Radiation therapy generates platelet-activating factor agonists.

Pro-oxidative stressors can suppress host immunity due to their ability to generate oxidized lipid agonists of the platelet-activating factor-receptor (PAF-R). As radiation therapy also induces reactive oxygen species, the present studies were designed to define whether ionizing radiation could generate PAF-R agonists and if these lipids could subvert host immunity. We demonstrate that radiation exposure of multiple tumor cell lines in-vitro, tumors in-vivo, and human subjects undergoing radiation therapy for skin tumors all generate PAF-R agonists. Structural characterization of radiation-induced PAF-R agonistic activity revealed PAF and multiple oxidized glycerophosphocholines that are produced non-enzymatically. In a murine melanoma tumor model, irradiation of one tumor augmented the growth of the other (non-treated) tumor in a PAF-R-dependent process blocked by a cyclooxygenase-2 inhibitor. These results indicate a novel pathway by which PAF-R agonists produced as a byproduct of radiation therapy could result in tumor treatment failure, and offer important insights into potential therapeutic strategies that could improve the overall antitumor effectiveness of radiation therapy regimens.

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 downregulates the expression of liver X receptor-α and its target genes in human neutrophils.

Liver X receptors (LXRs) are ligand-activated members of the nuclear receptor superfamily that regulate the expression of genes involved in lipid metabolism and inflammation, although their role in inflammation and immunity is less well known. It has been reported that oxysterols/LXRs may act as anti-inflammatory molecules, although opposite actions have also been reported. In this study, we investigated the effect of platelet-activating factor (PAF), a proinflammatory molecule, on LXRα signalling in human neutrophils. We found that PAF exerted an inhibitory effect on mRNA expression of TO901317-induced LXRα, ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and sterol response element binding protein 1c. This negative action was mediated by the PAF receptor, and was dependent on the release of reactive oxygen species elicited by PAF, as it was enhanced by pro-oxidant treatment and reversed by antioxidants. Current data also support the idea that PAF induces phosphorylation of the LXRα molecule in an extracellular signal-regulated kinase 1/2-mediated fashion. These results suggest that a possible mechanism by which PAF exerts its proinflammatory effect is through the downregulation of LXRα and its related genes, which supports the notion that LXRα ligands exert a modulatory role in the neutrophil-mediated inflammatory response.

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists.

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr -/- mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3(EGFP) transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

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.

The influence of platelet activating factor on the effects of platelet agonists and antiplatelet agents in vitro.

We assessed the effect of the intercellular mediator of inflammation, platelet activating factor (PAF), on platelet function. The interaction between PAF and the platelet agonists ADP, thrombin and convulxin was analyzed in vitro in whole blood with the use of flow cytometry and was further characterized with the use of receptor antagonists to PAF (ABT-491), P2Y1 (MRS-2179), and P2Y12 (cangrelor) as well as a monoclonal anti-PSGL-1 antibody (anti-CD162). Low concentrations of PAF (0.1 nM) synergistically augmented platelet activation induced by other agonists (P < 0.01). Augmentation by PAF was receptor mediated and did not require platelet-leukocyte interaction. With >99% inhibition of P2Y receptor-mediated platelet activation, greater than additive activation was still observed with the combination of ADP plus PAF. Accordingly, PAF synergistically augments platelet activation in response to ADP and thrombin, and the extent of inhibition exerted by P2Y receptor antagonists is decreased in the presence of PAF.

The phospholipid mediator platelet-activating factor mediates striatal synaptic facilitation.

The phospholipid mediator platelet-activating factor (PAF), an endogenous modulator of glutamatergic neurotransmission, can also be secreted by brain mononuclear phagocytes during HIV-1 infection. Platelet-activating factor can induce neuronal apoptosis by NMDA receptor-dependent and independent mechanisms. We now demonstrate that acute administration of sublethal doses of PAF to striatal slices augments synaptic facilitation in striatal neurons following high-frequency stimulation, which can be blocked by PAF receptor antagonists, suggesting that striatal synaptic facilitation can be augmented by PAF receptor agonism. We also demonstrate that repeated sublethal doses of PAF during tetanic stimulation can greatly increase the magnitude of postsynaptic potentials and action potentials, but a lethal dose of PAF destroys the capacity of corticostriatal synapses to achieve this augmented synaptic facilitation. Thus, the relative concentration and temporal pattern of PAF expression at glutamatergic synapses may govern whether it acts in a physiologic or pathophysiologic manner during striatal neurotransmission.

Synaptic activity becomes excitotoxic in neurons exposed to elevated levels of platelet-activating factor.

