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The Journal of Immunology, 2003, 170: 5697-5703.
Copyright © 2003 by The American Association of Immunologists

IL-1{alpha}, Innate Immunity, and Skin Carcinogenesis: The Effect of Constitutive Expression of IL-1{alpha} in Epidermis on Chemical Carcinogenesis1

Jo-Ellen Murphy, Romeo E. Morales2, Jordan Scott3 and Thomas S. Kupper4

Department of Dermatology, Harvard Skin Disease Research Center, Brigham and Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor promoters such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) are proinflammatory agents, and their mechanism of action in epithelial carcinogenesis has been linked to the release of IL-1{alpha} and the induction of chronic inflammation in skin. To test the role of IL-1{alpha} and inflammation in models of cutaneous carcinogenesis, we used our previously described FVB/N transgenic mice overexpressing 17-kDa IL-1{alpha} in the epidermis under the keratin 14 (K14) promoter. Strikingly, the K14/IL-1{alpha} mice were completely resistant to papilloma and carcinoma formation induced by a two-stage DMBA/TPA protocol, while littermate controls developed both tumor types. K14/IL-1{alpha} mice crossed with the highly sensitive TG.AC mice, constitutively expressing mutant Ha-Ras, also failed to develop papillomas or carcinomas. When the K14/IL-1{alpha} transgene was bred onto a recombinase-activating gene-2-deficient background, the resistance persisted, indicating that innate, but not acquired, mechanisms may be involved in the resistance to the initiation/promotion model. As an alternative approach, a complete carcinogenesis protocol using repetitive application of DMBA alone was applied. Surprisingly, although the IL-1{alpha} mice still did not develop papillomas, they did develop carcinomas de novo at an accelerated rate compared with controls. We conclude that constitutive IL-1{alpha} expression rendered FVB mice completely resistant to carcinomas that required evolution from prior papillomas, but facilitated carcinomas that did not evolve from papillomas, as in the complete carcinogenesis protocol. Thus, the role of IL-1{alpha} and, by extension that of other proinflammatory factors, in epithelial carcinogenesis are more complex than previously appreciated. These mice may provide a mechanism to investigate the validity of these models of human skin tumorigenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-1 is a pleiotropic cytokine that plays an important role in host defense and is thought to serve as a link between innate and acquired immune responses (1). While IL-1{beta} appears to be the dominant form of IL-1 produced by cells of hemopoetic origin, IL-1{alpha} predominates in epithelial cells, including keratinocytes of the skin (2). Functional IL-1{alpha} is stored in epidermal keratinocytes as a 31-kDa molecule that is biologically active and associates with the plasma membrane (3). IL-1{alpha} can also be cleaved to produce an active 17-kDa C-terminal polypeptide that is responsible for initiating the inflammatory cascade (4). A highly complex regulatory network of IL-1{alpha} agonists and antagonists has evolved in skin to ensure that the inflammatory response induced by IL-1 is tightly controlled (5). Previously, we tested the biological consequences of disrupting the natural balance of the IL-1 network by creating several transgenic (Tg)5 murine strains on the FVB/N background that individually overexpressed the IL-1 agonists or antagonists using the human keratin 14 (K14) promoter (6) to direct expression to the basal layer of the epidermis (7, 8, 9). As predicted, overexpression of 17-kDa IL-1{alpha} resulted in spontaneous inflammatory skin disease in the two lines of the K14/IL-1{alpha} Tg mice characterized (7). The Tg IL1.1 line had high level expression of IL-1{alpha} and showed spontaneous inflammatory skin disease, while the second line, Tg IL1.2, expressed lower levels of IL-1{alpha} and showed only subtle signs of inflammation. In both lines, constitutive up-regulation of IL-1-inducible cytokines and chemokines was demonstrated, confirming the potential for keratinocyte-derived IL-1{alpha} to induce downstream mediators of the inflammatory response (7). In the experiments described here we used the TgIL1.2 line to test the role of IL-1{alpha} in cutaneous chemical carcinogenesis.

