Francesca Mascia

Blockade of the EGF Receptor Induces a Deranged Chemokine Expression in Keratinocytes Leading to Enhanced Skin Inflammation

During inflammatory skin disorders such as psoriasis, atopic dermatitis, and allergic contact dermatitis, epidermal keratinocytes overexpress large amounts of soluble epidermal growth factor receptor ligands in response to tumor necrosis factor alpha and interferon gamma. These cytokines also promote de novo synthesis of numerous chemokines, including CCL2/MCP-1, CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, in turn responsible for the recruitment of different leukocyte populations. This study demonstrates that stimulation of EGFR down-regulates CCL2, CCL5, and CXCL10, while it increases CXCL8 expression in keratinocytes. Conversely, EGFR signaling blockade produces opposite effects, with increased CCL2, CCL5, and CXCL10, and reduced CXCL8 expression. In a mouse model of contact hypersensitivity, a single topical administration of a selective EGFR kinase blocker before antigen challenge results in a markedly enhanced immune response with increased chemokine expression and heavier inflammatory cell infiltrate. Targeting EGFR on epithelial cells may thus have profound impact on inflammatory and immune responses.

ERK1/2 Regulates Epidermal Chemokine Expression and Skin Inflammation

Resident cell populations of the skin contribute to the inflammatory response by producing an array of chemokines, which attract leukocytes from the circulation. TNF-α is a major inducer of proinflammatory mediators in keratinocytes. We have recently observed that epidermal growth factor receptor (EGFR) signaling affects TNF-α-driven chemokine expression in epidermal keratinocytes, and its functional impairment increases the levels of crucial chemoattractants such as CCL2/MCP-1, CCL5/RANTES, and CXCL10/IFN-γ-inducible protein-10. In this study, we report evidence that EGFR-dependent ERK1/2 activity is implicated in this mechanism. Abrogation of ERK1/2 activity with specific inhibitors increased chemokine expression in keratinocytes by enhancing mRNA stabilization. In mouse models, inflammatory response to irritants and T cell-mediated contact hypersensitivity were both aggravated when elicited in a skin area previously treated with an EGFR or a MAPK kinase 1/2 inhibitor. In contrast, impairment of p38αβ MAPK phosphorylation markedly attenuated these responses. Our data indicate that EGFR-dependent ERK1/2 activity in keratinocytes takes part to a homeostatic mechanism regulating inflammatory responses, and emphasize the distinct role of MAPKs as potential targets for manipulating inflammation in the skin.

EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo.

Recent advances in the knowledge of the EGFR pathway have revealed its contribution to distinct immune/inflammatory functions of the epidermis. The purpose of our study was to evaluate the role of EGFR in the regulation of keratinocyte GM-CSF expression. In cultured human keratinocytes, proinflammatory cytokines synergized with TGF-alpha to induce GM-CSF expression. Accordingly, high epidermal levels of EGFR activation are associated with enhanced expression of GM-CSF in lesional skin of patients with psoriasis or allergic contact dermatitis. In cultured keratinocytes, pharmacological inhibition of EGFR activity reduced GM-CSF promoter transactivation, whereas genetic inhibition of AP-1 reduced expression of GM-CSF. Furthermore, EGFR activation enhanced TNF-alpha-induced c-Jun phosphorylation and DNA binding, whereas c-Jun silencing reduced GM-CSF expression. Using two different mouse models, we showed that the lack of a functional EGFR pathway was associated with reduced cytokine-induced phosphorylation of ERK1/2, JNK1/2, c-Jun and reduced keratinocyte-derived GM-CSF expression both in vitro and in vivo. Finally, the analysis of GM-CSF expression in the skin of cancer patients treated with anti EGFR drugs showed an association between ERK activity, c-Jun phosphorylation, and epidermal GM-CSF expression. These data demonstrate that the EGFR pathway is critical for the upregulation of keratinocyte GM-CSF expression under conditions of cytokine stimulation.

