590 UVB generates microvesicle particles via Platelet-activating Factor-receptor signaling: a novel pathway by which a skin-specific stimulus exerts systemic effects

BACKGROUND MATERIALS & METHODS RESULTS RESULTS Cont’d 1. UVB generates MVP in vitro in a process involving PAF-R signaling. 2. UVB also generates MVP in human skin. 3. As MVP are thought to be functional signaling agents, these studies could provide a mechanism whereby UVB can generate systemic effects. SUMMARY For in vitro experiments, the human keratinocyte-derived cell line HaCaT was used. HaCaT cells were treated with UVB or the PAF-R agonist Carbamoyl-PAF (CPAF) or ethanol vehicle [3,5,7]. In some experiments HaCaT cells were preincubated for one hour with 0.5mM each of the antioxidants N-acetylcysteine and ascorbic acid before UVB/CPAF treatment. In some experiments the PAF-R-negative human epidermoid cell line KB cells transduced with functional PAF-Rs (KBP) or MSCV2.1 retroviral vector alone (KBM) were used [9]. At various times posttreatment supernatants were removed and MVP isolated using differential centrifugation and quantified using a NanoSight NS300 instrument using our previously published protocol [8,10]. For ex vivo experiments, we used skin discarded from human abdominoplasty experiments. The skin was warmed to ~37-38 degrees C and suction blisters separating epidermis from dermis were created using vacuum pumps and syringes (see Fig 5). Following blister formation, the blisters were treated with either sham or UVB or topical CPAF in ethanol:DMSO (1:9 v/v). Blister fluid was obtained and weighed in tared tubes. MVP were quantified as above. For in vivo experiments, the volar forearm of human volunteers underwent suction blistering and blisters were treated either sham or UVB. Again, blister fluid was removed and weighed and MVP quantified as outlined above. ACKNOWLEDGEMENTS These studies were supported in part by grants from the NIH (R01HL062996) and Basic Science VA Merit Award Platelet activating factor (PAF) is a lipid-derived 1-alkyl-2-acetly-glycerophosphocholine, produced in response to various stimuli that produces pronounced pro-inflammatory effects. These include marked vasodilatory effects, increased vascular permeability, bronchoconstriction, and platelet activation. PAF has also been implicated in sepsis [1,2]. PAF acts by binding to a cellular heterotrimeric G-protein coupled receptor PAF-R. This receptor is found on many cell types including granulocytes, B-cells, epithelial cells (keratinocytes), and some mesenchymal cells. PAF-R activation is known to induce intracellular calcium mobilization and is expressed on keratinocytes. Recent studies have demonstrated that PAF can be produced both enzymatically and through non-enzymatic oxidation of membrane glycerophosphocholines (ox-GPCs) [3]. Figure 1Biosynthesis of PAF through enzymatic and nonenzymatic pathways We and others have shown that in addition to its pro-inflammatory effects, PAF-R mediate pro-oxidative stressors including UVB-induced acute inflammation as well as delayed systemic immunosuppression [4-6]. Regarding the early acute effects of UVB, our previous studies have demonstrated that the PAFR plays an important role in the pain associated with sunburn [6], and mediates the increased UVB sensitivity in the Xeroderma Pigmentosum Complementation group A (XPA) knockout mouse [7]. Microvesicle particles (MVP) are small membrane-bound vesicles released by numerous cell types and can be found in the circulation. MVP can contain both nuclear and cytoplasmic constituents and are thought to provide a mechanism by which cells transmit cells systemically [8]. MVP are 200-1000nm in diameter and differ from smaller (40-200 nm) exosomes. Given that PAF is involved in UVB-mediated acute effects, these studies were designed to define if MVP are released from keratinocytes following UVB, and if PAF was involved in this process. Figure 3. Effect of UVB and CPAF on MVP release in HaCaT cells. HaCaT cells were either A) control (0) or UVB-irradiated at various fluences or B) vehicle control or various doses of CPAF. Four hours after treatment, the supernatants were removed and MVPs quantified as outlined in Methods. C) HaCaT cells were treated with 3.6 KJ/m2 UVB or 100 nM CPAF or control (CON), and were harvested 1 or 4 hrs post treatment. D). HaCaT cells were preincubated with 0.5 mM of N-aceytcysteine and 0.5 mM Vitamin C for one hour before treatment with 3.6 KJ/m2 UVB or 100 nM CPAF. The supernatants were removed and MVP measured 4 h later. The data presented are the mean ± SD. MVP numbers of duplicate values from a representative experiment of at least three performed. *Statistically (p changes from control values. NS: not statistically significant from control values. REFERENCES 1. Stafforini DM, McIntyre TM, Zimmerman GA, Prescott SM. Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes. Critical Reviews in Clinical Laboratory Sciences. 40(6):643-72, 2003. 2. Braquet P, Touqui L, Shen TY, Vargaftig BB. Platelet-activating Factor. Pharmacology Reviews. 39(2):97-145, 1987. 3. Marathe GK, Johnson C, Billings SD, Southall MD, Pei Y, Spandau D, Murphy RC, Zimmerman GA, McIntyre TM, Travers JB. Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage. Journal of Biological Chemistry. 280(42):35448-57, 2005 4. Walterscheid, J.P., Ullrich SE, Nghiem DX. Platelet-activating factor, a molecular sensor for cellular damage, activates systemic immune suppression. Journal of Experimental Medicine. 195: 171–179, 2002. 5. Zhang Q, Yao Y, Konger RL, Sinn AL, Cai S, Pollok KE, Travers JB. UVB radiation-mediated inhibition of contact hypersensitivity reactions is dependent on the platelet-activating factor system. Journal of Investigative Dermatology. 128(7):1780-7, 2008. 6. Zhang Q, Sitzman LA, Al-Hassani M, Cai S, Pollok KE, Travers JB, Hingtgen CM. Involvement of platelet-activating factor in ultraviolet B-induced hyperalgesia. Journal of Investigative Dermatology. 129(1):167-74, 2009. 7. Yao Y, Harrison K,. Al-Hassani M, Murphy RC, Rezania S, Konger RL, Travers JB. Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity. Journal of Biological Chemistry. 287(12): 9311-21, 2012. 8. Xiao X, Ma X, Liu L, Wang J, Bi K, Liu Y, Fan R, Zhao B, Chen Y, Bihl JC. Cellular Membrane Microparticles: Potential Targets of Combinational Therapy for Vascular Disease. Current Vascular Pharmacology. 13, 449-458, 2015. 9. Travers JB, Edenberg HJ, Zhang Q, Al-Hassani M, Yi Q, Baskaran S, Konger RL. Augmentation of UVB radiation- mediated early gene expression by the epidermal platelet-activating factor receptor. Journal of Investigative Dermatology. 128, 455-460, 2008. 10. Wang J et al., Analyses of Endothelial and Progenitor Cell Released Extracellular Microvesicles and Exosomes by Using Nanoparticle Tracking Analysis Method in Combination with Microbead and Q-dot Techniques. Science Reports 2016 in press. Figure 2. Picture of MVP released from a cell following stress UVB TREATMENT (KJ/m) M VP x 1 0 9 0 1 2 3 4 0 0.9 NS * *