One proven system for the study of skin carcinogenesis uses the application of the mutagen 7,12-dimethylbenzanthracene (DMBA), followed by repetitive application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). This two-stage protocol is predicated on the concepts of initiation and promotion (reviewed in Ref. 10). Initiation, as achieved by DMBA application, induces mutations in the Ha-ras gene at codon 61, which results in constitutive Ras activity (11, 12). Promotion with repetitive TPA treatment facilitates expression of the initiated phenotype by inducing proliferation and ultimately results in the early manifestation of benign papillomatous lesions (13). In the later phases of promotion, TPA-induced proliferation results in the accumulation of additional mutations, chromosomal deletions, and amplifications that strongly favor the progression of the benign papillomas to squamous cell carcinomas. An alternative protocol to generate skin carcinomas relies on the repetitive application of DMBA without TPA and is referred to as a complete carcinogenesis model (14). In this protocol, DMBA acts as both initiator and promoter, and carcinomas can form either from papillomas or de novo (15). This contrasts sharply with the two-stage model that reproducibly induces epidermal papillomas before carcinomas.

The tumor promoter TPA has been shown to induce phenotypic changes in epidermis similar to those observed in a cutaneous inflammatory response. The activity of TPA appears to directly mimic the natural response of the skin to injury, including the induction of both IL-1{alpha} release from keratinocyte stores and de novo IL-1 gene expression (16, 17). Thus, the cutaneous responses to TPA application including epidermal hyperplasia, release of growth factors and cytokines, and infiltration of leukocytes into the dermis, are similar to, and consistent with, a role for IL-1{alpha} as a primary cytokine in skin. This suggested that constitutive expression of IL-1{alpha} in skin would act as a promotional agent in the two-stage carcinogenesis protocol (18), potentially acting downstream of TPA to enhance tumor promotion. However, since IL-1{alpha} itself is known to have antitumor activity, we tested the sensitivity of K14/IL-1{alpha} Tg mice to cutaneous carcinogenesis to explore this controversy.

To our surprise, when the DMBA/TPA protocol was applied to K14/IL-1{alpha} mice both papilloma and carcinoma induction were completely abrogated. Cross-breeding of IL-1{alpha} mice to other Tg lines demonstrated that the protective effect was not dependent on the presence of T or B cells, nor was it due to a failure of the epidermis to respond to TPA. In contrast, K14/IL-1{alpha} mice were highly sensitive to the complete carcinogenesis protocol of repetitive DMBA alone. This treatment induced carcinomas without antecedent papilloma formation in K14/IL-1{alpha} mice, suggesting that the mechanism of abrogation in the two-stage protocol involved an inhibition of papillomagenesis, perhaps mediated by innate immune mechanisms. We propose a model in which constitutive IL-1{alpha} expression can suppress tumor formation that depends upon the rapid proliferation and expansion of initiated cells, as accomplished by application of TPA; however, IL-1’s antitumor effect can apparently be overcome by repetitive mutagenic events. This suggests that the outgrowth of mutated epithelial cells suffering from repeated genomic insults per se is not sensitive to IL-1 inhibition and may, in fact, be facilitated by this cytokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

DMBA and TPA were purchased from Sigma-Aldrich (St. Louis, MO), dissolved in acetone (Fisher Scientific, Pittsburgh, PA), and stored at -20°C as light-protected stock solutions that were diluted immediately before use.

Mice

As described previously (7), Tg mice overexpressing the 17-kDa cDNA of murine IL-1{alpha} (Tg1.2) from the human K14 promoter were generated in inbred FVB/N mice (19). Mice were genotyped by PCR using primers for the human K14 promoter and were grouped accordingly. The TG.AC strain, which expresses a mutated form of v-Ha-Ras from the {zeta}-globin promoter (20), was obtained from Charles River Laboratories (Wilmington MA). Recombinase-2-activating gene-deficient mice (RAG2 knockout) on the C57BL6 background (21) were backcrossed through 13 generations to FVB/N mice (B. Rich, unpublished observations) to produce a strain with the same permissive genetic FVB/N background as the K14/IL-1{alpha} Tg mice. Tg mice and their littermate controls were housed in a virus/Ab-free facility at Brigham and Women’s Hospital. All experimental protocols were approved by the Harvard medical area standing committee on animals.

Epidermal cultures were prepared from neonatal mouse skin by sequential digestion with dispase and trypsin and were cultured on collagen-coated dishes in medium containing 0.05 mM Ca2+ to allow maintenance of K14 expression, a technique used previously in the laboratory (8, 9).

