistent with these findings, there was no elevation of tissue levels for IL-1ß, IL-2, IL-4, and IL-12 in the skin of these transgenics, and only a moderate increase in IFN-{gamma} levels was observed. This is apparently in contrast to experiments performed previously that aimed at the elucidation of the role of GM-CSF in skin homeostasis in mice (6 , 8 , 10) . In two studies, the effect of both the application of recombinant GM-CSF and the application of a neutralizing antibody to GM-CSF prior to the treatment with tumor promoting agents had been evaluated. The systemic application of a neutralizing antibody to GM-CSF prior to the treatment with a tumor promoter greatly reduced the inflammatory response to the tumor promoter in the skin (6 , 8) . By contrast, systemic application of GM-CSF enhanced the effect of tumor promoters on keratinocyte proliferation and dermal leukocyte infiltration (6) . Similarly, the intradermal transgenic expression of GM-CSF using a replication-deficient adenovirus vehicle resulted in massive infiltration of neutrophils and macrophages as well as epidermal hyperplasia (40) .

To understand the different results between these studies as compared with our model, we must take into account the different modes of application for GM-CSF. Although the previous studies used different modes of application for GM-CSF, leading to highly elevated dermal or systemic levels of GM-CSF, the model described in our study exclusively addresses the question for the biological significance of keratinocyte-derived GM-CSF in vivo (6 , 8 , 10) . Although it had been shown previously that growth factors secreted by keratinocytes in transgenic animals can traverse the basal lamina and may be transported to the bloodstream (41 , 42) , this is not the case for GM-CSF in our transgenic mouse model. Tissue concentration of GM-CSF in the skin of our transgenics is constitutively elevated 2–10-fold over the respective concentrations measured in wt skin upon activation by 12-O-tetradecanoylphorbol-13-acetate (these authors; data not shown). Therefore, we conclude that keratinocyte-derived GM-CSF usually does not enter the bloodstream in detectable amounts and does not per se evoke massive inflammatory responses in the skin in the absence of additional stimuli.

GM-CSF and Keratinocyte Proliferation, Differentiation, and Apoptosis.
GM-CSF had been described as a stimulator of epidermal cell proliferation both in vitro and in vivo (5 , 9 , 10) . Consistent with these findings, we found an increase of mitotic indices in the interfollicular epidermis of the keratin 5/GM-CSF transgenics by a factor of approximately 2–3. However, the proliferating cells were still restricted to the basal layer of the epidermis, and we observed neither para- or hyperkeratosis nor pronounced hyperplasia. Also, the normal program of keratinocyte differentiation was not altered in these mice, which is reflected by the normal pattern of expression of keratin 14 and keratins 1 and 10 as well as the absence of keratin 6 in interfollicular epidermis. Again, these latter findings are apparently in contradiction with previous reports linking GM-CSF overproduction in the skin to hyperplasia, acanthosis, as well as the implementation of the alternative pathway of keratinocyte differentiation, as demonstrated by the induction of keratin 16 in interfollicular epidermis (5 , 9 , 10) .

The resolution of these discrepancies must be linked to the mode of application for GM-CSF, which involves additional stimuli in all previous studies, e.g., injection or adenoviral gene expression. However, our model analyzes the significance of the constitutive presence of keratinocyte-derived GM-CSF and without additional stimuli. Therefore, we conclude that GM-CSF released by keratinocytes is an autocrine stimulator of keratinocyte proliferation but does not alone evoke epidermal hyperplasia or acanthosis nor the implementation of the alternative program of keratinocyte differentiation that is characterized by the induction of keratins 6 and 16.

Because hyperproliferation in interfollicular keratinocytes appeared to be in contradiction to the lack of significant alterations of epidermal architecture and thickness, we wondered about the fate of the epidermal cells. Therefore, we analyzed apoptosis by the TUNEL assay and FACS analysis for annexin-V. Both the methods revealed an increase in numbers of apoptotic keratinocytes in the epidermis of the keratin 5/GM-CSF transgenic mice. Thus, we propose that standard epidermal thickness is maintained in the transgenic animals by increased rates of apoptosis. However, shortened transit times of keratinocytes may also contribute to the maintenance of normal homeostasis (43 , 44) .