Regulatory Role of Nitric Oxide on Monocyte-Derived Dendritic Cell Functions

Nitric oxide (NO) has an established role in the defense against bacterial infections and exerts multiple modulatory activities on both inflammatory and immune responses. However, the relevance of NO on dendritic cell (DC) functions has been poorly investigated. In this study, we found that addition of the NO donor S-nitrosoglutathione (GSNO) to monocyte-derived DCs matured by lipopolysaccharide (LPS) or soluble CD40 ligand led to a decreased capacity to activate naive allogeneic T cells but a more prominent Th1 polarization, with increased interferon-gamma (IFN-gamma) secretion and reduced interleukin-5 (IL-5) release. The presence of GSNO during maturation of DCs caused a reduced expression of surface CD86, whereas CD80, CD83, and MHC molecule expression was not affected. Moreover, GSNO induced a dose-dependent decrease of IL-10 and enhancement of tumor necrosis factor-alpha (TNF-alpha) release from mature DCs. In parallel, a marked reduced production of IL-12 p40 subunit but no significant perturbation of the bioactive IL-12 p70 production was observed. Finally, GSNO significantly reduced the release of IP-10/CXCL10 and RANTES/CCL5 but not IL-8/CXCL8 by mature DCs. Although GSNO can strengthen the capacity of mature DCs to induce type 1 polarization of T lymphocytes, our data suggest that it elicits distinct anti-inflammatory functions, eventually reducing T lymphocyte proliferation and recruitment.

Pathogenetic Mechanisms of Atopic Dermatitis

Atopic dermatitis (AD) is a chronic inflammatory disease which results from complex interactions between genetic and environmental mechanisms. An altered lipid composition of the stratum corneum is responsible for the xerotic aspect of the skin and determines a higher permeability to allergens and irritants. Keratinocytes of AD patients exhibit a propensity to an exaggerated production of cytokines and chemokines, a phenomenon that can have a major role in promoting and maintaining inflammation. Specific immune responses against a variety of environmental allergens are also implicated in AD pathogenesis, with a bias towards Th2 immune responses. In particular, dendritic cells expressing membrane IgE receptors play a critical role in the amplification of allergen-specific T cell responses. Cross-linkage of specific IgE receptors on dermal mast cells provokes the release and synthesis of a vast series of mediators. Following their recruitment and activation into the skin, eosinophils are also thought to contribute relevantly to tissue damage. Thus, a complex network of cytokines and chemokines contributes to establishing a local milieu that favors the permanence of inflammation in AD skin.

The Black Box Illuminated: Signals and Signaling

Unraveling the signaling pathways that transmit information from the cell surface to the nucleus has been a major accomplishment of modern cell and molecular biology. The benefit to humans is seen in the multitude of new therapeutics based on the illumination of these pathways. Although considerable insight has been gained in understanding homeostatic and pathological signaling in the epidermis and other skin compartments, the translation into therapy has been lacking. This review will outline advances made in understanding fundamental signaling in several of the most prominent pathways that control cutaneous development, cell-fate decisions, and keratinocyte growth and differentiation with the anticipation that this insight will contribute to new treatments for troubling skin diseases.

Keratinocytes in skin inflammation

Substantial evidence during the past years has indicated that keratinocytes play an active role in the generation and expression of protective immune responses and immunopathological reactions in the skin. In resting keratinocytes, immune mediators are barely detectable. Upon stimulation by environmental, physical or chemical stimuli, a significant increase in the release of pro-inflammatory mediators can be observed both in vitro and in vivo. The outcome of many skin reactions, however, critically depends on the cross-talk between infiltrating T cells and keratinocytes. Indeed, T-cell-derived lymphokines have been identified as the most effective triggers of keratinocyte expression of soluble and membrane molecules, which in turn are, themselves, responsible for the recruitment and local activation of T cells and other leukocytes, leading to the eventual amplification of the inflammatory reaction.