Tumor induction

Heterozygous transgenic mice from the K14/IL-1{alpha} strain were bred to nontransgenic siblings or to FVB/N wild-type mice to produce a synchronized delivery of a sufficient number of animals for carcinogenesis experiments. Mice ranging in age from 6–8 wk were grouped and anesthetized with metafane to minimize epidermal trauma during shaving. The dorsal skin was shaved 24 h before a single topical application of 25 µg of DMBA in 200 µl of acetone. One week after initiation, 5 µg of TPA in 200 µl of acetone was applied weekly for 20 wk in early experiments (15). In subsequent DMBA/TPA experiments, a biweekly promotion schedule was used. Tumor induction experiments involving the preinitiated TG.AC strain used an initial dose of 5 and 50 µg of TPA in 100 µl applied to shaved left and right dorsal sides, respectively. Biweekly applications of TPA (5 µg/200 µl acetone) were applied across both dorsal sides. For complete carcinogenesis experiments, an initial DMBA dose of 25 µg in 200 µl of acetone was followed by weekly doses of 5 µg in the same volume. All animals were observed regularly and reshaven as needed until lesions appeared; at that time, weekly observations were recorded. The number of mice, with tumors (incidence) and number of tumors per mouse (average burden), were recorded. A lesion was scored as a papilloma if it was >2 mm and was present for 2 consecutive wk. Suspected carcinomas were defined as lesions with elevated margins and ulceration and were measured weekly until they reached a diameter of 1 cm, at which time the animal was sacrificed. Tumors were excised, fixed in 10% buffered formalin, and stained with H&E for evaluation by a pathologist. All animals entered into a study were included in the final data analysis.

To assay the hyperproliferative response to TPA, the back skin of IL-1{alpha} Tg mice and FVB control littermates was shaved, and 24 h later applications of TPA (5 µg each) or acetone were initiated. Treatment was repeated at 3- to 4-day intervals. One day after the last application, animals were given an i.p. injection of bromodeoxyuridine (BrdU; 200 µg/g) before sacrifice, and the skin was removed for processing. Tissue sections were immunostained using an anti-BrdU mAb (Roche, Indianapolis, IN) according to the manufacturer’s instructions. The number of BrdU-positive cells in the epidermis was averaged for five fields from five mice per group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Two-stage carcinogenesis: papilloma and carcinoma formation

The response to the two-stage chemical carcinogenesis protocol (15) was tested on heterozygous Tg K14/IL-1{alpha} mice and their FVB control littermates. Female mice were initiated by topical application of DMBA, followed by a weekly TPA promotion schedule. As early as 15 wk postinitiation, small papillomas were noted in ~20% of the FVB control animals, while overexpression of IL-1{alpha} delayed papilloma formation (Fig. 1A). By wk 20, papilloma incidence level reached 50% for FVB mice and achieved 100% by wk 32. The peak papilloma burden observed for FVB mice (5.6 ± 1.2 SE papillomas/mouse) was similar to previous reports using the weekly promotion schedule (15). Unexpectedly, a nearly complete inhibition of papilloma formation was observed in the K14/IL-1{alpha} Tg littermates. This phenomenon extended out to wk 45, a point at which FVB mice had a 100% incidence of papillomas. At 52 wk postinitiation, only one of 19 IL-1{alpha} Tg females had developed lesions. Of the few papillomas observed in the course of repeated experiments, all appeared very late in the experiment (wk 45 or after) and remained small (~2 mm) for the life of the affected animal. Histological analysis of one late-developing papilloma indicated a distinctive sebaceous character, in contrast to the histology of the typical keratinized papillomas generated by the two-stage protocol (data not shown).



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  		FIGURE 1. Papilloma and carcinoma formation induced by the two-stage protocol are dramatically inhibited in mice overexpressing 17-kDa IL-1{alpha}. Heterozygous females of the K14/IL-1{alpha} (•) Tg strain and their FVB littermate controls ({circ}) were initiated with 25 µg of DMBA and promoted once a week for 20 wk with 5 µg of TPA. A, The number of papillomas was recorded weekly, and the average papilloma burden and incidence (inset) were calculated as a function of weeks after initiation. B, Consistent with the observed inhibition of papillomagenesis, both carcinoma burden and incidence (inset) were inhibited in K14/IL-1{alpha} Tg mice in the DMBA-TPA carcinogenesis protocol. C, For male mice, DMBA initiation followed by a biweekly TPA promotion schedule resulted in a higher papilloma yield in FVB littermates ({circ}), but the resistance of K14/IL-1{alpha} mice (•) was maintained.

