Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

Loss of the platelet activating factor receptor in mice augments PMA-induced inflammation and cutaneous chemical carcinogenesis.

Although platelet-activating factor (PAF) is a well-known acute inflammatory mediator, little is known regarding the role of PAF in chronic inflammation. Phorbol esters are known to stimulate PAF production. Moreover, the ability of repeated applications of phorbol esters to induce a sustained inflammatory response is crucial to their tumorigenic activity. We therefore examined whether PAF acts as a mediator of phorbol ester-induced inflammation and tumorigenesis. While PAF receptor knockout mice (PAFR(-/-)) showed an expected but modest reduction in the acute inflammatory response to phorbol 12-myristate 13-acetate (PMA), these mice exhibited a surprising increase in inflammation following chronic PMA application. This increased inflammation was documented by a number of findings that included: increased skin thickness, increased myeloperoxidase activity and expression and increased expression of known inflammatory mediators. Interestingly, vehicle-treated PAFR(-/-) mice also exhibited modest increases in levels of inflammatory markers. This suggests that the platelet activating factor receptor (PAFR) acts to suppress chronic inflammation in response to other stimuli, such as barrier disruption. The idea that chronic PAFR activation is anti-inflammatory was documented by repetitive topical PAFR agonist administration that resulted in reduced myeloperoxidase activity in skin. We next utilized a 7,12-dimethylbenz(a)anthracene/PMA carcinogenesis protocol to demonstrate that PAFR(-/-) mice exhibit significantly increased tumor formation and malignant progression compared with wild-type control mice. These studies provide evidence for two important, unexpected and possibly interrelated pathological roles for the PAFR: first, the PAFR acts to suppress PMA-induced chronic inflammation; secondly, the PAFR acts to suppress neoplastic development in response to chemical carcinogens.

MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock.

Septic shock is a leading cause of morbidity and mortality. However, genetic factors predisposing to septic shock are not fully understood. Excessive production of proinflammatory cytokines, particularly tumor necrosis factor (TNF)-alpha, and the resultant severe hypotension play a central role in the pathophysiological process. Mitogen-activated protein (MAP) kinase cascades are crucial in the biosynthesis of proinflammatory cytokines. MAP kinase phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAP kinase. Thus, we hypothesize that knockout of the Mkp-1 gene results in prolonged MAP kinase activation, augmented cytokine production, and increased susceptibility to endotoxic shock. Here, we show that knockout of Mkp-1 substantially sensitizes mice to endotoxic shock induced by lipopolysaccharide (LPS) challenge. We demonstrate that upon LPS challenge, Mkp-1-/- cells exhibit prolonged p38 and c-Jun NH2-terminal kinase activation as well as enhanced TNF-alpha and interleukin (IL)-6 production compared with wild-type cells. After LPS challenge, Mkp-1 knockout mice produce dramatically more TNF-alpha, IL-6, and IL-10 than do wild-type mice. Consequently, Mkp-1 knockout mice develop severe hypotension and multiple organ failure, and exhibit a remarkable increase in mortality. Our studies demonstrate that MKP-1 is a pivotal feedback control regulator of the innate immune responses and plays a critical role in suppressing endotoxin shock.

IL-4 regulates skin homeostasis and the predisposition toward allergic skin inflammation.

IL-4 promotes the development of Th2 cells and allergic inflammation. In atopic dermatitis lesions, IL-4 decreases the expression of multiple genes associated with innate defense, including genes in the epidermal differentiation complex (EDC) that regulate epidermal barrier function. However, it is not clear whether IL-4 also contributes to homeostatic control of EDC genes. In this report, we demonstrate that expression of EDC genes and barrier function is increased in the absence of endogenous IL-4. Mice that express a constitutively active Stat6 (Stat6VT) are prone to the development of allergic skin inflammation and have decreased expression of EDC genes. IL-4 deficiency protects Stat6VT transgenic mice from the development of allergic skin inflammation and decreased recovery time in barrier function following skin irritation, with a concomitant increase in EDC gene expression. These data suggest that IL-4 plays an important role in regulating epidermal homeostasis and innate barrier function.

