Pyrrophenone-elicited inhibition of AA release, LTB₄ and PGE₂ formation are pharmacologically indistinguishable. Cells were incubated for 120 min with GM-CSF/TNF in adenosine-free conditions, then stimulated with 100 nM A23187 for 10 min, alone or in presence of indicated concentrations of pyrrophenone. Arachidonate mobilization was determined by LC-MS, as described in the Experimental procedures section, whereas the generation of metabolites from the 5-LO or COX pathways were measured by RP-HPLC or ELISA. For each indicated metabolite, the amounts generated are shown as percentages, relative to samples stimulated in the absence of pyrrophenone (mean±SEM from n=3 experiments, each performed with different donors). Visually-determined IC₅₀ is indicated by a dotted line.

Exogenous arachidonate is utilized differentially by the 5-LO and COX pathways in neutrophils. (A & B) Cells were treated for 60 min with GM-CSF/TNF in adenosine-free conditions, then stimulated with indicated concentrations of arachidonate for 10 min. Generation of 5-HETE, 6-trans-LTB₄, and LTB₄ (A) was determined by RP-HPLC. PGE₂ generation (B) was assayed by ELISA. (C & D) Cells were treated as in (A), alone or in the presence of the COX-1/COX-2 inhibitor, indomethacin (INDO; 10 μM), or the non-specific EP receptor antagonist, AH 6809 (10 μM). Generation of 5-HETE (C) and of LTB₄ (D) was determined by RP-HPLC. In each panel, results are presented as the mean±SEM from n=3 experiments, each performed with different donors.

LTB₄ up-regulates COX-2 protein expression in neutrophils through engagement of its receptor. (A) Cells were incubated with indicated agonists (each used at a concentration of 100 nM) for the indicated times (B) Cells were incubated with LTB₄ or with its less-active analog 6-trans-LTB₄, (each used at a concentration of 100 nM) for the indicated times. (C) Cells were incubated with fMLP, alone or in combination with LTB₄ for the indicated times. Samples were processed for the determination of COX-2 by western immunoblotting, as described in the experimental procedures section. In each panel, shown are immunoblots obtained in one experiment, typical of three separate experiments, each performed with different donors. In panel C, COX-2 densitometry analyzes were performed as described in Experimental procedures; results represent the average (n=3, ±SEM) from data obtained with different 3 donors. (D) Cells were treated with saline, LTB₄ (100 nM) or fMLP (100 nM), then stimulated with AA or A23187. PGE₂ levels in cell-free supernatants were determined by ELISA. Results presented are the mean±SEM from n=3 experiments, each performed with different donors. *Significantly higher than in untreated cells.

Updates in the characterization of the COX-2 pathways in human neutrophils. In response to a stimulus, membrane-esterified arachidonic acid (AA) is mobilized by type IV cytosolic phospholipase A₂ (cPLA₂) in a Ca²⁺-dependent fashion. AA can be utilized either by 5-lipoxygenase (5-LO) or by the inducible cyclooxygenase isoform (COX-2). 5-LO pathway: 5-LO, a Ca²⁺-dependent enzyme, catalyzes two reactions: transformation of AA into 5-hydroperoxyeicosatetranoic acid (5-HPETE), and dehydration of the latter in leukotriene (LT)A₄. LTA₄ is metabolized in the active metabolite LTB₄ by the LTA₄ hydrolase. Neutrophils also express an omega carboxylase which sequentially transforms LTB₄ into less active metabolites: 20-OH- and 20-COOH-LTB₄. COX-2 pathway: COX-2, which can be up-regulated in neutrophils, also catalyses two reactions by which AA is transformed into prostaglandin (PG)H₂. PGH₂ can then be metabolized into the active metabolite PGE₂ by the microsomal PGE₂ synthase (mPGES)-1, or into TXA₂ by TXA₂ synthase (TXA₂S), both of which being constitutively-expressed in neutrophils. Thus, COX-2 expression is held to be the main limiting factor for the biosynthesis of prostanoids in these cells. Available information indicates that LTB₄, PGE₂ and TXA₂ are the main eicosanoids produced by human neutrophils, in vitro.

