Research Paper Mediators of Inflammation 2, 407-409 (1993)

LY 255283 [(1-(5-ethyl-2-hydroxy-4-(6-methyl-6-)1H-tetrazol-5-yl)-heptyloxy) phenyl)ethanone], a specific leukotriene B4 (LTB4) receptor antagonist, inhibited the production of LTB4 in human peripheral blood polymorphonuclear leukocytes (PMNL) and in monocytes activated by calcium ionophore A23187. In human monocytes activated by ionophore it inhibited also the production of thromboxane B2 (TXB2). The effect of LY 255283 on 5-lipoxygenase (5-LO) and LTA4 hydrolase activities which catalyse the production of LTB4 and LTA4 has not been studied yet. It is thought that LY 255283 may inhibit the production of LTB4 and TXA2 by antagonising the effect of ionophore-induced LTB4 on 5-lipoxygenase and cyclooxygenase in human peripheral blood PMNL and monocytes.


Introduction
LTB4, a 5-1ipoxygenase metabolite of arachidonic acid (AA), is an inflammatory mediator released from immunoinflammatory ceils, especially from neutrophils and monocytes. A variety of stimuli, including calcium ionophores, particulate (zymosan) and soluble stimuli (N-formyl-methionylleucyl-phenylalanine) and complement component CSa 1'2 can elicit the synthesis of LTB 4 from neutrophil and monocytes.
It has been shown that LTB 4

Materials and Methods
Ce// separation: Citrated blood was collected by venipuncture from healthy volunteers who had abstained from any drugs for at least 1 week before the sampling. A method described by Dewar was used for the isolation of human PMNL and monocytes. 1 Briefly, platelet rich plasma was discarded after the centrifugation of whole blood. Mononuclear cells and PMNL were separated by centrifugation on Ficoll-Paque cushions. The PMNL were separated from erythrocytes by dextran sedimentation and NH4C1 lysis. After the isolation procedure, the purity of PMNL was > 98% and viability determined by Trypan blue exclusion > 95%. Monocytes were further purified by adherence. 1

Results
Calcium ionophore A23187 (10 -6 M) elicited an increase in the synthesis of LTB4 and TXB2 in human PMNL and monocytes. This increase was  (Figs 1 and 2). In preliminary experiments, LY 255283 (ranging from 10 -9 to 10 -6 M) caused a dose-dependent inhibitory effect on LTB4 release (data not shown). LY 255283 also inhibited the synthesis of TXB2, the stable metabolite of TXA2 in monocytes (Fig. 2). However, LY 255283 did not significantly inhibit the synthesis of TXB2 in PMNL ( Fig. 1).

Discussion
It is well known that a variety of stimuli, including the calcium ionophore A23187, can elicit an increase in the synthesis of cyclooxygenase (prostaglandin E2, prostacylin, TXA2) and lipoxygenase (mainly LTB4) metabolites of AA in human Inhibitory effect of L Y 255283 on L TB4 and TXA e PMNL and monocytes. 1'2 In the present study, calcium ionophore A23187, produced an increase in the synthesis of LTB4 and TXB2 in these cells. The ionophore-induced synthesis of LTB 4 was more prominent than that of TXB2, a finding which supports previous observation of Moroney et al. 2 LTB 4 is also known as one of the activators of human PMNL and monocytes. It is therefore expected that LTB 4 may activate its own synthesis in these cells. McDonald et al. have described that 14,15-dideuterio-LTB 4 (D2-LTB4) activates 5lipoxygenase in PMNL and elicits an increase of LTB 4 synthesis. In this study, increased synthesis of LTB 4 by D2-LTB 4 in PMNL occurred when the cells were incubated with exogenous AA. It is well known that synthesis of LTB 4 from endogenous AA results from the activation of two independent calcium-dependent events. First, the release of AA from membrane phospholipids and second, the activation of the 5-LO. 3 In the present study, calcium ionophore A23187 increases Ca 2 + influx as well as intracellular Ca 2+ and therefore causes the release of AA from membrane phospholipids and activation of 5-LO.
Another interesting facet of the present study is that LY 255283 causes a decrease in the level of TXB 2 in monocytes. LTB 4 increases PGE2 and TXA2 synthesis in rat peritoneal macrophages elicited by carrageenin injection. 4 The results of the present study indicate that LY 255283 inhibited the synthesis of TXB2 of human peripheral monocytes, one of another mononuclear cell population. A possible explanation of this result is that the increase of LTB 4 induced by ionophore may activate the cyclooxygenase in human peripheral monocytes; therefore, this may increase the synthesis of TXA2. LY 255283 may decrease the level of TXB 2 in monocytes by inhibiting the synthesis of LTB 4. One can assume that the LTB 4 receptor blocker, LY 255283, may also suppress the synthesis of LTB4 by inhibiting 5-LO or LTA 4 hydrolase. Such an effect of the compound has not been described previously by other investigators yet and needs further studies using purified 5-LO or LTA4 hydrolase.
In conclusion, besides LTB4's activation of immunoinflammatory cells like human peripheral PMNL and monocytes, LTB 4 can also activate its own synthesis in these cells and activate the synthesis of TXB2 in human monocytes. LTB 4 receptor antagonists may provide an additional useful effect by antagonising the self-stimulatory effect of LTB4.