Phenotyping acute and chronic atopic dermatitis-like lesions in Stat6VT mice identifies a role for IL-33 in disease pathogenesis.

The Stat6VT mouse model of atopic dermatitis (AD) is induced by T-cell-specific expression of a constitutively active form of the protein signal transducer and activator of transcription 6 (STAT6). Although AD-like lesions are known to develop in Stat6VT mice, this study was designed to determine if these mice develop acute and chronic phases of disease similar to humans. To address this, AD-like lesions from Stat6VT mice were harvested at two different timepoints relative to their onset. Lesions harvested within 1 week after development were defined as acute lesions, and those present for 1 month or more were defined as chronic lesions. Acute and chronic AD-like lesions from Stat6VT mice exhibited histologic findings and cytokine expression patterns similar to acute and chronic AD lesions in humans. Further analysis revealed increased levels of interleukin (IL)-33 transcripts in AD-like lesions compared to Stat6VT nonlesional and wild-type skin controls. Immunofluorescence also revealed increased numbers of IL-33+ keratinocytes in Stat6VT lesional skin and localized IL-33+ keratinocytes to a keratin 5+ subset. Furthermore, AD-like disease was more severe in IL-33-deficient Stat6VT mice compared to IL-33-sufficient Stat6VT mice. These studies suggest that Stat6VT mice can serve as a model of acute and chronic AD and that IL-33 may attenuate inflammation in this system.

Topical application of a vitamin D analogue exacerbates atopic dermatitis and induces the atopic dermatitis-like phenotype in Stat6VT mice.

Calcipotriene is a topical vitamin D3 analogue approved for the treatment of plaque and scalp psoriasis. We report the case of a 2-year-old boy whose atopic dermatitis (AD) flared in response to application of calcipotriene 0.005% cream and solution for a mistaken diagnosis of plaque and scalp psoriasis. We investigated whether the patient's eruption was secondary to an allergic contact dermatitis. In the Stat6VT mouse model of AD we tested whether calcipotriene could induce the otherwise-spontaneous AD-like phenotype. Closed patch testing was done on the patient with calcipotriene solution and cream, moisturizing cream, and 51% isopropanol. Stat6VT and wild-type (WT) mice were treated for 7 days with calcipotriene solution or vehicle (isopropanol) applied to the right and left upper back skin, respectively, after which mice were followed longitudinally for 10 weeks. Biopsy specimens from prior treatment sites were then collected for histology and RNA isolation. RNA was analyzed for interleukin (IL-4) expression using quantitative polymerase chain reaction. Patch testing was negative. Stat6VT mice, in contrast to WT mice, developed a persistent eczematous dermatitis at sites of calcipotriene application. Clinical and histologic features and high IL-4 transcript levels were consistent with the spontaneous AD-like phenotype seen in Stat6VT mice. At sites of active disease, calcipotriene can worsen a flare of AD. In Stat6VT mice, calcipotriene can induce the AD-like phenotype.

Cigarette smoke exposure inhibits contact hypersensitivity via the generation of platelet-activating factor agonists.

Previous studies have established that pro-oxidative stressors suppress host immunity because of their ability to generate oxidized lipids with platelet-activating factor receptor (PAF-R) agonist activity. Although exposure to the pro-oxidative stressor cigarette smoke (CS) is known to exert immunomodulatory effects, little is known regarding the role of PAF in these events. The current studies sought to determine the role of PAF-R signaling in CS-mediated immunomodulatory effects. We demonstrate that CS exposure induces the generation of a transient PAF-R agonistic activity in the blood of mice. CS exposure inhibits contact hypersensitivity in a PAF-R-dependent manner as PAF-R-deficient mice were resistant to these effects. Blocking PAF-R agonist production either by systemic antioxidants or treatment with serum PAF-acetyl hydrolase enzyme blocked both the CS-mediated generation of PAF-R agonists and PAF-R-dependent inhibition of contact hypersensitivity (CHS) reactions, indicating a role for oxidized glycerophosphocholines with PAF-R agonistic activity in this process. In addition, cyclooxygenase-2 inhibition did not block PAF-R agonist production but prevented CS-induced inhibition of CHS. This suggests that cyclooxygenase-2 acts downstream of the PAF-R in mediating CS-induced systemic immunosuppression. Moreover, CS exposure induced a significant increase in the expression of the regulatory T cell reporter gene in Foxp3(EGFP) mice but not in Foxp3(EGFP) mice on a PAF-R-deficient background. Finally, regulatory T cell depletion via anti-CD25 Abs blocked CS-mediated inhibition of CHS, indicating the potential involvement of regulatory T cells in CS-mediated systemic immunosuppression. These studies provide the first evidence, to our knowledge, that the pro-oxidative stressor CS can modulate cutaneous immunity via the generation of PAF-R agonists produced through lipid oxidation.