Bcl-2 family members, important regulators of apoptosis (17, 18, 19 , 45 , 46) , are differentially expressed during the process of terminal differentiation of keratinocytes. Substantial epidermal expression of Bcl-2, Bcl-x, Bad, Bax, and Bak in normal epidermis had been demonstrated before (21, 22, 23, 24, 25) . However, we do not see prominent changes in the levels of expression of these factors in the skin of our keratin 5/GM-CSF transgenic mice. Instead, we see a massive induction of a novel regulator of programmed cell death, A1, which is hardly detectable in normal skin. Therefore, we conclude that the increase in apoptotic frequency in interfollicular epidermis of our transgenic mice does not result from a modulation of expression of the abundant regulators such as Bcl-2, Bcl-x, Bad, Bax, and Bak. Also, the induction of the normally infrequent Bcl-2 family member A1 is not an explanation for the increased frequency of apoptosis, because A1 has been described solely as antiapoptotic (47) . Therefore, a dominant mechanism must be involved rendering keratinocytes in our GM-CSF transgenics more susceptible to apoptosis. Because GM-CSF has been reported to prevent apoptosis in a variety of cell types (3 , 12 , 16) , this apoptosis-promoting mechanism in our transgenics may be indirect via the moderate elevation of IFN-{gamma} tissue levels observed in our transgenics. It had been shown that IFN-{gamma} can promote apoptosis in cultured keratinocytes by inducing the expression of predesquamin and Fas (26 , 27 , 48) . Whether this is the case in our transgenics is currently under investigation. We suggest that GM-CSF may play an important role in epithelial carcinogenesis because of its immunomodulating effect in the cutaneous system on one hand and because of its effects on cell proliferation, differentiation, and programmed cell death of keratinocytes after genotoxic stress.

Materials and Methods

Generation of Transgenic Mice.
The cDNA coding for murine GM-CSF (49) was inserted by blunt-end ligation into the end-filled SalI site of the keratin 5 expression vector (34 , 50) . The expression cassette was excised by KpnI digestion. The 10-kb construct-DNA was gel purified and used for pronuclear microinjection of fertilized eggs of mice of strain FVB/N, essentially as described (51) . Offspring were biopsied at ears or tails and analyzed by PCR using a bovine keratin 5-specific primer (5'-ATG AAG ACA GCG TTT GCA CCC-3'; position 1122–1142; GenBank accession no. Z32746; Ref. 52 ) 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. 49 ) or by Southern blot analysis using a bovine keratin 5-specific probe (52) .

Keratinocyte Culture.
Keratinocytes were derived from newborn mice and cultured as described previously (34) . Aliquots of the supernatants from 4-day-old cultures were used for ELISA to determine cytokine production.

RT-PCR Analysis.
RT-PCR analysis was performed using total cellular RNA from kidney, lung, heart, liver, dorsal skin, brain, and spleen. Total RNA was isolated using peqGOLD RNA Pure (Peqlab Biotechnologie, Erlangen, Germany). Reverse transcription was accomplished using a RT-PCR kit according to the manufacturer’s instructions (Stratagene, La Jolla, CA). Two hundred ng of total RNA were used as template for each assay, and PCR was performed using Taq-DNA polymerase (Boehringer Mannheim, Mannheim, Germany) in 35 cycles (94°C for 60 s; 61°C for 60 s; and 72°C for 60 s). Primer pairs for both endogenous BMP-6-specific RNA and transgene-derived RNA were flanking introns. Endogenous murine mRNA for BMP-6 was used as a standard as described previously (34 , 53) . The primer pair used to detect transgene-derived mRNA consisted of primer GM-exo-1 (5'-CTA CCA GAC ATA CTG CCC CCC-3'; position 222–242; GenBank accession no. X05906; Ref. 49 ), specific for the murine GM-CSF cDNA, and of the reverse primer GM-exo-2 (5'-GAG TAG AAG CTG CTA CTG CCG-3'; position 384–363; GenBank accession no. K03535) specific for the bovine keratin 5 splice site, as deduced from the sequence of the corresponding cDNA (54) . The primers produced a 364-bp diagnostic band at an annealing temperature of 60°C when cDNAs from appropriate transgenic tissues were used as templates.

