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• W1999333882 abstract "Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A4(LTA4) by another and this can then be exported for conversion to LTB4 or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC4 synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA4 products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA4 that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA4products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB4by the LTA4 hydrolase. This result was not because of saturation of the LTA4 hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC4 synthase), the LTA4 synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA4 for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism. Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A4(LTA4) by another and this can then be exported for conversion to LTB4 or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC4 synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA4 products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA4 that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA4products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB4by the LTA4 hydrolase. This result was not because of saturation of the LTA4 hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC4 synthase), the LTA4 synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA4 for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism. Enzymatic oxidation of arachidonic acid has long been implicated as a critical step in the production of mediators of inflammatory events (1Henderson W.R.J. Ann. Intern. Med. 1994; 121: 684-697Crossref PubMed Scopus (584) Google Scholar, 2Vane J.R. Botting R.M. Inflamm. Res. 1998; 47 (suppl.): 78-87Crossref Scopus (577) Google Scholar, 3Busse W.W. Am. J. Respir. Crit. Care Med. 1999; 157 (suppl.): 210-213Google Scholar). Cyclooxygenase, in particular COX-2 (4Smith W.L. DeWitt D.L. Arakawa T. Spencer A.G. Thuresson E.D. Song I. Thromb. Haemostasis. 1997; 78: 627-630Crossref PubMed Scopus (7) Google Scholar), as well as the leukotriene pathway (5Samuelsson B. Adv. Exp. Med. Biol. 1997; 433: 1-7Crossref PubMed Scopus (9) Google Scholar) are known to play important roles in this process. Human neutrophils express 5-lipoxygenase (6Ford-Hutchinson A.W. Gresser M. Young R.N. Annu. Rev. Biochem. 1994; 63: 383-417Crossref PubMed Scopus (416) Google Scholar), 5-lipoxygenase-activating protein (7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (650) Google Scholar), and LTA4 1The abbreviations used are:LTA4leukotriene A4cys-LTcysteinyl leukotrieneGM-CSFgranulocyte macrophage colony-stimulating factorfMLPformylmethionylleucylphenylalanineRP-HPLCreverse-phase high pressure liquid chromatographyPGB2prostaglandin B2 1The abbreviations used are:LTA4leukotriene A4cys-LTcysteinyl leukotrieneGM-CSFgranulocyte macrophage colony-stimulating factorfMLPformylmethionylleucylphenylalanineRP-HPLCreverse-phase high pressure liquid chromatographyPGB2prostaglandin B2 hydrolase (8Minami M. Ohno S. Kawasaki H. Radmark O. Samuelsson B. Jornvall H. Shimizu T. Seyama Y. Suzuki K. J. Biol. Chem. 1987; 262: 13873-13876Abstract Full Text PDF PubMed Google Scholar) and are, therefore, able to convert free arachidonic acid into the potent chemotactic and chemokinetic factor, LTB4. The importance of the leukotriene pathway in general is demonstrated by the significantly limited inflammatory response to specific stimuli seen in mice with a targeted disruption of the 5-lipoxygenase gene (9Chen X.S. Zhang Y.Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Neutrophils also express cytosolic phospholipase A2, which specifically releases arachidonic acid from glycerophosphocholine lipids for eicosanoid generation (10Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar), and targeted disruption of this gene also reduces inflammation (11Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar). However, the processes involved have, for the most part, been studied within a uniform cell population; in whole tissues eicosanoid biosynthesis, and specifically leukotriene biosynthesis, is much more complex resulting from the intricate interplay between different cells, cellular events, and