Treatment outcomes of secondarily impetiginized pediatric atopic dermatitis lesions and the role of oral antibiotics.

Patients with atopic dermatitis (AD) are predisposed to infection with Staphylococcus aureus, which worsens their skin disease; it has been postulated that the lack of antimicrobial peptides due to aberrant allergic inflammation in skin with AD could mediate this enhanced bacterial susceptibility. We sought to characterize the amounts of S. aureus and biological products found in infected AD lesions and whether treatment with topical corticosteroids and oral cephalexin as the only antimicrobial improved outcomes. Fifty-nine children with clinically and S. aureus-positive impetiginized lesions of AD were enrolled in this study. A lesion was graded clinically using the Eczema Area and Severity Index, and wash fluid was obtained from the lesion for quantitative bacterial culture and antibiotic sensitivities and measurement of bacterial products and cytokines. Subjects were re-evaluated 2 weeks after treatment. Improvement in the clinical and inflammatory characteristics of impetiginized lesions were noted, even in the 15% of lesions infected with Methicillin-resistant S. aureus (MRSA). In a subgroup of subjects whose lesions did not contain S. aureus 2 weeks after initiating treatment, beta-defensin levels were higher at both visits than in normal skin. Treatment of uncomplicated impetiginized pediatric AD with topical corticosteroids and cephalexin results in significant clinical improvement, even in subjects infected with MRSA. We propose that the inhibition of abnormal inflammation by the treatment regimen, resulting in the high levels of defensins, is involved in the improvement of AD and that systemic antibiotics do not appear to be necessary in secondary impetiginized AD.

Woringer-Kolopp disease mimicking foot dermatitis.

Woringer-Kolopp disease, also known as localized pagetoid reticulosis, is a rare cutaneous lymphoproliferative disorder classified as a solitary variant of mycosis fungoides (MF). Despite the indolent and benign nature of the disease, misdiagnosis and inappropriate treatment may result in years of debilitating symptoms and even loss of function. We present the case of a patient with long-standing Woringer-Kolopp disease that mimicked foot dermatitis. Histopathologic examination demonstrated epidermotropic infiltration of atypical lymphocytes that were CD3+ CD4- CD8-. The patient was successfully treated with topical keratolytics and bexarotene gel 1% with minimal residual lesions after 8 years of follow-up. We discuss the characteristics of this rare disease in contrast with localized MF as well as more aggressive forms of epidermotropic T-cell lymphoma.

Interleukin (IL)-4 inhibits IL-10 to promote IL-12 production by dendritic cells.

Interleukin (IL)-4 is known to be the most potent cytokine that can initiate Th2 cell differentiation. Paradoxically, IL-4 instructs dendritic cells (DCs) to promote Th1 cell differentiation. We investigated the mechanisms by which IL-4 directs CD4 T cells toward the Th1 cell lineage. Our study demonstrates that the IL-4-mediated induction of Th1 cell differentiation requires IL-10 production by DCs. IL-4 treatment of DCs in the presence of lipopolysaccharide or CpG resulted in decreased production of IL-10, which was accompanied by enhanced IL-12 production. In IL-10-deficient DCs, the level of IL-12 was greatly elevated and, more importantly, the ability of IL-4 to up-regulate IL-12 was abrogated. Interestingly, IL-4 inhibited IL-10 production by DCs but not by B cells. The down-regulation of IL-10 gene expression by IL-4 depended on Stat6 and was at least partly caused by decreased histone acetylation of the IL-10 promoter. These data indicate that IL-4 plays a key role in inducing Th1 cell differentiation by instructing DCs to produce less IL-10.

Dendritic cells produce inflammatory cytokines in response to bacterial products from Staphylococcus aureus-infected atopic dermatitis lesions.