In Situ Hybridization.
The transgene-specific cDNA probe derived from the splice and polyadenylation site of the bovine keratin 5 gene was generated by PCR. The primer pair consisted of primer K5-1 (5'-tata gaattc AGC AAG TTA CTG CCT CCC-3'; position 558–576; GenBank accession no. K03535) and of primer K5-2 (5'-tata aagctt ACC AGG CTA GAG ACT GGG-3'; position 736–717; GenBank accession no. K03535; Ref. 55 ), with EcoRI and HindIII restriction sites added at the 5' end. Fragments were cloned into pGEM-4Z (Promega Corp., Heidelberg, Germany), and digoxigenin-labeled sense and antisense RNAs were generated using in vitro transcription with SP6- and T7-RNA polymerases according to the supplier’s protocol (Boehringer Mannheim). The preparation of skin cryosections and stainings were performed as described previously (56) .

BrdUrd Labeling.
The number of nuclei in S-phase of the cell cycle was determined by BrdUrd labeling experiments using the In Situ Cell Proliferation Kit, FLUOS (Boehringer Mannheim). Mice received injections of 30 µg of BrdUrd i.p. per gram of body weight and were sacrificed after a labeling period of 2 h. Fixation and processing of the samples were performed according to the manufacturer’s instructions. The ratio of labeled nuclei in the basal layer of interfollicular epidermis to unlabeled cells was determined. For each experiment, values were obtained from at least three age- and sex-matched animals.

Cytokine Determinations.
Blood samples were obtained by cardiac puncture using 22-gauge needles. One half of the blood was mixed with 50 µl of 0.5 M EDTA for plasma preparation, and the other half was left at room temperature for 2 h for serum collection. After centrifugation at 6000 rpm (Sorvall; FA-Micro) for 10 min, the supernatants were used for ELISA. For skin extracts, mice were sacrificed, and 2-cm2 skin samples were taken from the shaved mid-dorsum. The tissue was pulverized under liquid nitrogen, weighed, and suspended in 600 µl of PBS. After the addition of 1 mg of protease inhibitor phenylmethylsulfonyl fluoride (Sigma Chemical Co., Deisenhofen, Germany), the samples were mixed intensively and centrifuged for 30 min with 12,000 rpm (Sorvall; FA-Micro) at 4°C. The supernatants were used for ELISA. IFN-{gamma}, IL-2, IL-4, IL-12, IL-1ß, and GM-CSF levels in serum, plasma, and skin extracts were evaluated using cytokine-specific ELISA kits (R&D Systems, Inc., Minneapolis, MN), according to the manufacturer’s instructions.

Immunohistology.
Mouse skin samples from age- and sex-matched animals were embedded in tissue freezing medium (Jung; Leica-Instruments, Nussloch, Germany), frozen immediately in liquid nitrogen, and stored at -80°C. Cryosections were cut at 5-µm thickness and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature or with acetone at 4°C for 15 min. After preblocking with 1% BSA, 5% serum, 0.5% Tween 20, and 100 mM MgCl2 in 10 mM Tris-HCl (pH 7.4) for 30 min at room temperature, the primary antibody was applied in the blocking solution at 4°C overnight. The secondary antibody incubation was performed at 37°C for 60 min. Signal detection was carried out using NBT/X-Phosphate or Fast Red (Boehringer Mannheim) as substrate. Endogenous alkaline phosphatase activity was inhibited by addition of 1 mM levamisol to the substrate solution. For fluorescence analysis, secondary antibodies conjugated with FITC and CyTM3 were used.

For the preparation of epidermal ear sheets and immunostaining, mice were sacrificed, and ears were removed. After dividing the ear in dorsal and ventral halves, the s.c. fat and cartilage were removed with the help of tweezers. The ear halves were incubated in 5 ml of 0.5 M NH4SCN solution in 0.1 M Na2HPO4, 0.1 M KH2PO4 buffer (pH 6.8) for 20 min at 37°C. Epidermis was separated from dermis and washed carefully in PBS. Fixation was carried out in ice-cold acetone for 40–45 min. After gently washing three times in PBS, the epidermal sheets were incubated with the primary antibody for 1 h at 4°C. After washing three times in PBS, the secondary antibody was added for 1 h at 4°C. The epidermis was spread out on a glass slide and examined under a fluorescence microscope (57) .

