Saveria Pastore

Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions

IL-10 is a critical cytokine that blocks the maturation of dendritic cells (DCs), but the relevance of autocrine IL-10 on DC functions has not been investigated. In this study, we found that immature monocyte-derived DCs released low but sizeable amounts of IL-10. After stimulation with bacteria, LPS, lipoteichoic acid, or soluble CD40 ligand, DCs secreted high levels of IL-10. Addition of an anti-IL-10-neutralizing Ab to immature DCs as well as to soluble CD40 ligand- or LPS-maturing DCs led to enhanced expression of surface CD83, CD80, CD86, and MHC molecules and markedly augmented release of TNF-α and IL-12, but diminished IL-10 mRNA expression. Moreover, DCs treated with anti-IL-10 Ab showed an increased capacity to activate allogeneic T cells and primed naive T cells to a more prominent Th1 polarization. DC maturation and IL-10 neutralization were associated with enhanced accumulation of the IL-10 receptor binding chain (IL-10R1) mRNA and intracellular IL-10R1 protein. In contrast, surface IL-10R1 and IL-10 binding activity diminished in mature DCs. These results indicate that autocrine IL-10 prevents spontaneous maturation of DCs in vitro, limits LPS- and CD40-mediated maturation, and increases IL-10 production by DCs. Moreover, IL-10R expression appears to be regulated by both transcriptional and posttranscriptional mechanisms. Endogenous IL-10 and IL-10R can be relevant targets for the manipulation of DC functions.

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.

Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis. Implications for sustained dendritic cell activation in the skin.

Lesional skin of atopic dermatitis (AD) harbors high numbers of dendritic cells with enhanced stimulatory capacity for T lymphocytes. In this study, lesional AD skin was shown to stain heavily in both epidermal and dermal compartments for GM-CSF, a cytokine crucial to dendritic cell functions. Keratinocyte cultures established from uninvolved skin of AD patients exhibited markedly increased spontaneous and PMA-stimulated release of GM-CSF compared with keratinocytes from nonatopic controls. Correspondingly, keratinocytes from AD patients showed higher constitutive as well as PMA-induced GM-CSF gene expression. Larger amounts of GM-CSF were produced by AD keratinocytes, also in response to IL-1alpha, but not after stimulation with LPS, lipoteichoic acid, or staphylococcal enterotoxin B. Hydrocortisone reduced GM-CSF gene expression and protein release in both atopic and control keratinocytes. Supernatants from atopic keratinocytes were able to strongly stimulate PBMC proliferation in a GM-CSF-dependent manner. Moreover, conditioned medium from PMA-treated AD keratinocytes, together with exogenous IL-4, could support phenotypical and functional maturation of peripheral blood precursors into dendritic cells. Enhanced production of GM-CSF by keratinocytes may contribute relevantly to the establishment and chronicity of AD lesions, in particular to the increased number, sustained activation, and enhanced antigen-presenting functions of dendritic cells.

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.

Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NFκB and AhR and EGFR-ERK pathway.

Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA+UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50μM resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR.

CXCR2 ligands and G-CSF mediate PKCα-induced intraepidermal inflammation

Transgenic mice overexpressing PKCα in the epidermis (K5-PKCα mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCα is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model of cutaneous inflammation was used to define mediators of skin inflammation that may have clinical relevance. Activation of cutaneous PKCα increased the production of the chemotactic factors cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein 2 (MIP-2) in murine plasma. TPA treatment of cultured K5-PKCα keratinocytes also released KC and MIP-2 into culture supernatants through an NF-κB–dependent pathway. MIP-2 and KC mediated the infiltration of neutrophils into the epidermis, since this was prevented by ablating CXCR2 in K5-PKCα mice or administering neutralizing antibodies against KC or MIP-2. The neutrophilia resulted from PKCα-mediated upregulation of cutaneous G-CSF released into the plasma independent of CXCR2. These responses could be inhibited by topical treatment with a PKCα-selective inhibitor. Inhibiting PKCα also reduced the basal and TNF-α– or TPA-induced expression of CXCL8 in cultured psoriatic keratinocytes, suggesting that PKCα activity may contribute to psoriatic inflammation. Thus, skin can be the source of circulating factors that have both local and systemic consequences, and these factors, their receptors, and possibly PKCα could be therapeutic targets for inhibition of cutaneous inflammation.

