Platelet-activating factor and oxidized phosphatidylcholines do not suppress endotoxin-induced pro-inflammatory signaling among human myeloid and endothelial cells

Platelet-activating factor (PAF) and related phospholipid oxidation products termed oxidized phospholipids (OxPLs) promote inflammation. PAF is made in response to bacterial endotoxin-lipopolysaccharide (LPS) that is recognized by Toll-like receptor-4 (TLR-4) whose activation leads to translocation of transcription factor NF-ΚB to the nucleus—a key regulator of multiple pro-inflammatory genes including COX-2 and IL-8. Paradoxically, PAF and OxPLs are claimed to inhibit LPS-mediated signaling, questioning the very pro-inflammatory roles of PAF and OxPLs and anti-inflammatory nature of PAF-acetylhydrolase (PAF-AH), an enzyme that attenuates both PAF and OxPLs signaling. We investigated the effect of PAF and representative OxPLs: 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn- glycero-3-phosphocholine (PGPC) and 1-alkyl-2-butanoyl-sn-glycero-3-phosphocholine PAF (C4 PAF) on LPS-induced expression of NF-ΚB mediated inflammation in isolated human myeloid cells: polymorphonuclear leukocyte (PMNs), monocytes and human umbilical vein endothelial cells (HUVECs). Using intracellular calcium transients, we show that POVPC and PGPC dose-dependently activate the PAF-receptor (PAF-R) in PMNs, that can beblocked by the PAF-R antagonist WEB-2086 and rPAF-AH pre-treatment. All the three cell types express minute or no detectable COX-2 when stimulated with either PAF (0.1 µM) or OxPLs (0.1 µM) alone. While LPS (100 ng/mL) induced expression of COX-2 in all the cell types, pre-activation of PAF-R with PAF (0.1 µM) or OxPLs (0.1 µM) did not suppress LPS (100 ng/mL)-induced COX-2 expression and in fact we obresved incereased PGE2 levels in an NS-398 sensitive manner. In addition, pre-activation of PAF-R significantly augmented LPS (100 ng/mL)-induced IL-8 production in PMNs. Thus, PAF and OXPLs do not suppress the ability of LPS to exert its pro-inflammatory effects in isolated human vascular cells

Lipopolysaccharide Cross-Tolerance Delays Platelet-Activating Factor-Induced Sudden Death in Swiss Albino Mice: Involvement of Cyclooxygenase in Cross-Tolerance.

Lipopolysaccharide (LPS) signaling through Toll-like receptor-4 (TLR-4) has been implicated in the pathogenesis of many infectious diseases. Some believe that TLR-mediated pathogenicity is due, in part, to the lipid pro-inflammatory mediator platelet-activating factor (PAF), but this has been questioned. To test the direct contribution of PAF in endotoxemia in murine models, we injected PAF intraperitoneally into Swiss albino mice in the presence and absence of LPS. PAF alone (5 μg/mouse) caused death within 15-20 min, but this could be prevented by pretreating mice with PAF-receptor (PAF-R) antagonists or PAF-acetylhydrolase (PAF-AH). A low dose of LPS (5 mg/kg body wt) did not impair PAF-induced death, whereas higher doses (10 or 20 mg/kg body wt) delayed death, probably via LPS cross-tolerance. Cross-tolerance occurred only when PAF was injected simultaneously with LPS or within 30 min of LPS injection. Tolerance does not appear to be due to an abundant soluble mediator. Histologic examination of lungs and liver and measurement of circulating TNF-α and IL-10 levels suggested that the inflammatory response is not diminished during cross-tolerance. Interestingly, aspirin, a non-specific cyclooxygenase (COX) inhibitor, partially blocked PAF-induced sudden death, whereas NS-398, a specific COX-2 inhibitor, completely protected mice from the lethal effects of PAF. Both COX inhibitors (at 20 mg/kg body wt) independently amplified the cross-tolerance exerted by higher dose of LPS, suggesting that COX-derived eicosanoids may be involved in these events. Thus, PAF does not seem to have a protective role in endotoxemia, but its effects are delayed by LPS in a COX-sensitive way. These findings are likely to shed light on basic aspects of the endotoxin cross-tolerance occurring in many disease conditions and may offer new opportunities for clinical intervention

Dose-dependent effect of PAF on mortality in Swiss albino mice.

<p>The indicated amounts of PAF were aliquoted from a stock of 5 mg/mL in methanol, evaporated under a stream of nitrogen, reconstituted in phosphate buffered saline (PBS) containing 0.1% albumin, and sonicated. The animals were divided into 6 groups, each containing 6 animals. Each mouse was intraperitoneally injected with the designated dose of PAF in a total volume of 0.5 mL of PBS containing 0.1% albumin. Control animals received 0.5 mL PBS containing 0.1% albumin. The animals were monitored for survival for up to 6 days. The results are representative of 3 independent trials.</p

Specific COX-2 inhibitor confers complete protection against both PAF-induced lethality and delayed death due to PAF and hyperactivated TLR-4.