Preparation of Epidermal Cell Suspensions for FACS Analysis.
Mice were sacrificed, and ears were removed. After disinfection with 70% ethanol, ears were split into dorsal and ventral halves using fine tweezers. After removal of s.c. fat and cartilage, ear halves were incubated in 0.5% Trypsin/0.2 mM EDTA for 20 min at 37°C. The digestion reaction was stopped by the addition of excess FCS. The epidermis was removed from the dermis, and single-cell suspension was obtained by pressing the epidermis through a nylon mesh (58) .

Flow Cytometric Analysis.
After washing the cells twice with PBS, indirect immunofluorescence staining was performed. The cells were incubated with each primary monoclonal antibody diluted to the optimal concentration for immunostaining at 4°C for 20 min. After two washes with cold medium (PBS, 2% FCS, and 0.5 mM EDTA), cells were incubated with phycoerythrin- and FITC-conjugated secondary antibodies at 4°C for 20 min and washed twice in PBS. About 5,000–10,000 cells were collected by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). Debris and dead cells were gated out, and live cells were plotted using the FACScan Lysis II software (Becton Dickinson).

Antibodies.
The following antibodies at the mentioned dilutions were used for immunohistochemistry in this study: rat monoclonal antibody CD11b (1:200; PharMingen), CD4 (1:50; PharMingen; FITC-conjugated), CD8a (1:50; PharMingen; FITC-conjugated), DEC205 (NLDC145, BMA), CD117 (1:50; PharMingen), cytokeratin 10 (1:1600; Sigma), GM-CSF (1:50; PharMingen), and rabbit polyclonal antibody against murine cytokeratin 6 (59) and cytokeratin 14 (34) .

Secondary antibodies used in different dilutions in this study were CyTM3-conjugated donkey antimouse IgG (Dianova), CyTM3-conjugated goat antirat IgG (Dianova), AP-conjugated rabbit antirat IgG (Sigma), and AP-conjugated sheep antirabbit IgG (Boehringer Mannheim).

Antibodies used for flow cytometric analysis in this study were as follows: mouse monoclonal antibody against murine MHC class II (KH118; I-Aq; PharMingen), DEC205 (NLDC145, BMA), and AnnexinV-FITC (PharMingen). Isotype-matched controls were obtained from PharMingen (San Diego, CA).

Quantification of Apoptosis.
Detection and quantification of programmed cell death in the epidermis were performed using In Situ Cell Death Detection Kit, FLUOS (Boehringer Mannheim). Fresh cryosections from different mouse lines were treated as recommended by the supplier, counterstained for cytokeratin 14, and analyzed by fluorescence microscopy. The number of apoptotic cells in the basal epidermal layer was determined and related to 100 total basal cell nuclei (4',6-diamidino-2-phenylindole). The number of nuclei per visualized field was calculated by an average value obtained from 50 fields. Nuclei were counted from at least three age- and sex-matched animals of each line.

Histology.
For histopathological diagnosis, skin specimens were fixed overnight in neutral buffered 4% formalin, processed for paraffin embedding, sectioned, and stained routinely with H&E. For mast cell detection, skin cryosections were stained with May-Grünwald/Giemsa stain. For quantification of mast cells, at least three age-matched animals of each line were analyzed.

Northern Blot Analysis.
Total RNA from wt and transgenic skin was isolated using peqGOLD RNA Pure (Peqlab Biotechnologie). Twenty-five µg of RNA were electrophoresed on 1% agarose formaldehyde gels and subsequently blotted onto nylon membranes (Hybond N, Amersham, Braunschweig, Germany). After hybridization with 32P-labeled cDNA probes, the filters were processed at high stringency as described (60) .