Neurologic impairment in HIV-1-associated dementia (HAD) and other neuroinflammatory diseases correlates with injury to dendrites and synapses, but how such injury occurs is not known. We hypothesized that neuroinflammation makes dendrites susceptible to excitotoxic injury following synaptic activity. We report that platelet-activating factor, an inflammatory phospholipid that mediates synaptic plasticity and neurotoxicity and is dramatically elevated in the brain during HAD, promotes dendrite injury following elevated synaptic activity and can replicate HIV-1-associated dendritic pathology. In hippocampal slices exposed to a stable platelet-activating factor analogue, tetanic stimulation that normally induces long-term synaptic potentiation instead promoted development of calcium- and caspase-dependent dendritic beading. Chemical preconditioning with diazoxide, a mitochondrial ATP-sensitive potassium channel agonist, prevented dendritic beading and restored long-term potentiation. In contrast to models invoking excessive glutamate release, these results suggest that physiologic synaptic activity may trigger excitotoxic dendritic injury during chronic neuroinflammation. Furthermore, preconditioning may represent a novel therapeutic strategy for preventing excitotoxic injury while preserving physiologic plasticity.

Platelet-activating factor inhibits the secretion of platelet-activating factor acetylhydrolase by human decidual macrophages.

To clarify the role of platelet-activating factor (PAF) in parturition, the effects of PAF on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by decidual macrophage populations were examined. The cells were isolated from human decidual tissue by enzymatic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The nonhydrolyzable agonist of PAF, carbamyl-PAF (C-PAF), inhibited the secretion of PAF-AH by either decidual cells or flow cytometrically purified decidual macrophages. A specific PAF receptor antagonist, WEB 2086, blocked the C-PAF-induced inhibition. Lyso-PAF, a metabolite of PAF, had no effect on the enzyme secretion. An intracellular calcium channel blocker, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, partially blocked the inhibition by C-PAF, whereas extracellular calcium channel blockers, nifedipine and verapamil, were without effect. The inhibitory effect of C-PAF was also partially blocked by protein kinase C (PKC) inhibitors, sphingosine and H-7. A PKC activator, 12-O-tetradecanoylphobol 13-acetate, decreased the secretion of PAF-AH. The decrease was abolished by the addition of sphingosine and H-7. It is suggested that PAF inhibits the PAF-AH secretion by decidual macrophages and that the inhibitory action is mediated by a signal transduction mechanism involving intracellular calcium and PKC.

Specific PAF antagonist WEB-2086 induces terminal differentiation of murine and human leukemia cells.

A pharmacological approach to neoplasia by differentiation therapy relies on the availability of cytodifferentiating agents whose antitumor efficacy is usually assayed first on malignant cells in vitro. Using murine erythroleukemia cells (MELCs) as the model, we found that WEB-2086, a triazolobenzodiazepine-derived PAF antagonist originally developed as an anti-inflammatory drug, induces a dose-dependent inhibition of MELC growth and hemoglobin accumulation as a result of a true commitment to differentiation. MELCs treated for 5 days with 1 mM WEB-2086 show greater than or equal to 85% benzidine-positive cells, increased expression of alpha- and beta-globin genes, and down-regulation of c-Myb. This differentiation pattern, which does not involve histone H4 acetylation and is abrogated by the action of phorbol 12-myristate 13-acetate, recalls the pattern induced by hexamethylene bisacetamide (HMBA). In addition to MELCs, human erythroleukemia K562 and HEL and myeloid HL60 cells are massively committed to maturation by WEB-2086 and, with some differences, by its analog, WEB-2170. This suggests that WEB-2086, structurally distant from other known inducers, might be a member of a new class of cytodifferentiation agents active on a broad range of transformed cells in vitro and useful, prospectively, for anticancer therapy due to their high tolerability in vivo.

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

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 100x 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 100x less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous diseases.

Effects of interleukin (IL)-3 and IL-5 on human eosinophil degranulation induced by complement components C3a and C5a.

It is suggested that eosinophils (Eos) play an important role in the pathogenesis of bronchial asthma by releasing cytotoxic cationic eosinophil granule proteins and damaging bronchial epithelial cells. However, the exact nature of the actual inducer of eosinophil degranulation in vivo is unclear. We examined eosinophil cationic protein (ECP) release from human Eos in response to soluble agonists such as C5a, C3a, platelet-activating factor, and FMLP with or without interleukin (IL)-3 or IL-5 priming. Eosinophil degranulation induced by these soluble agonists required the pretreatment of Eos by cytochalasin B even in IL-3 priming. Among four agonists, C5a was the most effective stimulus of ECP release either with or without IL-5 priming. IL-3 and IL-5 remarkably enhanced ECP release in Eos triggered by C3a and C5a. The enhancement of ECP release by IL-3 and IL-5 occurred at 0.1-0.3 ng/ml and became maximal at 10-30 ng/ml, concentration-dependently. The enhancement of ECP release by cytokines became optimal at an interval of 10 min. Our data support the importance of complement components and cytokines in eosinophil degranulation in vivo.