Atopic Dermatitis (AD) patients often acquire secondary skin infections resulting in increased inflammation. The increased inflammation occurs through the activation of multiple cell types including dendritic cells (DC). In this study, we investigated the activity of soluble products present in infected AD lesions by measuring the ability of patients' wash fluids from a quantitative culture of lesions to activate DC. We found that wash fluid derived from AD lesions induced cytokine production by murine bone marrow-derived DC, including IL-1ß, IL-6, ΙL-10, and tumor necrosis factor-α. The lipoprotein lipoteichoic acid (LTA) from Staphylococcusaureus was implicated as a potent stimulus in the wash fluids as only wash fluid samples that contained LTA exerted this activity, and exogenous LTA triggered similar DC cytokine activation. Wash fluid- and LTA-stimulated DC cytokine production required MyD88, but not the platelet-activating factor receptor (PAF-R), despite the ability of LTA to function through this receptor in keratinocytes. Thus, our results support a role for DC in the worsening of AD inflammation due to secondary bacteria infections.

Ultraviolet B radiation generated platelet-activating factor receptor agonist formation involves EGF-R-mediated reactive oxygen species.

Recent studies have implicated the lipid mediator platelet-activating factor (PAF) in UVB-mediated systemic immunosuppression known to be a major cause for skin cancers. Previously, our group has demonstrated that UVB irradiation triggers the production of PAF and oxidized glycerophosphocholines that act as PAF-receptor (PAF-R) agonists. The present studies explored the mechanisms by which UVB generates PAF-R agonists. UVB irradiation of human epidermal KB cells resulted in both increased levels of reactive oxygen species (ROS) and PAF-R agonistic activity. Pretreatment of KB cells with antioxidants vitamin C and N-acetylcysteine or the pharmacological inhibitor PD168393 specific for the epidermal growth factor receptor all inhibited UVB-induced ROS as well as PAF-R agonists, yet had no effect on fMLP-mediated PAF-R agonist production. In addition, in vivo production of PAF-R agonists from UVB-irradiated mouse skin was blocked by both systemic vitamin C administration and topical PD168393 application. Moreover, both vitamin C and PD168393 abolished UVB-mediated but not the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine-mediated immunosuppression as measured by the inhibition of delayed type contact hypersensitivity to the chemical dinitrofluorobenzene. These studies suggest that UVB-induced systemic immunosuppression is due to epidermal growth factor receptor-mediated ROS which results in PAF-R agonist formation.

The landscape of EGFR pathways and personalized management of non-small-cell lung cancer.

Two classes of anti-EGF receptor (EGFR) agents, monoclonal anti-EGFR antibodies and small-molecule EGFR tyrosine kinase inhibitors, have been used for the treatment of non-small-cell lung cancer (NSCLC). However, only a subset of patients will benefit from EGFR-targeted therapy. The discovery of biomarkers that select the appropriate patients for the therapy and predict the responses to the therapy is urgently needed. Molecular genetic analyses provide new insights into EGFR pathway alterations and demonstrate promise for predicting the clinical outcome of patients with NSCLC. In this article, we summarize the latest available knowledge on the clinical impact of EGFR mutations, gene copy number, EGFR overexpression, phosphorylation expression and the alteration of the EGFR pathway downstream factors in predicting the response to EGFR-targeted therapy in NSCLC patients. The role of KRAS and BRAF mutations and ALK rearrangement in lung cancer-targeted therapy, are also reviewed.

Infected atopic dermatitis lesions contain pharmacologic amounts of lipoteichoic acid.