The probes were generated by PCR (35 cycles; 94°C for 30s; 58°C for 45 s; and 72°C for 55 s) with spleen cDNA serving as template and using the following primer pairs: A1 (A1–1: 5'-GCC TCC AGA TAT GAT TAG GG-3'; position 28–48; GenBank accession no. L16462) and (A1–2: 5'-CTG ATA ACC ATT CTC GTG GG-3'; position 698–678; GenBank accession no. L16462; Ref. 47 ); ß-actin (act-1: 5'-GTG GGC CGC TCT AGG CAC CA-3'; position 183–203; GenBank accession no. X03672) and (act-2: 5'-CTG TGC CCA TCT ACG AGG GCT A-3'; position 578–565; GenBank accession no. X03672; Ref. 61 ); Bad (Bad-1: 5'-CAG GGA GGT GTC ATT AAC CC-3'; position 378–398; GenBank accession no. L37296) and (Bad-2: 5'-CCA GGA CTG GAT AAT GCG CG-3'; position 1041–1021; GenBank accession no. L37296; Ref. 17 ); Bak (Bak-1: 5'-ATT CAG GTG ACA AGT GAC GG-3'; position 212–232; GenBank accession no. Y13231) and (Bak-2: 5'-GAG AGA GGT TTA GTC CAG CC-3'; position 940–920; GenBank accession no. Y13231; Ref. 62 ); Bax (Bax-1: 5'-ATG GAC GGG TCC GGG GAG CA-3'; position 1–21; GenBank accession no. L22472) and (Bax-4: 5'-CTT CTT CCA GAT GGT GAG CG-3'; position 571–551; GenBank accession no. L22472; Ref. 63 ); Bcl-2 (Bcl2–1: 5'-AGA ACA GGG TAT GAT AAC CG-3'; position 1836–1856; GenBank accession no. M16506) and (Bcl2–2: 5'-TAT CCT GGA TCC AGG TGT GC-3'; position 2386–2366; GenBank accession no. M16506; Ref. 64 ); Bcl-x (Bcl-x-1: 5'-GCT AAA CAC AGA GCA GAC CC-3'; position 166–186; GenBank accession no. L35049) and (Bcl-x-2: 5'-GAG ATC CAC AAA AGT GTC CC-3'; position 825–805; GenBank accession no. L35049; Ref. 65 ). All PCR products were verified by sequence analysis.

Acknowledgments

We are indebted to Karin Nicol and Martina Protschka for excellent technical assistance and Prof. Meyer zum Büschenfelde and Prof. Galle for constant encouragement and support. We thank R. Kastelein (DNAX) for providing us with a cDNA coding for murine GM-CSF.

Footnotes

1 This work was funded by Grant SFB-432/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 To whom requests for reprints should be addressed, at Johannes Gutenberg University, SFB-311, I. Medical Department, Obere Zahlbacher Strasse 63, D-55131 Mainz, Germany. Phone: 49-6131-3933357; Fax: 49-6131-3933364; E-mail: Blessing{at}mail.uni-mainz.de Back

4 The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; LC, Langerhans cell; AD, atopic dermatitis; IL, interleukin; RT-PCR, reverse transcription-PCR; wt, wild type; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling; FACS, fluorescence-activated cell sorter; BMP, bone morphogenic protein. Back

5 G. Müller, M. Blessing, A. Mann, K. Breuhahn, K. Steinbrink, C. Szalma, J. Bergmann, J. Knop, and A. E. Enk. Mice overexpressing GM-CSF in skin are resistant to tolerance induction by UV-light or low-zone application of contact allergens, manuscript in preparation. Back

Received for publication 9/15/99. Revision received 12/ 3/99. Accepted for publication 1/ 5/00.

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Up- and Down-Regulation of Granulocyte/Macrophage-Colony Stimulating Factor Activity in Murine Skin Increase Susceptibility to Skin Carcinogenesis by Independent Mechanisms
Cancer Res., March 1, 2001; 61(5): 2311 - 2319.
[Abstract] [Full Text]

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M Li, H Chiba, X Warot, N Messaddeq, C Gerard, P Chambon, and D Metzger
RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations
Development, January 3, 2001; 128(5): 675 - 688.
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