Ultraviolet B radiation of human skin generates platelet-activating factor receptor agonists.

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor-alpha (TNF-alpha), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB-mediated production of PAF agonists is due primarily to the pro-oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF-receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB-generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF-alpha) production, these studies indicate that this novel activity could be involved in human disease.

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53.

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21(CDKN1A). Furthermore, IGF-1R-dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R-dependent premature senescence.

Augmentation of UVB radiation-mediated early gene expression by the epidermal platelet-activating factor receptor.

UVB radiation (UVB) is a known inducer of many biological changes in human skin, and triggers the production of glycerophosphocholines that act as platelet-activating factor (PAF) agonists. To gain a better insight into the role of the epidermal PAF receptor (PAF-R) in UVB-mediated gene expression, Affymetrix oligonucleotide microarrays were used to compare mRNA expression in the PAF-R-negative epithelial cell line KB-expressing PAF-Rs (KBP) with that in KB cells transduced with a vector control (KBM). Total RNA was isolated from KB cells 1 hour after treatment with a PAF-R agonist or UVB irradiation. Treatment of KBP with PAF agonist resulted in altered expression of 220 genes, including cytokines and growth factors. UVB irradiation of KB cells resulted in an increased expression of genes in both cell types. A panel of genes including cytokines CCL20 (MIP3alpha) and tumor necrosis factor-alpha (TNF-alpha) were upregulated selectively in KBP cells and are also selectively upregulated in response to PAF agonist. Consistent with these in vitro findings, UVB irradiation resulted in increased levels of epidermal CCL20 and TNF-alpha mRNA in wild-type over PAF-R-deficient mice in vivo. These studies provide evidence that the epidermal PAF-R can modulate UVB-mediated early gene expression.

Involvement of P38 MAP kinase in the augmentation of UVB-mediated apoptosis via the epidermal platelet-activating factor receptor.

Platelet-activating factor (PAF) is a group of phosphocholines with various biological effects, which are mediated by the PAF receptor (PAF-R). Our previous studies have demonstrated that ultraviolet B radiation (UVB) is a potent stimulus for PAF production, and that the presence of the PAF-R on epithelial cells results in an augmentation of UVB-induced apoptosis. Inasmuch as PAF-R activation results in numerous signal transduction pathways, the present study was designed to characterize the signal transduction pathway responsible for PAF-R-mediated enhanced UVB-induced cytotoxicity. Using a model system of PAF-R-negative and -positive epithelioid KB cells, we demonstrate that inhibitors of p38 MAP kinase block the augmentation of UVB-mediated apoptosis seen in PAF-R-positive KB cells. In contrast, pharmacological and/or molecular inhibition of other pathways linked to PAF-R activation including ERK MAP kinase and NFkappaB do not affect PAF-R-mediated cytotoxicity. This study demonstrates the important role that p38 MAP kinase plays in PAF-R-mediated augmentation of UVB cytotoxicity.

Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor.

Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor-positive and -negative (PAF-R-positive and -negative) cells and PAF-R-deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R-positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R-deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R-expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.

Augmentation of chemotherapy-induced cytokine production by expression of the platelet-activating factor receptor in a human epithelial carcinoma cell line.

In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-alpha induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-kappaB were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NFkappaB pathway using a super-repressor IkappaBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-kappaB-dependent process.

The epidermal platelet-activating factor receptor augments chemotherapy-induced apoptosis in human carcinoma cell lines.

Most chemotherapeutic agents exert their cytotoxic effects in part through the induction of apoptosis. In addition, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors, it is not known whether PAF is involved in chemotherapeutic agent-induced apoptosis. These studies examined the role of the PAF system in chemotherapy-mediated cytotoxicity using model systems created by retroviral mediated transduction of the PAF receptor-negative human epidermal carcinoma cell line KB with the human PAF receptor (PAF-R) and ablation of the endogenous PAF-R in the carcinoma cell line HaCaT with a retroviral mediated inducible antisense PAF-R vector. The presence of the PAF-R in these models resulted in an augmentation of apoptosis induced by chemotherapeutic agents etoposide and mitomycin C but not by tumor necrosis factor-related apoptosis-inducing ligand or by C(2) ceramide. Oxidative stress and the transcription factor nuclear factor kappaB (NF-kappaB) are found to be involved in this augmentative effect because it was blocked by antioxidants and inhibition of the NF-kappaB pathway using a super-repressor form of inhibitor B. These studies provide evidence for a novel pathway whereby the epidermal PAF-R can augment chemotherapy-induced apoptotic effects through an NF-kappaB-dependent process.