Novel acquisitions on the immunoprotective roles of the EGF receptor in the skin

The EGF receptor (EGFR) and its ligands represent one of the most powerful and complex signaling networks in higher vertebrates. This system exerts an unusually wide spectrum of diverse bioregulatory functions. Numerous indications have been collected in the past decades, essentially based on mouse models, that emphasize its central role in the development and homeostasis of the skin. In particular, the multiple phenotypes of mouse models with distinct dysregulations of the EGFR pathway indicate clearly that this system exerts a major impact on keratinocyte proliferation and differentiation and, eventually, on the process of wound healing, hair follicle morphogenesis and malignant transformation [1]. Notably, despite a long history of robust investigation into the diverse biological outcomes deriving from its possible dysfunctions in vivo in the mouse, a novel and, at least in part, unprecedented compendium of the role of EGFR in human skin is now emerging from the widespread use of EGFR inhibitors in the therapy of EGFR-dependent tumors. Currently, a humanized anti-EGFR monoclonal antibody (cetuximab), a fully human monoclonal antibody (panitumumab) and two smallmolecule EGFR tyrosine kinase inhibitors (gefitinib and erlotinib) are available for the treatment of four metastatic epithelial cancers: non-small-cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer and pancreatic cancer [2]. In patients treated with these drugs, a common adverse effect is a cutaneous inflammatory rash, characterized by papular pustular or acneiform eruption, which can be severe enough to lead to treatment modification or cessation [3]. These skin lesions are frequently pruritic and mostly affect the face and the upper trunk, although they may affect areas such as the dorsal arms and lower legs, and respond to anti-inflammatory drugs. Data from a large number of clinical trials suggest that the presence and severity of this cutaneous toxic effect are the most important positive correlates and predictors of the efficacy of anti-EGFR therapy in terms of progression-free survival [2,3]. Nonetheless, there is a serious need for an improved understanding of this side

Abstract 18: Targeted epidermal ablation of EGFR causes local and systemic inflammation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC EGFR is overexpressed or mutated in a variety of malignancies, and it plays a fundamental role in tumor development and progression, in the angiogenetic process and in the metastatic spread of tumors. As a result investigators have developed approaches to inhibit EGFR activation, with the aim of blocking tumor growth and invasion. The most common adverse effect seen with EGFR inhibition therapy is a pronounced skin inflammatory response (papulopustular rash) that can affect different areas of the body with variable severity. Interestingly, in vitro and in vivo experiments demonstrate that the pharmacological blockade of EGFR or ERK1/2, induces chemokine expression in epidermal keratinocytes and in turn leads to worsening of skin inflammatory conditions. Recent findings suggest a link between chemokine expression and EGFR status in the skin lesions of patients undergoing EGFR therapy and during tumor progression in BCC (basal cell carcinoma) and SCC (squamous cell carcinoma) skin lesions. Given these previous findings we believe that the study of the regulatory functions of the EGFR-ERK1/2 pathway on skin inflammatory mediators will help in understanding not only the adverse effect of this broad class of antitumor drugs but will possibly help to understand if and how they can modify the tumor microenvironment. In this study a mouse model was developed to better characterize the role of EGFR in the control of skin inflammation. By crossing Keratin 5 driven Cre recombinase transgenics with EGFR floxed mice, EGFR was ablated in the epidermis. During the first week of life, double transgenics started to display a strong skin phenotype with aberrant hair growth. During the following weeks the skin of these mice became progressively more inflamed with abundant neutrophilic infiltrate, scaly, dry and less elastic and underwent cycles of hair growth and hair loss. Double transgenic mice experienced a strong pruritus through their entire life, and their scratching behavior caused skin erosions. Tissue samples isolated from skin of adult double transgenics contained high levels of CD45 antigen and increased mRNA levels of a subset of inflammatory mediators namely CCL17, CCL20, CCL2, TNF-α, IL-6, IL-17, IL-18, and G-CSF. Targeted knockout mice also displayed hematological abnormalities with a higher number of circulating CD11b/Gr1 positive cells, extramedullary hematopoiesis and higher plasma levels of G-CSF, IL-1ra, IL-17, IL-6 and CCL2. These data indicate that the local absence of EGFR triggers local and systemic inflammatory responses. Therefore, this model represents a valuable tool to further investigate the control of EGFR in the maintenance of a balanced immuno-homeostasis of the skin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 18.