Mohammed Al-Hassani

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.

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.

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.

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-α 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-κB 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 NFκB pathway using a super-repressor IκBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-κB-dependent process.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC α and ε, the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

Chemotherapeutic Agents Subvert Tumor Immunity by Generating Agonists of Platelet-Activating Factor

Oxidative stress suppresses host immunity by generating oxidized lipid agonists of the platelet-activating factor receptor (PAF-R). Because many classical chemotherapeutic drugs induce reactive oxygen species (ROS), we investigated whether these drugs might subvert host immunity by activating PAF-R. Here, we show that PAF-R agonists are produced in melanoma cells by chemotherapy that is administered in vitro, in vivo, or in human subjects. Structural characterization of the PAF-R agonists induced revealed multiple oxidized glycerophosphocholines that are generated nonenzymatically. In a murine model of melanoma, chemotherapeutic administration could augment tumor growth by a PAF-R-dependent process that could be blocked by treatment with antioxidants or COX-2 inhibitors or by depletion of regulatory T cells. Our findings reveal how PAF-R agonists induced by chemotherapy treatment can promote treatment failure. Furthermore, they offer new insights into how to improve the efficacy of chemotherapy by blocking its heretofore unknown impact on PAF-R activation.

Platelet-activating factor receptor agonists mediate Xeroderma Pigmentosum A photosensitivity

Background: Deficiency of the nucleotide excision repair protein xeroderma pigmentosum type A (XPA) is characterized by photosensitivity. Results: XPA-deficient cells and mice generate increased platelet-activating factor (PAF), oxidized glycerophosphocholines, and skin inflammation following ultraviolet B radiation with the skin inflammation blocked by PAF antagonists. Conclusion: PAF-R signaling mediates photosensitivity seen in XPA deficiency. Significance: These studies provide important insights into the mechanisms of 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.

The enhancement by wortmannin of protein kinase C-dependent activation of phospholipase D in vascular endothelial cells

Phosphatidic acid generation by phospholipase D (PLD) activation has been implicated in agonist- and oxidant-mediated endothelial cell signal transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. Pretreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100 microM), and bradykinin- (1 microM) induced [32P]PEt formation, an index of PLD activation. However, wortmannin by itself had no effect on PLD activity. The potentiating effect of wortmannin on TPA-induced PLD activation was dose- (1-10 microM) and time-dependent (5-30 min) and was inhibited by bisindoylmalemide, an inhibitor of protein kinase C (PKC). Furthermore, down-regulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuated the wortmannin effect. This effect of wortmannin was specific for TPA- or agonist-induced PLD activation as no potentiation of [32P]PEt formation was observed with H2O2 (1 mM) or ionomycin (1 microM). The effect of wortmannin was not due to activation of PKC alpha as determined by western blot analysis of PKC alpha in the cytosol and membrane fractions. Also, genistein, an inhibitor of tyrosine kinases, did not attenuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involvement of protein tyrosine phosphorylation. These results indicate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells.

Identification of Staphylococcal Protein A in Infected Atopic Dermatitis Lesions

Staphylococcus aureus (S. aureus) infection is a known trigger for skin inflammation and can modulate immune responses. Atopic dermatitis (AD), a chronic inflammatory pruritic skin disease, affects 10–20% of children and 1–3% of adults (De Benedetto et al., 2009). Due to the loss of skin integrity by scratching, as well as decreased levels of antimicrobial peptides in comparison to normal skin or other inflammatory diseases such as psoriasis (Leung, 2003; Ong et al., 2002), patients with AD are particularly susceptible to staphylococcal skin infections, which can further worsen their skin disease (Bieber, 2008). Studies have suggested several underlying mechanisms for staphylococcus-mediated inflammation, which include production of inflammatory cytokines following either direct infection of keratinocytes or immune cells by the bacteria, or indirectly by bacterial products (Baker, 2006; Leung, 2003; Sasaki et al., 2003; Travers et al., 2001). We have demonstrated previously that lipoteichoic acid (LTA), a gram-positive bacterial lipoprotein, may be an important component of the ability of S. aureus to exacerbate AD lesions (Travers et al., 2010). In the present study, we report that staphylococcal protein A (SPA) could also contribute to this process.

Progression of Toxic Epidermal Necrolysis After Tanning Bed Exposure

BACKGROUND In addition to recreational tanning bed use, UV radiation exposures are sometimes sought to self-treat skin conditions. The ability of tanning bed radiation exposure to trigger toxic epidermal necrolysis has not been reported. OBSERVATIONS A young woman attempted to treat a self-limiting drug hypersensitivity reaction via tanning bed radiation exposure, which resulted in a systemic toxic epidermal necrolysis-like reaction. Studies with cultured keratinocytes and an epithelial cell line reveal that UV-A radiation can synergize with other stimuli such as phorbol esters or interleukin 1 to produce large amounts of tumor necrosis factor, providing a potential mechanism for this exaggerated reaction. CONCLUSION In addition to inducing photodamage and skin cancer, tanning bed radiation exposure can trigger a toxic epidermal necrolysis-like reaction, possibly via the exaggerated production of keratinocyte cytokines such as tumor necrosis factor.