BACKGROUND: Bacterial infection with Staphylococcus aureus is a known trigger for worsening of atopic dermatitis (AD); the exact mechanisms by which bacterial infection worsens dermatitis are unknown. OBJECTIVE: We sought to characterize the amounts of the biologically active bacterial lipoprotein lipoteichoic acid (LTA) in infected AD lesions. METHODS: Eighty-nine children with clinically impetiginized lesions of AD were enrolled in this study. A lesion was graded clinically by using the Eczema Area and Severity Index (EASI), wash fluid obtained from the lesion for quantitative bacterial culture, and measurement of LTA and cytokines. The staphylococcal isolate was tested for antibiotic susceptibilities. The patients were treated with a regimen that included topical corticosteroids and systemic antibiotics, and the lesion was reanalyzed after 2 weeks. RESULTS: S aureus was identified in 79 of 89 children enrolled in the study. The bacterial colony-forming unit (CFU) counts correlated with the EASI lesional score (P = .04). LTA levels as high as 9.8 mug/mL were measured in the wash fluid samples, and the amounts correlated with the lesional EASI scores (P = .01) and S aureus CFU (P < .001). Approximately 30% of clinically impetiginized AD lesions contained greater than 1 mug/mL LTA, amounts that exert effects on various cell types in vitro. Moreover, injection of skin tissue ex vivo with amounts of LTA found in AD lesions resulted in epidermal cytokine gene expression. CONCLUSION: Pharmacologic levels of LTA are found in many infected atopic dermatitis lesions.

Vaccinia virus infection modulates the hematopoietic cell compartments in the bone marrow.

Successful proliferation and differentiation of hematopoietic progenitor cells in bone marrow (BM) is essential to generate all mature blood cell types, including those involved in the immune response. Although vaccinia virus (VV) is known to induce a strong immune response, the effect of VV infection on hematopoiesis remains largely unknown. Here, we showed that in vivo VV infection results in the expansion of c-Kit(hi)Sca-1+Lin- (KSL) hematopoietic stem cells. The in vivo expansion of the KSL population requires MyD88 that is a critical adaptor for Toll-like receptor-mediated signaling. Moreover, in BM of VV-infected mice, common myeloid progenitors (CMP) was decreased because of the rapid differentiation of CMP to more mature cells. However, the CMP compartment was not affected by VV infection in the absence of MyD88. The common lymphoid progenitor (CLP) cell population was increased regardless of MyD88 status, suggesting the independent regulation of CMP and CLP compartments by VV infection. VV infection also enhanced the potential of progenitors that preferentially induce the programming of dendritic cell (DC) development toward plasmacytoid DC. Therefore, the host immune response is gearing toward antiviral responses as early as at the precursor level upon VV infection.

Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects.

Ultraviolet light radiation (UVR) has profound effects upon human skin. Yet, the exact targets for UVR are unclear. Inasmuch as UVR is a known pro-oxidative stressor, one potential target for UVR could be oxidatively modified glycerophosphocholines (GPC). Importantly, recent studies demonstrate that these oxidized GPCs (ox-GPC) are potent agonists for the platelet-activating factor receptor and peroxisome proliferator-activated receptor gamma. This review discusses these new biologically active lipids and their down-stream receptor targets that provide a unique system of biosensors for detecting and responding to UVR photo-oxidation.

Role of PKCdelta in IFN-gamma-inducible CIITA gene expression.

The class II transactivator (CIITA) is a key regulatory factor for MHC class II expression. Here, we demonstrate that PKCdelta plays an important role in regulating IFN-gamma-inducible CIITA gene expression in macrophages. Inhibition of PKCdelta by either a PKCdelta inhibitor or a dominant negative (DN) mutant form of PKCdelta led to down-regulation of CIITA expression. The decrease in CIITA expression by PKCdelta inhibition was in part due to the reduced recruitment of serine 727-phosphorylated Stat1 and histone acetyltransferases to the CIITA promoter. As a result, IFN-gamma induced histone acetylation at the CIITA promoter is also compromised. However, inhibition of PKCdelta did not affect IRF-1 expression or IRF-1 binding to the CIITA promoter. Therefore, we report, for the first time, that PKCdelta is an essential signaling molecule to achieve the maximal expression of CIITA in response to IFN-gamma in macrophages. In addition, although IRF-1 is a key transcription factor to activate the IFN-gamma inducible CIITA promoter, the effect of PKCdelta on CIITA expression is mediated primarily by serine phosphorylation of Stat 1.