enzymatic reactions. For example, contact between cooperating cells can lead to the multidirectional transfer of arachidonate or reactive intermediates of this fatty acid such as LTA4.Adhesion of neutrophils to vascular endothelial cells is a characteristic feature of inflammation involving numerous adhesion proteins that regulate their transmigration across the vascular endothelial barrier (12Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar). The microenvironment at the interface between adherent neutrophils and endothelial cells is suggested to represent a strategic site for exchange, transcellular synthesis, and metabolism of lipid mediators. Neutrophils by themselves do not synthesize cys-LT but, in cooperation with endothelial cells (or platelets), lead to the production of these mediators by the exchange of newly synthesized LTA4 to the endothelial cell or platelet that contains constitutively active leukotriene synthase (13Feinmark S.J. Cannon P.J. J. Biol. Chem. 1986; 261: 16466-16472Abstract Full Text PDF PubMed Google Scholar, 14Maclouf J.A. Murphy R.C. J. Biol. Chem. 1988; 263: 174-181Abstract Full Text PDF PubMed Google Scholar). This cooperative formation of cys-LT has been shown to cause coronary vasoconstriction and severe inflammatory changes in the rabbit heart (15Sala A. Rossoni G. Buccellati C. Berti F. Folco G. Maclouf J. Br. J. Pharmacol. 1993; 110: 1206-1212Crossref PubMed Scopus (54) Google Scholar, 16Sala A. Aliev G.M. Rossoni G. Berti F. Buccellati C. Burnstock G. Folco G. Maclouf J. Blood. 1996; 87: 1824-1832Crossref PubMed Google Scholar). Furthermore, pharmacological manipulations aimed at altering the neutrophil-endothelial cell adhesion process, such as the application of nitric oxide and prostacyclin, have also been shown to reduce not only the cys-LT production but also, secondarily, to alter coronary vascular reactivity and cardiac function (17Rossoni G. Sala A. Berti F. Testa T. Buccellati C. Molta C. Muller-Peddinghaus R. Maclouf J. Folco G.C. J. Pharmacol. Exp. Ther. 1996; 276: 335-341PubMed Google Scholar, 18Buccellati C. Rossoni G. Bonazzi A. Berti F. Maclouf J. Folco G. Sala A. Br. J. Pharmacol. 1997; 120: 1128-1134Crossref PubMed Scopus (17) Google Scholar).Previous studies from our group have suggested that the transcellular biosynthesis process is even more complicated and may often involve an additional exogenous supply of arachidonate from the platelet or endothelial cell, which is taken up by the neutrophil for conversion to the LTA4, that is then handed back to the donor cell for final conversion to cys-LT (19Antoine C. Murphy R.C. Henson P.M. Maclouf J. Biochim. Biophys. Acta. 1992; 1128: 139-146Crossref PubMed Scopus (31) Google Scholar). To gain further insight into the biochemistry of this neutrophil-endothelial cell interaction for generation of bioactive leukotrienes within the context of a functioning organ system, we investigated the metabolic profile of LTA4-derived metabolites in isolated neutrophils as well as when perfused through a spontaneously beating, isolated rabbit heart. The study demonstrates that the metabolic fate of arachidonate released from the endogenous pool of neutrophil phospholipids is different from that of arachidonate derived from exogenous sources.RESULTSInitial experiments evaluated the profile of 5-lipoxygenase products formed after the challenge of neutrophils alone,i.e. utilizing endogenous sources of arachidonate. Human neutrophils (107 ml−1 in suspension) were primed with GM-CSF and stimulated with fMLP. Significant amounts of LTA4 metabolites were synthesized with maximal production at concentrations higher than 0.1 μm (Fig.1). The most abundant metabolite observed was 20-COOH-LTB4 with lower amounts of 20-OH-LTB4 and LTB4. Nonenzymatic LTA4 metabolites were not seen, and in the absence of endothelial cells or platelets, no LTC4 was produced (Fig. 2 A).Figure 2RP-HPLC analysis of LTA4 metabolites produced by isolated rabbit hearts perfused with GM-CSF-primed human neutrophils. Representative chromatograms are shown from: (A) isolated human neutrophils (107cells) challenged with fMLP (0.3 μm) after priming with GM-CSF (1 nm, 30 min); (B) the entire recirculating perfusate obtained 60 min after the fMLP (0.3 