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

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

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

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

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

Sepsis: in search of cure

Introduction Sepsis is a complex inflammatory disorder believed to originate from an infection by any types of microbes and/or their products. It is the leading cause of death in intensive care units (ICUs) throughout the globe.The mortality rates depend both on the severity of infection and the host’s response to infection.Methods Literature survey on pathobiology of sepsis in general and failure of more than hundred clinical trialsconducted so far in search of a possible cure for sepsisresulted in the preparation of this manuscript.FindingsSepsis lacks a suitable animal model thatmimics human sepsis. However, based on the resultsobtained in animal models of sepsis, clinical trials con-ducted so far have been disappointing. Althoughinvolvement of multiple mediators and pathways in sepsishas been recognized, only few components are being tar-geted and this could be the major reason behind the failureof clinical trials.ConclusionInability to recognize a single critical medi-ator of sepsis may be the underlying cause for the poortherapeutic intervention of sepsis. Therefore, sepsis is stillconsidered as a disease—in search of cu