 
In the later phases of promotion in the two-stage model, the number of benign lesions declines as tumors undergo a progression phase and malignant tumors emerge as squamous cell carcinomas. For the FVB controls, carcinoma formation was first noted at wk 24, and a 50% incidence level was reached at 36 wk (Fig. 1B). The average tumor burden achieved at the point of 100% tumor incidence was similar to previous reports for FVB mice (1.3 ± 0.1 SE carcinomas per mouse). Consistent with the remarkable inhibition of papilloma formation, carcinoma induction was completely abrogated (100%) in the K14/IL-1{alpha} Tg mice.

Since male mice are reported to have a higher sensitivity to the DMBA/TPA protocol (22), we tested the response of K14/IL-1{alpha} Tg males to two-stage carcinogenesis. A biweekly promotion schedule was used since it has been shown to induce a larger papilloma burden than the weekly schedule. Indeed, the male FVB littermates exhibited an increased peak papilloma burden of 9.6 papillomas/mouse, while the K14/IL-1{alpha} Tg males failed to develop papillomas and carcinomas, confirming the abrogation of TPA-induced carcinogenesis by epidermal IL-1{alpha} expression (Fig. 1C).

Integrity of the promotion response in IL-1{alpha} Tgs

Possible explanations for the suppression of papilloma and carcinoma formation in IL-1{alpha} Tg mice included failure of DMBA-mediated initiation or of TPA-mediated promotion. To investigate further, K14/IL-1{alpha} mice were cross-bred to the TG.AC Tg strain that constitutively expresses an activated Ha-ras gene (20). These mice develop carcinomas after cutaneous injury or TPA promotion without prior DMBA initiation (20, 22, 23) and are thus sometimes referred to as preinitiated mice. If the resistance to two-stage carcinogenesis observed for the K14/IL-1{alpha} mice was due to failed initiation, then expression of the IL-1{alpha} transgene in TG.AC mice was predicted to reverse this effect. Using a biweekly promotion schedule, the TG.AC mice presented with large papilloma burdens as early as 7 wk after the first application of TPA (Table I), consistent with previous reports describing their increased sensitivity (20). Many of these lesions rapidly progressed to carcinomas. In contrast, K14/IL-1{alpha}// TG.AC mice developed only occasional small papillomas that regressed spontaneously; no carcinomas were identified. Thus, even in the presence of mutant, activated Ha-Ras, overexpression of IL-1{alpha} suppressed papilloma formation and abrogated carcinoma induction, demonstrating that tumor promotion, rather than initiation, was the variable being influenced by IL-1{alpha} expression.


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  		Table I. Response to the two-stage chemical carcinogenesis protocol of K14/IL-1{alpha} Tg mice cross-bred to Ras-overexpressing TG.AC micea

 
A trivial explanation of this phenomenon is that the IL-1{alpha} transgene rendered mice insensitive to TPA-induced inflammation and keratinocyte proliferation. This possibility was tested by assaying the ability of the mice to develop a hyperproliferative epidermal response following repeated TPA treatment. After four TPA applications, both the thickness of the epidermis and the number of proliferating cells in the basal layer were similar for the K14/IL-1{alpha} Tg mice and FVB littermate controls (Fig. 2). This result indicated that the resistance of the IL-1{alpha} mice to papilloma and carcinoma induction was not simply due to a failure of keratinocytes in transgenic epidermis to respond to the tumor promoter.



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  		FIGURE 2. Abrogation of papillomas and carcinomas in K14/IL-1{alpha} Tg mice is not due to a failure to respond to the tumor promoter TPA. Four applications of TPA (5 µg each) or acetone were applied to the shaved back skin of the K14/IL-1{alpha} Tgs and their FVB control littermates at 3- to 4-day intervals. One day after the final application, the mice were injected with BrdU (200 µg/g) and sacrificed, and the skin was processed for histological analysis. Immunohistochemical detection of BrdU in representative sections of FVB (left) and K14/IL-1{alpha} Tg backskin (right) is shown. No significant differences were detected in the average number of BrdU-immunoreactive cells between the Tg and control animals.

