Tumor necrosis factor release by murine macrophages stimulated by the cytotoxic ether lipid 1-O-hexadecyl-2-O-methyl-SN-glycero-3-phosphorylcholine (ET-18-O-OCH3).

The cytotoxic ether lipid 1-O-hexadecyl-2-O-methyl-SN-glycero-3-phosphorylcholine (ET-18-O-OCH3) is a structural analog of the mediator of inflammation platelet-activating factor (PAF). Recent studies demonstrated that ET-18-O-OCH3 activates human monocytes selectively at non-cytotoxic concentrations. The current studies determined the capacity of ET-18-O-OCH3 to stimulate release of TNF alpha by murine peritoneal macrophages. Macrophage receptors for ET-18-OCH3 and PAF were also assessed. ET-18-O-OCH3 and PAF stimulated TNF alpha release by resident BALB/c macrophages in the presence of LPS but not in the absence of this co-factor. In contrast, both ET-18-O_OCH3 and PAF stimulated TNF alpha release by thioglycollate-elicited macrophages in the absence of LPS although release was greater in the presence of this co-stimulus. Optimal stimulation of TNF alpha release occurred at 10(-14)-10(-11) M ET-18-O-OCH3 and PAF. Elicited macrophages and splenic macrophages from C57Bl/6 mice, unlike those from BALB/c mice, did not respond to 10(-15)-10(-8) M ET-18-O-OCH3 or PAF without or with LPS. Scatchard analysis of [3H]PAF binding to elicited BALB/c macrophages revealed the existence of high affinity receptors for PAF. In contrast, there was no evidence for receptors for ET-18-O-OCH3. ET-18-O-OCH3 did not compete with PAF for binding; macrophage activation by ET-18-O-OCH3 was not stereospecific; and, binding studies using [3H]ET-18-O-OCH3 did not reveal saturable binding characteristic of binding to specific receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of tumour necrosis factor release by cytotoxic analogues of platelet-activating factor.

The capacity of cytotoxic analogues of platelet-activating factor (PAF) to stimulate tumour necrosis factor-alpha (TNF-alpha) synthesis and release by human monocytes was determined. Cell-associated TNF-alpha was quantified by protein immunoblotting and released TNF-alpha was quantified by cytotoxicity bioassay. Picomolar concentrations of methoxyPAF, SDZ 62-759, SDZ 68-826, SDZ 62-434 and SRI 62-834 induced a two- to fivefold increase in cell-associated and released TNF-alpha. These compounds were as potent as PAF for stimulating monocytes. In contrast, they lacked direct platelet-activating activity and inhibited platelet aggregation induced by PAF selectivity. The analogues inhibited PAF binding to platelets but not to monocytes. The PAF binding antagonists kadsurenone, BN52021 and WEB2086 inhibited TNF-alpha release induced by 10(-11) M PAF or methoxyPAF by a maximum of only 30-60% whereas they inhibited platelet aggregation by 10(-8) M PAF completely. Monocyte receptors for methoxyPAF were evaluated. Scatchard analysis of [3H]methoxyPAF binding to monocytes revealed large numbers of relatively low affinity receptors (Kd = 5.9 +/- 0.5 x 10(-7) M; 9.1 +/- 4.2 x 10(7) sites/monocyte). These values do not correspond to binding constants of monocyte receptors for PAF and do not account for monocyte activation by picomolar concentrations of methoxyPAF. Cytotoxic analogues of PAF stimulate TNF-alpha synthesis and release but they do not stimulate monocytes by interacting with PAF receptors.

Tumor necrosis factor release by human monocytes stimulated with platelet-activating factor.

Platelet-activating factor (PAF) is a low molecular weight phospholipid which enhances human monocyte cytotoxicity for tumor cells. In the current studies, the capacity of PAF to stimulate release of tumor necrosis factor alpha (TNF alpha) by human monocytes was assessed. PAF induced maximal TNF alpha synthesis 2-3 hr after monocyte stimulation as assessed by dot blotting of cell-associated TNF alpha using polyclonal anti-TNF antibody. Maximal net release of TNF alpha occurred 5-16 hr after monocyte stimulation, as assessed by a specific ELISA. Dose-response studies revealed that a maximal two- to three-fold increase in release of TNF alpha occurs at 10-100 pM PAF. LysoPAF and the optical isomer of PAF did not stimulate release of TNF alpha, suggesting that stimulation is mediated by specific PAF receptors. Scatchard analysis of [3H]PAF binding to monocyte membranes revealed 651 +/- 495 binding sites/monocyte with a Kd of 4.7 +/- 4.2 x 10(-10) M. PAF is a structurally unique activator of monocytes whose interactions with TNF alpha and other cytokines may be critical to host defense against tumors.

Selective activation of human monocytes by the platelet-activating factor analog 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine.

The capacity of platelet-activating factor (PAF) and its 2-O-methyl analog (methoxy-PAF) to activate human monocytes, neutrophils and platelets were compared. Both PAF and methoxy-PAF increased monocyte cytotoxicity toward WEHI 164 cells with a maximal increase in cell killing at 100 pM to 1 nM. Methoxy-PAF was slightly, but significantly, more potent than PAF for increasing cytotoxicity. PAF and methoxy-PAF increased monocyte release of TNF two- to three-fold above control release with no difference in their potency. Methoxy-PAF increased cell-associated TNF maximally after 2 to 3 h of incubation and increased TNF release maximally after 5 to 18 h of incubation. PAF induced release of the neutrophil granule enzyme beta-glucuronidase with maximal net release of 15 to 20% at 100 nM PAF whereas methoxy-PAF did not induce release of beta-glucuronidase. Similarly, 10 nM PAF induced 30% platelet aggregation whereas methoxy-PAF induced aggregation only at 1000-fold higher concentrations. Analysis of PAF and methoxy-PAF metabolism by monocyte and serum acylhydrolases indicates that methoxy-PAF is substantially more resistant than PAF to degradation by these enzymes. These observations indicate that methoxy-PAF activates monocytes selectively and suggest that this phospholipid or a related compound could be used for in vivo immunotherapy.

Platelet-activating factor binding to human platelet membranes.

Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor.