The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor

The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 ± 10% (mean ± S.E.M., n = 13) and 182 ± 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b β2-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.

However, both IL-8 and GM-CSF increase 5-LO activity in response to other agonists, 8'12'6'18 and this may contribute to the priming effect of these cytokines on the respiratory burst. 6 This is because the products of 5-LO, 5-hydroxyeicosatetraenoic acid (5-HETE) 9 and LTB420'21 themselves potentiate superoxide production. A recent study of respiratory burst priming showed that partial inhibition of 5-LO, did not prevent respiratory burst priming by either TNFz or GM-CSF, .2 but because LTB4 release was inhibited by less than 70%, these data are inconclusive.
Cells were stimulated with either 1/IM formylmethionyl-leucyl-phenyalanine (fMLP) or 1.67/.zM 12-Otetradecanoylphorbol- 13 with occasional mixing. Under these conditions approximately 80% of the isotope was taken into the cells. The radiolabelled cells were centrifuged (180 x g, 5 min) and the supernatant containing unincorporated isotope was removed. The cell pellet was washed four times in PBSG and finally resuspended to 2 x 106 cells/ml PBSG. Control experiments measuring superoxide production showed that this labelling procedure did not itself cause priming of neutrophils. 0.5 ml aliquots were equilibrated to 37C, mixed with either diluent, IL-8 (100 ng/ml), or GM-CSF (10 ng/ml) and incubated for 30 min at 37C. Arachidonate metabolism was stimulated with Ca 2+ ionophore A23187 (1 #M) for 7 min and the reaction was terminated by placing the samples on ice. The samples were centrifuged (12 000 x g', 2 min) and 0.4 ml aliquots of the supernatants were assayed for radioactivity by liquid scintillation spectroscopy. The cell pellets were lysed with 0.1% Triton-X-100, made up to 0.5 ml volume, and 0.4ml aliquots were also assayed for radioactivity.  Table 1 shows the effects of the cytokines, IL-8 and GM-CSF on the release of radioactivity from both resting and ionophore-stimulated neutrophils. The release of radioactivity stimulated by IL-8 Confirmation of the inhibition of 5-1ipoxygenase in GM-CSF primed cells by piriprost and MK-886 using RP-HPLC" The authors have previously shown using RP-HPLC that total cellular leukotriene synthesis and extracellular release by ionophore-stimulated neutrophils is completely inhibited by 87/M piriprost. 26 In the present study total eicosanoid production was similarly measured in ionophorestimulated cells by extracting both the cells and supernatants together as described in the methods and then separating the leukotrienes by RP-HPLC. Neutrophils were incubated with either 100 nM MK-886 or 0.1% DMSO for 5 min before exposure to GM-CSF (10 ng/ml) or diluent for 30 min. All samples were stimulated with 1 HM A23187 for 10 min. In samples not exposed to MK-886, peaks were identified that had the spectral characteristics of LTB4 and its oxidation products, however the peptidoleukotrienes, LTC4, LTD4 and LTE4 were not detected. Figure 2 shows the data for GM-CSF primed neutrophils and similar data (not shown) were obtained with unprimed cells. Figure 2 also shows the complete inhibition of leukotriene production in GM-CSF primed neutrophils preincubated with 100 nM MK-886. Similar inhibition of leukotriene synthesis by MK-886 was seen in unprimed neutrophils (data not shown).
The eject of 5-go inhibitors on the activity of IL-8 and GM-CSF a) Priming of fmLP-stimulated superoxide generation: Neutrophil superoxide production was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome c. Initial experiments showed that the 5-LO inhibitors, piriprost (87/M) and MK-886 (100 nM), did not inhibit respiratory burst activity stimulated by 1.67 #M TPA, demonstrating that the doses used were not toxic to the cells and that these inhibitors did not inhibit protein kinase c (data not shown). Neither did piriprost nor MK-886 inhibit the generation of superoxide stimulated by 1/M fMLP (Table 2).  Figure 3 shows that IL-8 rapidly stimulated the increased expression of CD11b antigen on purified neutrophils. CD11b expression was upregulated more slowly by GM-CSF, reaching a maximum by 20 min. When cells were pre-incubated with either 100nM MK-886 or 87/M piriprost before exposure to cytokines, there was no change in either the kinetics or magnitude of the response (Fig. 3).   trophils, however this fungal metabolite grossly alters neutrophil phospholipid metabolism and is itself a priming agent, iv Using specific inhibitors of arachidonate 5lipoxygenase, piriprost, 22 and the indole derivative, MK-886, 2 which specifically binds to the membrane protein FLAP, inhibiting translocation of 5-LO and leukotriene production in intact cells, 28 it has been demonstrated that the release of [H]arachidonate metabolites from unprimed or IL-8 primed neutrophils could be blocked by greater than 90%. That these compounds inhibited both the intracellular synthesis and extracellular release of leukotrienes in unprimed and GM-CSF primed cells was confirmed by RP-HPLC. In this study the Ca 2+ ionophore A23187 was used to demonstrate the efficacy of these compounds because it is the most potent stimulus for neutrophil leukotriene production. The inhibition observed with these compounds was not due to any toxic et:fect because at the same concentrations neither inhibited the generation of superoxide stimulated by phorbol ester or fMLP.
The authors sought to determine whether leukotrienes mediate the signal transduction or priming activities of GM-CSF and IL-8. Previous studies have shown little evidence for the involvement of 5-LO in IL-8 signalling, but studies of Rapoport et al. 6 have suggested a role for this enzyme in GM-CSF signal transduction. Our data exclude the possibility that leukotrienes function in this respect, as the upregulation of cell adhesion molecules directly stimulated by IL-8 and GM-CSF was not altered by complete inhibition of 5-LO. Priming of the fMLP stimulated respiratory burst by IL-8 and GM-CSF was similarly unaffected by inhibition of 5-LO.
It is concluded that leukotriene synthesis does not mediate either the direct activity or the priming eects of IL-8 and GM-CSF on mature neutrophils. The study shows that while therapeutic treatment of inflammation with 5-1ipoxygenase inhibitors will inhibit leukotriene production by neutrophils exposed to cytokines, it is unlikely to inhibit other activities of these cytokines such as those associated with upregulation of cell adhesion proteins, degranulation, adhesion and migration, or increased oxygen free-radical production.