Platelet-activating factor does not mediate UVB-induced local immune suppression.

The lipid mediator Platelet-activating factor (PAF) and oxidized glycerophosphocholine PAF agonists produced by UVB have been demonstrated to play a pivotal role in UVB-mediated systemic immunosuppression. Importantly, employing the ability of distant UVB irradiation to inhibit contact hypersensitivity (CHS) responses to the chemical antigen dinitrofluorobenzene (DNFB) to an area of unirradiated murine skin, we and others have demonstrated that UVB-mediated systemic immunosuppression was only observed in PAF-R expressing wild type (WT) mice and not in PAF-R-knockout (Pafr-/-) mice. As it is not known if PAF is involved in UVB-mediated local immunosuppression, these studies compared local UVB on CHS responses in WT versus Pafr-/- mice. We demonstrate that the application of DNFB onto UVB-exposed (locally) area of mouse skin resulted in a similar significant inhibition of subsequent CHS responses in both WT and Pafr-/- mice compared to sham-irradiated control mice. Furthermore, the expression of langerin, a marker for the presence of Langerhans cells was substantially reduced equally in the epidermal ears of UVB-irradiated WT and Pafr-/- mice compared to their respective sham control groups. These findings indicate that the PAF-R is not involved UVB-induced local immunosuppression.

Epidermal platelet-activating factor receptor activation and ultraviolet B radiation result in synergistic tumor necrosis factor-alpha production.

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production which has been implicated in photoaggravated dermatoses. In addition to cytokines such as tumor necrosis factor-alpha (TNF-alpha), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous studies have demonstrated that UVB-mediated production of keratinocyte TNF-alpha is in part due to PAF. The current studies use a human PAF-receptor (PAF-R) negative epithelial cell line transduced with PAF-Rs and PAF-R-deficient mice to demonstrate that activation of the epidermal PAF-R along with UVB irradiation results in a synergistic production of TNF-alpha. It should be noted that PAF-R effects are mimicked by the protein kinase C (PKC) agonist phorbol myristic acetate, and are inhibited by pharmacological antagonists of the PKC gamma isoenzyme. These studies suggest that concomitant PAF-R activation and UVB irradiation results in a synergistic production of the cytokine TNF-alpha which is mediated in part via PKC. These studies provide a novel potential mechanism for photosensitivity responses.

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 radiation-mediated inhibition of contact hypersensitivity reactions is dependent on the platelet-activating factor system.

Through its ability to both induce immunosuppression and act as a carcinogen, UVB radiation plays a major role in cutaneous malignancies. Recent studies have indicated that UVB-mediated inhibition of delayed-type hypersensitivity reactions is mediated, in part, by the lipid mediator platelet-activating factor (PAF). The objective of this study was to further define the mechanism by which UVB inhibits contact hypersensitivity (CHS) reactions. UVB irradiation resulted in an inhibition of subsequent CHS to the chemical DNFB in wild-type, but not in PAF-R-deficient mice. UVB-mediated inhibition of CHS was also blocked by a cyclooxygenase-2 (COX-2) inhibitor or a neutralizing antibody directed against IL-10. UVB irradiation upregulated IL-10 mRNA levels in lymph nodes and spleen only to significant levels in PAF-R-expressing mice. Bone marrow transplantation studies demonstrated that UVB-mediated immunomodulatory effects were dependent on PAF-R-positive bone marrow. These studies suggest that UVB irradiation results in epidermal production of PAF agonists, which then act on PAF-R-positive bone marrow-derived cells to upregulate IL-10 through COX-2-generated prostaglandins.