μm) challenge of isolated rabbit heart with no neutrophils; and (C) the entire recirculating perfusate of isolated rabbit heart in the presence of GM-CSF-primed human neutrophils (107 cells) 60 min after the challenge with fMLP (0.3 μm). PGB2 (25 ng) was used as an internal standard. UV absorbance spectra of peaks a–f eluting at the characteristic retention time of 20-COOH-LTB4, 20-OH-LTB4, LTB4LTC4, LTD4 and LTE4, respectively, are shown in insets. Chromatographic peaks labeled withx represent uncharacterized background UV-absorbing material resulting from the extraction of the large volumes of recirculating buffer in the heart perfusion experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In agreement with previous results (16Sala A. Aliev G.M. Rossoni G. Berti F. Buccellati C. Burnstock G. Folco G. Maclouf J. Blood. 1996; 87: 1824-1832Crossref PubMed Google Scholar), an fMLP challenge of the same number of neutrophils in the reperfused, spontaneously beating, isolated rabbit heart resulted in the production of large amounts of cys-LT (Fig. 2 C), presumably as a result of neutrophil-endothelial cell cooperative synthesis of cys-LT (see Ref.23Sala A. Maclouf J. Folco G. Murphy R.C. Samuelsson B. Inhibitors of Leukotrienes. Birkhauser, Basel, Switzerland1999: 113-124Google Scholar). Whereas no LTA4 products were seen in the perfusate of fMLP-challenged isolated rabbit hearts alone, quantitative analysis revealed that in the neutrophil-perfused hearts the amount of LTA4 metabolites detected was greatly increased and, importantly, was much more that that seen when the same number of neutrophils were stimulated in vitro (Fig.3). The data suggested that neutrophil interaction with the coronary vasculature of the heart not only led to the new synthesis of cys-LTs (transcellular biosynthesis) but also to a 4-fold increase in total LTA4 metabolites, likely because of additional sources of externally derived (nonneutrophil) arachidonate.Figure 3Increased production of LTA4 metabolites by the combination of GM-CSF primed human neutrophils and isolated rabbit hearts. Effect of challenge with fMLP (0.3 μm) on the production of LTA4 metabolites by isolated rabbit hearts, by neutrophils in suspension, and by neutrophils perfusing the isolated rabbit heart under recirculating conditions. Purified human neutrophils (107 cells) were primed with GM-CSF (1 nm, 30 min) prior to perfusion. LTA4 metabolites were measured by RP-HPLC. Values are expressed as mean ± S.E. (n = 3–5). ***,p < 0.001. PMNL, neutrophils.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Under the working hypothesis that arachidonate released from endothelial cells (therefore “exogenous” arachidonate) might represent a privileged precursor responsible for both the increase in LTA4 metabolites observed and also the preferential conversion of LTA4 into products of transcellular metabolism, the metabolic fate of exogenous arachidonic acid supplied to human neutrophils was investigated. To minimize the potential homotypic transcellular metabolism occurring during neutrophil activation in suspension (20Sala A. Bolla M. Zarini S. Muller-Peddinghaus R. Folco G. J. Biol. Chem. 1996; 271: 17944-17948Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), the number of neutrophils was limited to 2 × 106, and cells were allowed to adhere to a plastic surface at the density of 2 × 105cm−2 for 5 min prior to the challenge. Simultaneous evaluation of the metabolic fate of endogenous and exogenous arachidonate was carried out using GM-CSF-primed neutrophils challenged with fMLP in the presence of d8-arachidonate (5 μm). The results shown in Fig.4 indicate a significantly increased abundance of deuterium label (increasedd8/d0 ratio) in nonenzymatic LTA4 hydrolysis products (e.g.Δ6-trans-LTB4 isomers and 5,6-dihydroxyeicosatetraenoic acids) when compared with LTA4 hydrolase metabolites (e.g.LTB4, 20-OH-LTB4 and 20-COOH-LTB4). The LTA4 derived from exogenous arachidonate therefore seemed to be preferentially excluded from the enzymatic conversion to LTB4 and its subsequent metabolites. Relatively low levels of 20-OH-LTB4 and 20-COOH-LTB4 were seen in this experiment compared with Fig. 1 (data not shown) probably reflecting the absence of cell-cell contact and the transcellular metabolism needed for ω-oxidation of LTB4 (20Sala A. Bolla M. Zarini S. Muller-Peddinghaus R. Folco G. J. Biol. Chem. 1996; 271: 17944-17948Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 24Cluzel M. Undem B.J. Chilton F.H. J. Immunol. 1989; 143: 3659-3665PubMed Google Scholar).Figure 4Differential metabolism of exogenousversus endogenous arachidonate in adherent human neutrophils. Relative ratio of d8-labeled to unlabeled LTA4 metabolites in adherent human neutrophils challenged with fMLP in the presence ofd8-arachidonate (5 μm). LTA4 metabolites were separated by RP-HPLC and analyzed using a triple quadrupole mass spectrometer as described under “Materials and Methods.” Values are expressed as mean ± S.E. (n = 3). *, p < 0.05. AA, arachidonic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)One possible explanation for the observed shift with exogenous arachidonate toward a preferential release of LTA4, rather than conversion into LTB4, was that the LTA4hydrolase might have been saturated with substrate. Accordingly, the relative ratio of enzymatic to nonenzymatic metabolites of LTA4 resulting from the addition of increasing amounts of arachidonate in unprimed neutrophils was investigated. As shown in Fig.5, this ratio remained unchanged even when the concentration of exogenous arachidonate was varied (1–10 μm). A comparison of LTA4 product level showed that administration of 2–5 μm exogenous arachidonate together with fMLP in unprimed neutrophils led to amounts of metabolites not different from that observed in the GM-CSF-primed, fMLP-stimulated cells (Fig. 6). In keeping with the results observed with deuterium-labeled arachidonate, when unprimed neutrophils were challenged with fMLP and exogenous arachidonate (2–5 μm) more nonenzymatic LTA4metabolites were produced than seen with GM-CSF-primed cells stimulated with fMLP in the absence of added arachidonate (Fig.7 A), and the ratio of nonenzymatic to enzymatic products showed a 2-fold increase (Fig.7 B). To determine if the increased production of nonenzymatic metabolites reflected an increased availability of intact LTA4 for transcellular metabolism, GM-CSF-primed neutrophils and platelets were co-incubated at a ratio of 1:40 (neutrophil:platelet) and challenged with fMLP (which does not activate the platelets) in the presence of deuterium-labeled arachidonate (5 μm). This resulted in the preferential formation of deuterium-labeled LTC4 (ratio ofd8-LTC4/LTC4 is 2.3) when compared with enzymatic metabolites of LTA4-hydrolase (ratio ofd8-20-OH-LTB4/20-OH-LTB4is 1.2) (Fig. 8).Figure 5Relative amounts of LTA4 escaping LTA4-hydrolase metabolism at different concentrations of exogenous arachidonate. Ratio of nonenzymatic to enzymatic metabolites of LTA4 in adherent human neutrophils upon a challenge with fMLP (0.1 μm) in the presence of exogenous arachidonate (1–10 μm). LTA4 metabolites were measured by RP-HPLC. Values are expressed as mean ± S.E. (n = 3). AA, arachidonic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Total LTA4 metabolites produced by GM-CSF-primed human neutrophils compared with unprimed neutrophils supplied with arachidonate. fMLP induced production of LTA4 metabolites in human neutrophils either after priming with GM-CSF or in the absence of priming but with increasing concentrations of arachidonate (1–5 μm). LTA4 metabolites were measured by RP-HPLC. Values are expressed as mean ± S.E. (n = 3).n.d., not detectable; *, p < 0.05versus GM-CSF-primed neutrophils; AA, arachidonic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Nonenzymatic LTA4 metabolites produced by GM-CSF-primed human neutrophils compared with unprimed neutrophils supplied with arachidonate. fMLP induced production of nonenzymatic LTA4 metabolites (A) and the ratio of enzymatic to nonenzymatic products (B) in human neutrophils either after priming with GM-CSF or in the absence of priming but in the presence of exogenous arachidonate (2 and 5 μm). LTA4 metabolites were measured by RP-HPLC. Values are expressed as mean ± S.E. (n = 3). *, p < 0.05 versus GM-CSF-primed neutrophils; AA, arachidonic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Differential metabolism of exogenousversus endogenous arachidonate in human neutrophils