Type IV cPLA₂ is chiefly involved in the release of arachidonate transformed in PGE₂ through the COX-2 pathway in neutrophils. Cell suspensions, incubated for 120 min with GM-CSF/TNF (1.5 nM/100 nM) in adenosine-free conditions, were stimulated with 100 nM A23187 (or its diluent:DMSO) for 10 min, in absence or presence of the specific cPLA₂ inhibitor pyrrophenone (100 nM), an inhibitor of the GIIA, GV and GX sPLA₂s, LY 311727 (LY 311; 50 μM), or the specific COX-2 inhibitor, NS 398 (20 μM). (A) Generation of metabolites from the 5-LO (5-HETE and LTB₄) pathways were measured by RP-HPLC, as described in the Experimental procedures section. (B) In addition to the conditions described in A), cells were stimulated with exogenous AA (10 μM). PGE₂ levels were determined in cell-free supernatants by ELISA, as described in the Experimental procedures section. Results are presented as the mean±SEM, from n=3 separate experiments, each performed with different donors. *Significantly different from cells incubated in absence of pyrrophenone. **Significantly different from cells incubated in absence of NS 398.

Pyrrophenone inhibits COX-2-derived PGE₂ production elicited by inflammatory mediators involving receptor engagement. Cells were treated for 120 min with saline or GM-CSF/TNF in adenosine-free conditions, then stimulated with 100 nM fMLP or opsonized zymosan (Zop), for 15 min. Stimulations were performed in absence or presence of pyrrophenone (100 nM), LY 311727 (50 μM), or NS 398 (20 μM). PGE₂ levels were determined in cell-free supernatants by ELISA. Results are expressed as percentages of production, relative to the value obtained in AA-stimulated cells (10 μM; 100%=14.5±3.3 ng/5×10⁶ cells). Results presented are the mean±SEM from at least n=3 experiments, each performed with different donors. *Significantly higher than in saline-treated cells.

Elevation in intracellular Ca²⁺ is required for AA availability, but not for COX-2 up-regulation. (A) Cell suspensions were incubated either for 60 min with GM-CSF/TNF in normal, or in Ca²⁺-free (incubated in HBSS supplemented with 3 mM EGTA) conditions. Cells were stimulated with arachidonic acid (AA) or with A23187 for 10 min (left panel), or with 100 nM fMLP (right panel). PGE₂ levels in cell-free supernatants were determined by ELISA. Results presented are the mean±SEM from n=3 experiments, each performed with different donors. *Significantly lower than in normal conditions. Insert: neutrophils were incubated with GM-CSF/TNF in normal, or Ca²⁺-free conditions then processed for the determination of COX-2 protein levels by western immunoblotting. (B) Monocytes were treated with 2 μg/ml LPS for 16 hr in normal, or Ca²⁺-free (medium supplemented with 5 mM EGTA) conditions. PGE₂ levels in cell-free supernatants were determined by ELISA. AA stimulation: cells were washed twice with PBS, fresh medium (normal or Ca²⁺-free) was added, then cells were stimulated with 10 μM AA for an additional 60 min. Results presented are the mean ± SEM from n = 3 experiments, each performed with different donors. *Significantly lower than in normal conditions. Insert: monocytes incubated with LPS in normal, or Ca²⁺-free conditions, then processed for the determination of COX-2 protein levels by western immunoblotting.

Microsomal PGES-1 is constitutively expressed in neutrophils and co-localizes with cyclooxygenase-2. (A) Cells were incubated with saline, or with GM-CSF/TNF for indicated times, than processed for the determination of COX-1, COX-2 and mPGES-1 mRNA expression by real-time PCR, as described in the Experimental procedures section. Results are expressed in fold-variations of mRNA expression, compared to that observed in saline-treated cells, and values are normalized for GAPDH. Results are presented as the mean±SD from two experiments performed with different donors. (B) Cell suspensions were incubated for 120 min with fMLP, or LPS, or GM-CSF/TNF, and samples were processed for the determination of mPGES-1 by western immunoblotting, as described in the Experimental procedures section. Shown is one immunoblot, typical of three experiments, each performed with different donors, as well as densitometric measurements (mean±SEM) obtained from these 3 donors. (C) Cell suspensions were incubated for 120 min with GM-CSF/TNF, alone or in combination with adenosine deaminase (ADA) and with the specific A_2A receptor agonist, CGS 21680 (CGS). Samples were processed for the determination of COX-2 and mPGES-1 by western immunoblotting. Shown is one immunoblot, typical of three experiments, each performed with different donors, as well as densitometric measurements (mean±SEM) obtained from these 3 donors. (D) Cells were incubated for 120 min with GM-CSF/TNF, then subjected to cell cavitation by the nitrogen bomb technique and separated into 18 fractions by centrifugation on Percoll gradient, as described in the Experimental procedures section. Samples processed for the determination of COX-2, mPGES-1 and thromboxane (TX)-Synthase expression by western immunoblotting. GRP-78 was used as a marker of ER and Golgi. Fractions 7 to 15 are shown.