 
To investigate the possibility that some biological difference existed in the epidermal cells themselves as a consequence of transgene expression, keratinocytes were isolated from neonatal mice of the K14/IL-1{alpha} strain and their control littermates. The cells were cultured under conditions that sustained constitutive transgene expression in vitro. Extensive comparative analyses of the cells in culture did not reveal significant differences in the protein expression levels of a panel of genes in response to TPA, including protein kinase C{eta}, NF-{kappa}B, I{kappa}B, IL-1R-associated kinase, TNFR-associated factor 6, calpain, mitogen-activated protein kinase, JunB, and ornithine decarboxylase, or in the DNA binding activity of NF-{kappa}B (data not shown). These results suggest that the epidermal keratinocytes did not exhibit an identifiable change in these signaling pathways as a consequence of transgene expression or the site of integration.

Analysis of the role of the acquired immune response in the observed resistance

Recent experiments with TCR-deficient mice have demonstrated a role for T cells in cutaneous chemical carcinogenesis (24). To test whether the resistance of the K14/IL-1{alpha} mice involved enhanced activity of the acquired immune system in general and induction of T cells in particular, the IL-1{alpha} Tg mice were cross-bred to mice deficient in RAG2. RAG2 encodes the enzyme necessary to initiate the processes of V(D)J rearrangement of the TCR and Ig genes that are required for functional lymphocyte maturation (21). Following application of the two-stage protocol with a biweekly TPA promotion schedule, mice expressing the K14/IL-1{alpha} transgene on a RAG2-deficient background were resistant to papilloma and carcinoma formation, while the RAG2-/-FVB littermates were not (Fig. 3). Consistent with the reported increased sensitivity to the two-stage protocol of mice deficient in {gamma}{delta}T cells (24), the RAG2-deficient FVB mice exhibited an increased papilloma burden compared with wild-type FVB control mice (expressing RAG2+/+) (J. Murphy and T. S. Kupper, unpublished observations). Thus, the protective effect of Tg IL-1{alpha} expression against papilloma and carcinoma generation using TPA promotion is not a function of the adaptive immune response involving mature T and B cells and suggests a role for innate immunity.



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  		FIGURE 3. The protective effect of IL-1{alpha} overexpression does not involve the acquired immune system. The K14/IL-1{alpha} Tg mice were cross-bred to RAG2-deficient mice on an FVB background to test the role of T and B cells in the protective effect of IL-1{alpha} in the two-stage carcinogenesis model. The RAG-deficient mice express neither T nor B cell Ag receptors due to a lack of the critical RAG. Following application of the two-stage carcinogenesis protocol with a biweekly TPA promotion schedule, IL-1{alpha}-expressing, RAG2-deficient mice (•) were completely protected, while their RAG2-deficient littermate controls ({circ}) developed papillomas and carcinomas.

 
Complete carcinogenesis

As an alternative approach, the sensitivity of K14/IL-1{alpha} Tg mice to a complete carcinogenesis protocol was tested. The mice were treated with weekly applications of DMBA, a protocol described previously for carcinoma induction in the FVB strain (15). Papillomas developed on FVB littermates by wk 22 (Fig. 4), although the incidence (50%) and burden (0.63/mouse) were low, relative to those observed with the two-stage DMBA/TPA model. No papillomas were observed for the K14/IL-1{alpha} Tg mice following repeated DMBA application, similar to the dramatic inhibition observed with the two-stage protocol. However, carcinomas did arise de novo from treated skin of the K14/IL-1{alpha} Tg mice by wk 22; they preceded the appearance of carcinomas in the FVB mice, which became apparent at wk 25. At wk 28, the carcinoma incidence for IL-1{alpha} Tg mice was 5 times greater than that for the FVB controls (p < 0.04), suggesting that transgene expression was actually enhancing the rate of tumor formation. The median carcinoma incidence for the K14/IL-1{alpha} Tg animals (26 wk) occurred 7 wk earlier than that for the FVB controls (33 wk). However, this difference did not reach statistical significance, and the overall tumor incidence was not different ultimately in the two strains. Thus, in contrast to the abrogation of tumor formation observed in the two-stage carcinogenesis protocol, K14/IL-1{alpha} mice readily formed carcinomas in response to repeated application of the initiator in this complete carcinogenesis model, and they appeared to do so at an earlier time point. This result also indicated that the mice overexpressing 17-kDa IL-1{alpha} were not resistant to the mutagenic activity of DMBA.



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  		FIGURE 4. Overexpression of 17-kDa IL-1{alpha} in the epidermis fails to confer resistance to a complete carcinogenesis protocol. K14/IL-1{alpha} Tg mice (•) and FVB littermates ({circ}) were treated with repetitive DMBA applications alone, including an initiating dose of 25 µg, followed by 20 weekly applications of 5 µg each. Tumor incidence was analyzed using Kaplan-Myers test. In contrast to the two-stage carcinogenesis protocol, treatment with DMBA alone resulted in more rapid lesion formation in K14/IL-1{alpha} Tg animals compared with FVB controls. No difference was detected in the histology of tumors from Tg and control animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have demonstrated that mice expressing IL-1{alpha} in the epidermis are sensitive to a complete carcinogenesis protocol of repeated DMBA application, but are completely resistant to papilloma and carcinoma formation following application of a two-stage DMBA/TPA protocol. This result is even more striking since the FVB/N background strain of the K14/IL-1{alpha} Tg mice is one of the most sensitive to the two-stage protocol for papilloma and carcinoma induction (15). The resistance of K14/IL-1{alpha} mice was not due to an insensitivity to the tumor promoter TPA, as this agent induced both inflammation and epidermal hyperproliferation when applied to Tg skin. Also, no abnormalities were detected in the epidermal keratinocytes themselves when the cells were cultured under conditions where K14-driven transgene expression was maintained. This abrogation of papilloma and carcinoma formation persisted even when K14/IL-1{alpha} mice were crossed with TG.AC mice, which carry a constitutive mutated H-ras gene that endows them with a preinitiated phenotype, requiring only injury or TPA application to generate skin tumors. By crossing K14/IL-1{alpha} mice with the acquired immune-deficient RAG2 knockout mice, we were also able to exclude a role for T and B cells in the protection from papillomas and carcinomas. Although RAG2-deficient FVB mice are also hypersensitive to the DMBA/TPA tumorigenesis protocol, they were rendered papilloma and carcinoma resistant through expression of the IL-1{alpha} transgene in the epidermis.

Other Tg and knockout mouse models have been reported that display resistance to the two-stage initiation/promotion protocol, as evidenced by a decrease in papilloma yield. These include strains of TNF-{alpha}-deficient mice (25, 26) and those with targeted suppression of AP-1 activity, including the K14/TAM67 dominant-negative line (27) and the c-Jun N-terminal kinase 2-deficient strain (28). While it is difficult to compare our carcinogenesis results to the outcomes of these reports due to differences in strains and promotion regimens, none has resulted in an inhibition of papilloma formation as dramatic as that reported here for the K14/IL-1{alpha} Tg mice on the FVB/N background.

The finding that basal layer IL-1{alpha} overexpression inhibited two-stage chemical carcinogenesis was unexpected. Since it has been demonstrated previously that anti-inflammatory agents suppress TPA-induced tumor formation in this model, we hypothesized that increased inflammation would enhance two-stage carcinogenesis. Certainly, chronic inflammation is thought to predispose to squamous cell carcinomas in some tumor models. Yet, while K14/IL-1{alpha} mice express inflammatory cytokines and chemokines in skin and develop spontaneous skin inflammation, as characterized by a mixed dermal infiltrate (7), these mice produced only a minimal number of papilloma-like lesions that appeared very late in the experiments (after wk 40), remained small (~2 mm), and did not progress to carcinomas. Although we have ruled out a role for the acquired immune system in the resistance of K14/IL-1{alpha} Tg mice to the two-stage protocol, the possibility that the resistance could be attributed to cutaneous innate immunity remains uncertain. Interestingly, resistance to the two-stage carcinogenesis protocol has also been reported for mice selectively bred to have a maximal acute inflammatory response (29). Although the specific mechanism of tumor inhibition in these mice is similarly unknown, it was proposed to operate at the level of the innate immune response and to involve neutrophil infiltration (30). IL-1{alpha} has been shown to induce neutrophilia in the absence of activation (31) and is known to at least partially mediate neutrophil migration into the skin (16). In addition, low level constitutive expression of gro-{alpha}, the murine homologue of IL-8 and a chemoattractant for neutrophils, was detected in the skin of IL-1{alpha} Tg mice (8). The idea that IL-1{alpha}-mediated induction of gro-{alpha} could contribute to the condition of innate tumor immunity observed for the IL-1{alpha} Tg mice is supported by proposals that early infiltration of neutrophils into tumors can be beneficial (32) and that neutrophils can play a role in immune surveillance (33).