incubated with platelets. Selected ion chromatograms (negative ions) of LTA4 metabolites resulting from stimulating surface-adherent human neutrophils with fMLP in the presence ofd8-arachidonate (5 μm) and human platelets (in a ratio of 1:40, neutrophil:platelet). A,20-hydroxy-LTB4 derived from endogenous arachidonate (9.6 min, m/z 351); B,20-hydroxy-LTB4 derived from exogenous arachidonate (9.5 min, m/z 359); C, leukotriene C4 derived from endogenous arachidonate (13.9 min,m/z 624); and D, leukotriene C4 derived from exogenous arachidonate (13.8 min,m/z 632). Each individual panel is normalized to the abundance of the corresponding arachidonate metabolite, and the ratio of the arbitrary abundance units (relative intensity) provides the relative ratio of endogenous to exogenous source of arachidonate involved in the biosynthesis of the leukotriene. LTA4 metabolites were separated by RP-HPLC and analyzed using a triple quadrupole mass spectrometer as described under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONStudies with isolated neutrophil preparations have illustrated the mechanisms underlying 5-lipoxygenase activation and the production of biologically relevant amounts of leukotrienes. In addition, however, transcellular metabolic events involving cooperation between different cell species in the synthesis of eicosanoids critically determine the overall quantity, as well as profile, of arachidonate metabolites generated by cell activation at the organ level. We and others (25Maclouf J. Murphy R.C. Henson P. Blood. 1989; 74: 703-707Crossref PubMed Google Scholar, 26Brady H.R. Serhan C.N. Biochem. Biophys. Res. Commun. 1992; 186: 1307-1314Crossref PubMed Scopus (79) Google Scholar) have previously reported transcellular synthesis of cys-LT in mixed cell systems and more recently, the functional consequences of this process, in a model of a spontaneously beating, neutrophil-perfused rabbit heart (15Sala A. Rossoni G. Buccellati C. Berti F. Folco G. Maclouf J. Br. J. Pharmacol. 1993; 110: 1206-1212Crossref PubMed Scopus (54) Google Scholar, 20Sala A. Bolla M. Zarini S. Muller-Peddinghaus R. Folco G. J. Biol. Chem. 1996; 271: 17944-17948Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).In the present study we addressed the qualitative and quantitative changes in the profile of arachidonate metabolites produced by the activation of human neutrophils during perfusion of the isolated rabbit heart. In this environment, fMLP stimulation of GM-CSF-primed human neutrophils resulted in the production of very large amounts of cys-LT. In the absence of neutrophils the isolated perfused hearts did not generate detectable LTA4 products. In sharp contrast, the same number of GM-CSF-primed neutrophils stimulated with fMLP in vitro produced significantly lower amounts of overall LTA4 metabolites and only LTA4hydrolase-derived products, namely LTB4 and its ω-oxidized derivatives, whereas LTC4 as well as nonenzymatic LTA4 metabolites could not be observed. This observation led to the hypothesis that additional arachidonate was provided to the neutrophils by rabbit heart cells, likely endothelial cells, thereby significantly enhancing the production of leukotrienes. The alternative suggestion that the leukotrienes came from the rabbit cells has been previously ruled out, showing that their formation is completely dependent on neutrophil 5-lipoxygenase (15Sala A. Rossoni G. Buccellati C. Berti F. Folco G. Maclouf J. Br. J. Pharmacol. 1993; 110: 1206-1212Crossref PubMed Scopus (54) Google Scholar). Furthermore it appeared likely that this additional arachidonate supplied to neutrophils was undergoing a preferential metabolism into a pool of LTA4 that was readily exported outside of the neutrophil and therefore available for transcellular metabolism into LTC4 by adjacent endothelial cells.The suggestion that exogenous arachidonate plays a critical role in this process was tested in GM-CSF-primed isolated neutrophils challenged in the presence of exogenous, deuterium-labeled arachidonate. This allowed a distinction between metabolites derived from endogenous or exogenous arachidonate. The relative amount of labeled and unlabeled enzymatic (LTA4 hydrolase) metabolites of LTA4 was compared with that of