Original ArticlesIdentification and pharmacological characterization of platelet-activating factor and related 1-palmitoyl species in human inflammatory blistering diseases
Introduction
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a family of sn-2 acetyl phosphocholines with diverse and significant pathophysiological effects 1, 2. PAF is a potent chemoattractant and activator of granulocytes, yet also has trophic effects on fibroblasts and lymphocytic cell lines 3, 4. Although PAF can be metabolized to potentially biologically active neutral lipid or phosphatidic acid species 5, 6, the majority of PAF effects are thought to be mediated through a G-protein-coupled membrane associated receptor (PAF-R) (7). Consistent with the myriad of responses linked to PAF, activation of the PAF-R stimulates numerous signal transduction systems, including phospholipase C (PLC), PLA2, PLD, and MAP-kinase.
1-Alkyl-sn-2 acetyl-GPCs are the best characterized ligands for the PAF-R. However, other natural products utilize this receptor. These other ligands include oxidized phospholipids and low-density lipoproteins 8, 9, lipopolysaccharide and protein A (10), lipotechoic acid moieties on Streptococcus species (11) and 1-acyl 2-acetyl-GPCs 12, 13. Whereas 1-alkyl are the most abundant sn-2 acetyl GPC species produced by neutrophils and macrophages 14, 15, 1-acyl sn-2 acetyl-GPC species comprise the majority sn-2 acetyl GPC synthesized by activated endothelial cells, mast cells, and B cell lines 13, 16, 17, 18 in vitro. Various cell types have been shown to synthesize these species in vitro, yet 1-acyl PAF analogs have not been demonstrated to be produced in large quantities in vivo.
The pharmacological activities of 1-acyl PAF analogs are unclear, as both agonistic and antagonistic activities on the PAF-R have been reported. 1-Palmitoyl-2-acetyl GPC (PAPC) has been shown to have PAF-like effects including intracellular calcium mobilization 18, 19, 20, 21, 22, MAP-kinase activation (18), and leukotriene C4 synthesis (22). In these model systems, PAPC is 100–1000 times less potent than 1-hexadecyl-2-acetyl-GPC as a direct agonist. However, PAPC in nanomolar concentrations can prime PMNs for significantly enhanced superoxide production after stimulation with C5a or fMLP 23, 24. PAF-R antagonists can inhibit PAPC effects, providing indirect evidence that PAPC mediates these effects through binding to the PAF-R.
In addition to these agonistic/priming effects, PAPC treatment of neutrophils and basophils has been reported to inhibit subsequent PAF-mediated actions 19, 22. Again, in contrast to its weak potency as a putative agonist of the PAF-R, PAPC has been reported to be much more potent as an inhibitor of subsequent PAF-induced effects 19, 22. These findings suggest the possibility that PAPC may act as a partial agonist. Furthermore, PAPC is readily metabolized to biologically active lipids, including the protein kinase C activator diacylglycerol and lysophosphatidylcholine (25). Of note, lysophosphatidylcholine has been reported to induce calcium mobilization in human vascular smooth muscle cells through a PAF-R-independent mechanism (26).
The objectives of this study were twofold. The first was to assess blister fluid samples obtained from patients with inflammatory or noninflammatory (suction-induced) cutaneous blistering diseases for sn-2 acetyl GPCs. Our finding that PAPC was the most abundant species found in cutaneous bullous diseases prompted the second objective, to assess the pharmacological activity of PAPC. Using retroviral-mediated gene transduction to express the human wild-type PAF-R in the PAF-R-negative epidermoid cell line KB 21, 27 we present evidence indicating that PAPC is a weak PAF-R agonist because it binds with low affinity to the PAF-R. The ability of PAPC to induce intradermal inflammation in Wistar Rats also was examined. These findings suggest that 1-acyl PAF analogs are a source of PAF-R agonist in vivo.
Section snippets
Reagents
Solvents (HPLC grade ethanol, methanol, chloroform, hexane, and acetic acid) and reagent grade diethyl ether were obtained from Fisher (Fair Lawn, NJ). Perdeutero acetyl chloride, perdeutero acetic acid, dimethylaminopyridine, and pentafluorobenzoyl chloride were obtained from Aldrich (Milwaukee, WI). 1-O-hexadecyl-2-acetylGPC, the lyso-PAF species with 1-O-hexadecyl and 1-O-octadecyl alkyl chains were obtained from Bachem (Bubendorff, Switzerland). The lyso GPC species, 1-palmitoyl-2-lyso-GPC,
AcetylGPC production in blistering disorders
Previously published data had indicated the presence of a PAF-like activity in the autoimmune blistering disease bullous pemphigoid (35). To investigate the molecular species of sn-2 acetyl-GPC found in this disease that resulted in this previously described PAF-like activity, a sensitive and specific GC/MS technique was used to detect the presence of sn-2 acetyl-GPC species in fresh blister fluid from blistering diseases. This procedure permitted structural characterization of closely related
Discussion
Because of its pro-inflammatory and trophic effects, PAF and PAF-R have been implicated in a wide range of pathophysiological processes ranging from asthma to septic shock. Structural identification of PAF 42, 43, cloning of PAF-R (44) and, more recently, the production of PAF-R overexpressing transgenic mice (45) have all served to increase our understanding of the importance of this family of lipid mediators. Similarly, defining both the species of PAF-R ligands found in vivo and their
Acknowledgements
The authors wish to acknowledge the technical assistance of Drs. J. Clark Huff and Loren Golitz, and thank Drs. Ginat Mirowski, Charles Lewis, and Patrick Logan for providing blister fluid samples from their patients. This research was supported in part by grants from The Dermatology Foundation, The Pharmaceutical Manufacturer’s Research Association, The Showalter Memorial Foundation, and the National Institutes of Health grants K08AR1993, HL34303, and AR074011.
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2003, Journal of Investigative DermatologyCitation Excerpt :This system utilizes the human epidermal cell line KB, which, unlike normal human keratinocytes, does not express functional PAF-R (Travers et al, 1995;Pei et al, 1998). A PAF-R-positive KB cell line was created by transducing KB cells with the replication-defective MSCV2.1 retrovirus containing the entire human PAF-R cDNA (Travers et al, 1998a;Pei et al, 1998). By comparing the effects of α-toxin on both PAF-R-positive (KBP) and PAF-R-negative (transduced with empty MSCV 2.1 retrovirus; KBM) KB cells, the role of the PAF-R on signaling and arachidonic acid release could be readily assessed.
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2000, Journal of Investigative DermatologyCitation Excerpt :These studies provide evidence that the epidermal PAF-R may be a pharmacologic target for UVB, and thus involved in UVB-induced cytokine production. The study of PAF/PAF-R has been limited by the rapid metabolism of PAF and because PAF metabolites can exert biologic activity independent of the PAF-R (Wilcox et al. 1987;Travers et al. 1990). The model system used in these studies was developed to overcome some of the current limitations in the study of PAF/PAF-R, and to account for the diverse ligands recognized by the PAF-R (Smiley et al. 1991;Triggiani et al. 1991;Nakamura et al. 1992;Cundell et al. 1995).
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