In contrast, these mice were sensitive to a complete carcinogenesis protocol that relies solely on repeated mutagen application to model multiple genetic insults. This suggests that epidermal IL-1{alpha} expression per se did not make mice resistant to DMBA-mediated mutagenesis. Interestingly, the repetitive DMBA treatment did not induce papillomas in K14/IL-1{alpha} mice, and the observed carcinomas evolved de novo from nonpapillomatous skin. This result is consistent with oral carcinogenesis experiments on the K14/IL-1{alpha} Tg mice that used repetitive application of the mutagen 4-nitroquinoline oxide to the oral cavity, a complete carcinogenesis protocol for oral epithelium. Since the K14 promoter drives transgene expression in stratified squamous epithelium other than the skin (6, 34), it effectively produces Tg mice that can function as disease models for multiple tissue types, including the oral cavity. Application of the 4-nitroquinoline oxide protocol to the K14/IL-1{alpha} Tgs resulted in aggressive squamous cell carcinomas in 100% of the Tg mice, while their FVB littermates were resistant (S. Dayan and T. S. Kupper, manuscript in preparation). Thus, complete carcinogenesis protocols using two different mutagens on two different epithelial tissues resulted in similar findings.

Based on these results, we propose that K14/IL-1{alpha} mice provide an opportunity to dissect the critical events affecting clonal progression of initiated cells to malignant carcinomas in two distinct models of human epidermal cancer. Under conditions that simulate the host experiencing one primary mutagenic insult and multiple subsequent exposures to promotional agents, as in the two-stage model, IL-1{alpha} appears to inhibit the growth of initiated cells, perhaps through activation of the innate immune system. However, under conditions where the host experiences repeated genetic insults and accumulation of multiple genetic mutations (as in the complete protocol), high levels of IL-1{alpha} do not inhibit carcinoma formation and can apparently lead to slightly enhanced tumor cell growth, as evidenced by the increased sensitivity of IL-1{alpha} Tg mice to repeated DMBA treatment. This may be a consequence of activation of accumulated inflammatory cells which could contribute to genetic instability and progression (35) or of an autocrine mitogenic signal of IL-1{alpha} on the tumor cells (36).

Whether one of these models is a more accurate reflection of human skin tumorigenesis is uncertain and may, in fact, be dependent on the individual. In either case, therapeutic targeting of IL-1{alpha} signaling may be important in blocking the early expansion of damaged epithelial cells. Documented IL-1{alpha} antitumor activity in human melanoma (37, 38), breast (39) and cervical carcinoma cell lines (14), and mouse tumor models (40, 41, 42, 43) led to clinical trials of IL-1{alpha} (44, 45, 46, 47, 48); however, unacceptable side effects were reported, and many trials were stopped before efficacy could be assessed. As cancer research shifts its focus toward preventative measures (49), the ability to understand and manipulate both the genetic and environmental events leading to IL-1{alpha} induction and the evolution of transformed epithelial cell proliferation in an inflammatory microenvironment may prove useful.


   Acknowledgments
 
We thank all past and present colleagues in the Department of Dermatology who have assisted with this project, including Richard Groves, Ifor Williams, and Tamara Rauschmeyer-Kopp. We are grateful to Denise Long-Woodward for expert histological assistance, to Dr. Benjamin Rich for the recombinase-2-deficient mice (RAG2-/-) backcrossed on the FVB/N background, and to Dr. Scott Granter for reviewing the histopathology and offering helpful suggestions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AR40124 and AR42689-8. Back

2 Current address: United Medical Associates, Vestal, NY 13850. Back

3 Current address: Pediatrics Department, University of Massachusetts Medical School, Worcester, MA 02115. Back

4 Address correspondence and reprint requests to Dr. Thomas S. Kupper, Harvard Skin Disease Research Center, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: tskupper{at}rics.bwh.harvard.edu Back

5 Abbreviations used in this paper: Tg, transgenic; BrdU, 5-bromo-2'-deoxyuridine; DMBA, 7,12-dimethylbenzanthracene; K14, keratin 14; RAG, recombinase-activating gene; SCC, squamous cell carcinoma; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back

Received for publication January 2, 2003. Accepted for publication March 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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