nonenzymatic metabolites as an index of exported LTA4. The results obtained showed that significantly larger amounts of the exogenous arachidonate underwent preferential metabolism to an LTA4pool that was not apparently available to cytosolic LTA4hydrolase, thus resulting in export and nonenzymatic hydrolysis. As predicted, in the presence of a limited number of platelets, the LTA4 arising from metabolism of exogenous arachidonate was preferentially converted into LTC4 by the platelets (rather than into LTB4) as a result of transcellular cooperation between neutrophils and platelets, as described previously.One possible explanation for the different pathway exhibited for exogenous versus endogenous arachidonate was that the LTA4 hydrolase was readily saturated and/or suicide inactivated so that excess arachidonate supplied from outside the neutrophil was oxidized to LTA4 but was unable to be enzymatically hydrated. However, the arachidonate dose-response experiments suggested that this was not the case in that external arachidonate supplied to fMLP-stimulated, unprimed neutrophils at a concentration that yielded the same amount of total LTA4metabolites as that seen from only endogenous sources (fMLP with GM-CSF-primed neutrophils with no added arachidonate) still resulted in preferential production of nonenzymatic products.The data led to the suggestion that exogenous arachidonate is metabolized to leukotrienes by a pathway that is physically separate in the cell involved in conversion of arachidonate from endogenous sources. The simplest version of this concept is that a portion of the 5-lipoxygenase is located at (or translocated to) the plasma membrane and there preferentially metabolizes arachidonate reaching the cell from the outside. LTA4 produced at this site would be available for export and may have some difficulty gaining access to the cytosolic LTA4 hydrolase. More complex pathways may exist but again would require maintenance of separate pools of arachidonate and protection of the exogenous arachidonate-derived LTA4from the hydrolase. These possibilities are currently under investigation. Whatever the mechanism, the observations suggest that this unique pathway of arachidonate metabolism and eicosanoid generation occurs with physiologic cell stimuli not just with deliberately added arachidonate but also with arachidonate derived from adjacent cells and can also be extended to an intact organ, the isolated perfused heart. In this system, the complex transcellular metabolism event(s), namely arachidonate from endothelial cells to neutrophils and LTA4 back to the endothelial cells, seems to result in preferential production of cysteinyl leukotrienes and, as we have shown previously, significant physiologic consequences to the organ as a whole (16Sala A. Aliev G.M. Rossoni G. Berti F. Buccellati C. Burnstock G. Folco G. Maclouf J. Blood. 1996; 87: 1824-1832Crossref PubMed Google Scholar). Enzymatic oxidation of arachidonic acid has long been implicated as a critical step in the production of mediators of inflammatory events (1Henderson W.R.J. Ann. Intern. Med. 1994; 121: 684-697Crossref PubMed Scopus (584) Google Scholar, 2Vane J.R. Botting R.M. Inflamm. Res. 1998; 47 (suppl.): 78-87Crossref Scopus (577) Google Scholar, 3Busse W.W. Am. J. Respir. Crit. Care Med. 1999; 157 (suppl.): 210-213Google Scholar). Cyclooxygenase, in particular COX-2 (4Smith W.L. DeWitt D.L. Arakawa T. Spencer A.G. Thuresson E.D. Song I. Thromb. Haemostasis. 1997; 78: 627-630Crossref PubMed Scopus (7) Google Scholar), as well as the leukotriene pathway (5Samuelsson B. Adv. Exp. Med. Biol. 1997; 433: 1-7Crossref PubMed Scopus (9) Google Scholar) are known to play important roles in this process. Human neutrophils express 5-lipoxygenase (6Ford-Hutchinson A.W. Gresser M. Young R.N. Annu. Rev. Biochem. 1994; 63: 383-417Crossref PubMed Scopus (416) Google Scholar), 5-lipoxygenase-activating protein (7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (650) Google Scholar), and LTA4 1The ab" @default.
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• W1999333882 title "Differential Metabolism of Exogenous and Endogenous Arachidonic Acid in Human Neutrophils" @default.
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