Identification and pharmacological characterization of platelet-activating factor and related 1-palmitoyl species in human inflammatory blistering diseases

Abstract

Through its pro-inflammatory effects on leukocytes, endothelial cells, and keratinocytes, the lipid mediator platelet-activating factor (PAF) has been implicated in cutaneous inflammation. Although the 1-alkyl PAF species has been considered historically the most abundant and important ligand for the PAF receptor (PAF-R), other putative ligands for this receptor have been described including 1-acyl analogs of sn-2 acetyl glycerophosphocholines. Previous bioassays have demonstrated a PAF-like activity in lesions of the autoimmune blistering disease bullous pemphigoid. To assess the actual sn-2 acetyl glycerophosphocholine species that result in this PAF agonistic activity, we measured PAF and related sn-2 acetyl GPCs in fresh blister fluid samples from bullous pemphigoid and noninflammatory (suction-induced) bullae by mass spectrometry. We report the presence of 1-hexadecyl as well as the 1-acyl PAF analog 1-palmitoyl-2-acetyl glycerophosphocholine (PAPC) in inflammatory blister fluid samples. Because PAPC is the most abundant sn-2 acetyl glycerophosphocholine species found in all samples examined, the pharmacological effects of this species with respect to the PAF-R were determined using a model system created by transduction of a PAF-R-negative epidermoid cell line with the PAF-R. Radioligand binding and intracellular calcium mobilization studies indicated that PAPC is approximately 100× less potent than PAF. Though a weak agonist, PAPC could induce PAF biosynthesis and PAF-R desensitization. Finally, intradermal injections of PAF and PAPC into the ventral ears of rats demonstrated that PAPC was 100× less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous 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.