<p>Mice were divided into 7 groups containing 6 animals each. Three groups received an intraperitoneal injection of NS-398 (20 mg/kg body wt) 30 min before receiving PAF, LPS, or a combination of both. NS-398 (20 mg/kg body wt) completely abolished PAF-induced sudden death and enhanced the cross-tolerance effect exerted by the high dose of LPS (20 mg/kg). The animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

Biologically inactive structural analogs of PAF are not lethal to Swiss albino mice.

<p>Aliquots of lysoPAF and lysoPC were taken from a stock of 5 mg/mL in methanol, dried under nitrogen, reconstituted in PBS containing 0.1% albumin, and sonicated. The indicated doses of each were brought up to a total volume of 0.5 mL with PBS containing 0.1% albumin and intraperitoneally injected into the mice. Animals injected with the vehicle (PBS containing 0.1% albumin) received the same volume. PAF (5 μg/mouse) was used as a positive control; all the animals injected with PAF died within 15–20 min. No mortality was observed for the animals intraperitoneally injected with the lyso analogs at doses equivalent to that used for PAF or 10-fold higher (50 μg/mouse). All the animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

Aspirin partially protects Swiss albino mice from PAF-induced lethality and amplifies the cross-tolerance exerted by hyperactivated TLR-4.

<p>Mice were divided into 11 groups. Three groups were intraperitoneally injected with a 10 mg/kg dose of aspirin 30 min before being injected with the indicated doses of PAF, LPS, or both. Three other groups were intraperitoneally injected with a 20 mg/kg dose of aspirin 30 min before receiving PAF, LPS, or both. The 20 mg/kg dose of aspirin partially protected the animals from PAF-induced sudden death, whereas it completely protected the animals from delayed death due to PAF (5 μg/mouse) plus a high dose of LPS (20 mg/kg). The animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

PAF induces sudden death in Swiss albino mice.

<p>Swiss albino mice were divided into 9 groups containing 6 animals each. The indicated amounts of WEB-2086 or BN-52021 were aliquoted from a stock in DMSO, brought up to a total volume of 0.5 mL in PBS, and injected intraperitoneally into animals 30 min before PAF (5 μg/mouse) was injected. Animals that received rPAF-AH (25 μg/mouse) were intraperitoneally injected with it 30 min before being injected with PAF (5 μg/mouse), as were the animals that received the vehicle for rPAF-AH (sodium citrate, sucrose, pluronic, and Tween-80). All the animals injected with PAF alone (5 μg/mouse) died within 15–20 min. In contrast, all the animals that received either a PAF-R antagonist or rPAF-AH 30 min before being injected with a lethal dose of PAF survived. All animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

LPS cross-tolerance to PAF-induced lethality in Swiss albino mice is temporal.

<p>Mice were divided into 6 groups containing 6 animals per group. PAF (5 μg/mouse) was intraperitoneally injected together with LPS (20 mg/kg body wt) or at 30 min, 3 h, or 6 h after LPS was injected. All the animals injected with PAF alone (5 μg/mouse) or at 3 h or 6 h after receiving LPS (20 mg/kg) died in 15–20 min. In the group that received PAF concomitantly with LPS (20 mg/kg body wt), 33.4% of the animals died within 24 h, and 66.6% survived for 6 days. In the group that received PAF (5 μg/mouse) 30 min after LPS (20 mg/kg) was administered, the time until death was delayed to 12–24 h. All the animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

LPS cross-tolerance is not transferable.

<p>Mice were divided into 4 groups containing 4 animals each. In the initial phase of this experiment, animals were intraperitoneally injected with 0.5 mL of PBS or with LPS (20 mg/kg) + PAF (5 μg/mouse). These animals were then anesthetized, and their blood was collected 1 h after treatment. In the second phase of this experiment, 100 μL of the serum obtained from the mice treated with PBS or LPS (20 mg/kg body wt) + PAF (5 μg/mouse) was intraperitoneally injected into the other 2 groups of mice 30 min before they were injected with PAF (5 μg/mouse). Half of the mice in the group that originally received PAF (5 μg/mouse) + LPS (20 mg/kg) survived, but the serum from these animals failed to protect the naive animals injected with PAF (5 μg/mouse) 30 min after receiving the serum. The results are representative of 3 individual trials.</p

Strain-specific effects of PAF in murine species.

<p>Five-microgram aliquots of PAF were taken from a stock of 5 mg/mL in methanol, evaporated under a stream of nitrogen, reconstituted to a final volume of 0.5 mL in phosphate buffered saline containing 0.1% albumin, and sonicated. Then, the 5 μg doses of PAF were intraperitoneally injected into 3 strains of mice: Swiss albino, C57BL/6J, and BALB/c (n = 6 per strain). The mice were monitored for survival for up to 6 days. All of the Swiss albino mice that received PAF died within 15–20 min, whereas none of the C57BL/6J or BALB/c mice injected with PAF died. The results are representative of 3 individual trials.</p