Interferon-γ promotes exaggerated cytokine production in keratinocytes cultured from patients with atopic dermatitis

Abstract Recent studies suggest that skin keratinocytes from patients with atopic dermatitis (AD) and nonatopic subjects differ in their intrinsic ability to respond to proinflammatory stimuli. In this study keratinocyte cultures established from the normal-looking skin of six adult patients with AD and six healthy, nonatopic control subjects were compared in their response to interferon (IFN)-γ, a potent proinflammatory lymphokine whose expression is increased in chronic AD lesions. Basal expression of IFN-γ receptor as well as IFN-γ–induced membrane expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 were evaluated by flow cytometry. Keratinocyte release of IL-1α, IL-1 receptor antagonist (IL-1ra), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α were measured by ELISA on culture supernatants after treatment with IFN-γ or medium alone. Expression of membrane IFN-γ receptor was similar in keratinocytes cultured from nonatopic subjects and subjects with AD. IFN-γ (10 to 500 U/ml) induced comparable levels of membrane HLA-DR and ICAM-1 in both groups of keratinocytes. In contrast, spontaneous release of IL-1α, IL-1ra, GM-CSF, and TNF-α was increased in the supernatants of unstimulated keratinocytes from patients with AD compared with keratinocytes from control subjects, with IL-1ra and GM-CSF reaching statistically significant difference. Moreover, IFN-γ–induced release of all the cytokines tested was much higher for keratinocytes from patients with AD, but the IL-1ra/IL-1α ratio for the two groups of keratinocytes was not substantially different, either basally or after IFN-γ stimulation. The results indicate that keratinocytes from patients with AD are hyperresponsive to IFN-γ in terms of cytokine release.

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.

Plant Polyphenols Regulate Chemokine Expression and Tissue Repair in Human Keratinocytes Through Interaction with Cytoplasmic and Nuclear Components of Epidermal Growth Factor Receptor System

AIMS To evaluate mechanisms underlying modulation of inflammatory chemokines in primary human keratinocytes (normal human epidermal keratinocytes) and repair-related processes in wound models by plant polyphenols (PPs) with antioxidant and superoxide scavenging properties (verbascoside [Vb], resveratrol [Rv], polydatin [Pd], quercetin [Qr], and rutin). RESULTS Epidermal growth factor receptor (EGFR)-controlled chemokines CXCL8/interleukin 8 (IL-8), CCL2/monocyte chemotactic protein-1 (MCP-1), and CXCL10/interferon gamma-produced protein of 10 kDa (IP-10) were modulated by transforming growth factor alpha (TGF-α) and by the tumor necrosis factor alpha/interferon gamma combination (T/I). EGFR phosphorylation, nuclear translocation, and downstream cytoplasmic signaling pathways (extracellular regulation kinase [ERK]1/2, p38, STAT3, and PI-3K) were studied. All PPs did not affect TGF-α-induced STAT3 phosphorylation, whereas they suppressed T/I-activated NFkappaB and constitutive and T/I-induced but not TGF-α-induced ERK1/2 phosphorylation. Vb and Qr suppressed total EGFR phosphorylation, but they synergized with TGF-α to enhance nuclear accumulation of phosphorylated EGFR. Vb strongly inhibited TGF-α-induced p38 phosphorylation and T/I-induced NFkappaB and activator protein-1 (AP-1) binding to DNA. Vb was an effective inhibitor of T/I-stimulated chemokine synthesis, and it accelerated scratch wound healing in vitro. Anti-inflammatory and wound healing activities of Vb were confirmed in vivo in the full-thickness excision wound. Although Pd and Rv did not affect EGFR activation/translocation, they and Qr synergized with TGF-α and T/I in the induction of IL-8 transcription/synthesis while opposing enhanced MCP-1 and IP-10 transcription/synthesis connected with pharmacologically impaired EGFR functioning. INNOVATION PPs perturb the EGFR system in human keratinocytes, and this effect may be implicated in the regulation of inflammatory and repair-related processes in the skin. CONCLUSION Anti-inflammatory and wound healing effects of PPs depend on their interaction with EGFR-controlled cytoplasmic and nuclear pathways rather than on their direct redox properties.

Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes

BACKGROUND Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined. OBJECTIVES We sought for genetic, immunological, and metabolic markers in MCS. METHODS We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST, glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined. RESULTS Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased. CONCLUSIONS Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.