Cell–Cell Interaction of Macrophages and Vascular Smooth Muscle Cells in the Synthesis of Leukotriene B4

Biosynthesis of LTB4 during cell-cell interaction between vascular smooth muscle cells (SMC) and alveolar macrophages (AM) has been investigated by use of both high-pressure Hquid chromatography (HPLC) and radtoimmunoassay (RIA). Both interleukin-β (IL-β) and tumour necrosis factor-α (TNFα) induced a time- and dose-dependent synthesis of 15-, and 5-hydroxyeicosatetraenoic acids (HETEs) from cultured SMC. However, neither TNFα nor IL-1β induced a significant LTB4 production in SMC alone or AM alone after 24 h of incubation. Addition of IL-1β and TNFα simultaneously to SMC resulted in a dose-dependent synergistic increase of HETEs. Macrophages dose-dependently transformed extremely low concentrations of exogenous LTA4 into LTB4. Incubation of vascular SMC with various numbers of AM in the presence of IL-1β (5 units/ml) and TNFα (10 units/ml) induced a great increase of LTB4 synthesis in comparison with the detectable levels of LTB4 produced by macrophages alone. Pretreatment of SMC with NDGA, cycloheximide, and actinomycin not only inhibited IL-1 and TNT induced HETEs synthesis but also abolished LTB4 production when co-incubated with macrophages. These results suggest that LTB4 in a mixture of SMC and macrophages could originate from a transcellular metabolism, i.e. macrophages transforming SMC-derived LTA4 into LTB4.


Introduction
The generation of leukotrienes (LTs) by lipoxygenase catalysed reactions is associated with a wide range of cell types that are involved in both physiological and pathological events. The transcellular metabolism of lipoxygenase derived metabolites has recently been reported not only to amplify the level of eicosanoids within a local milieu but also to stimulate the generation of biologically active metabolites with functions different from those in its original cells. For example, the presence of erythrocytes can greatly induce LTB formation from neutrophil-derived LTA4; co-incubation of endothelial cells, smooth muscle cells or platelets with neutrophils, can result in transcellular LTC4 synthesis. Perfusion of blood free rabbit lung and isolated pulmonary artery with neutrophils induced an overall Lwg4, Ewe and LTD release. The transfer of unstable metabolic intermediates has been considered to be a biochemical basis for these phenomena?- 5.9a0 Macrophages are well known to play a central role in the modulation of inflammatory and immune processes, as well as in tissue injury and repair. 1 Macrophages have 5-1ipoxygenase activity that inserts molecular oxygen into arachidonic acid to form an unstable peroxide 5-HPETE leading to the generation of 5-HETE, or through the epoxide intermediate, LTA4, to the .generation of LTB4 by hydrolase; or conjugation with glutathione by glutathione-Stransferase to form the cysteine LTC4, D and E4. 2'1 Recently, many studies have indicated that macrophages are a potent source of arachidonate metabolites generated via lipoxygenase pathways. 1-1s In vivo, SMC and macrophages have been noted to interact at the site of thrombosis, vessel injury and inflammation by secreting interleukin-1 (IL-1), tumour necrosis factor (TNF), prostaglandins (PG) and leukotrienes. 15a6 This investigation was designed to study whether or not a shift in the metabolic profile of arachidonate products generated by activated macrophages or vascular SMC could be induced under circumstances that may be related more closely to those operative inflammatory reactions in vivo.

Materials and Methods
Reagents: Human recombinant IL-1]] and TNF0t were obtained from Sigma (Saint Quentin Fallavier, France). Labelled H-AA and radioactive standards were from Amersham (Aylesbury, UK). Synthetic LTA4 methyl ester from Sigma was hydrolysed to yield the free acid according to the methods described by Maycock. 17 Octadecyl silica Sep-Pak cartridges were obtained from Millipore/Waters (Les Ulis, France). Media, sera and reagents for cell cultures were obtained, if not further specified, from Gibco (Paisley, UK). All solvents used in chromatographic systems were of HPLC grade. Vascular SMC culture:. Vascular SMC cells were obtained by dissociation of tissues from rat abdominal aorta with 0.05% EDTA and 0.1% trypsin in HAM F10 medium. During the first 2 weeks, the cells were cultured at 37C, with 5% CO,. in HAM F10 medium supplemented with 20% foetal calf serum. When a monolayer was obtained, the cells were removed by trypsin-EDTA (0.05-0.02%) and cultured in 25 ml flasks in HAM F10 medium, supplemented with 10% foetal calf serum and 1% penicillin-streptomycin. Culture SMC grew in typed 'hill and valley' formation. Cells up to the 25th passage were used for the experiments.
Alveolar macrophage isolation and culture: Respiratory disease-free 125 to 150 g female Wistar rats were housed under pathogen-free condition. Rats were anaesthetized with i.p. sodium pentobarbital and lungs were exercised and washed as described previously. TM Bronchoalveolar lavage cells were 96% AM by microscopic examination of cytocentrifuge preparations stained with a modified Wright-Giemsa stain (Diff-Qick, American Scientific Products, IL). Bronchoalveolar cells (5 x 106) suspended in 10 ml of M199 were plated in 25 ml flasks and cultured at 37C in a humidified atmosphere of 5% CO,. in air. After 2 h, nonadherent cells were removed by washing twice with the medium. Cell monolayers were then cultured in M199 containing 10% heat inactivated newborn calf serum before experimental incubation.
3H-arachidonic acid metabolism in macrophages and SMC: Monolayer vascular SMC cells (5 x 106) or macrophages (5 xl06) were plated in a fresh culture medium. The cells were incubated with 1 btM 3H-AA for 18 h and then the medium was harvested. The cells were washed three times with PBS containing 0.25% fatty acid free bovine serum albumin (BSA) to eliminate non-incorporated AA. These cells were covered with 7 ml of serum free medium. IL-1]] (5 units/ml) and TNF (10 units/ml) were added and the cells were incubated at 37C for 24 h. Then the culture medium was harvested and centrifuged. The supernatants were stored at-80C for eicosanoid assay.
Co-culture of macrophages and SMC: Biosynthesis of gWB during cell-cell interaction was studied by incubating fixed concentrations of H-AA labelled SMC with various numbers of alveolar macrophages. For co-culture experiments, H-labelled SMC (5x 106 cells) were further incubated with macrophages in the presence of IL-113 (5 units/ml) and TNF (10 units/ml) at 37C for 24 h following the ratios of SMC to macrophages: SMC alone, 100:1, 10:1, 1:1, 1:10, 1:100, macrophages alone, and macrophages alone but in the presence of IL-I and TNF. After the incubation, the supernatants were collected, centrifuged and stored at-80C until eicosanoid assay. Transformation of lra4 by macrophages: Macrophages (lx 106 cells) in 35 mm wells were allowed to equilibrate for 5 min at 37C in 1 ml HBSS/BSA and then incubated at 37C for 15 min with LTA4 at concentrations described in the figure legends. At the end of the incubation, 1 ml of icecold methanol was added and the medium was harvested, centrifuged and kept at-20C for analysis by RIA for LTB4. HPLC analysis: The HPLC system utilized was from Waters associated (Milford, MA) using two pumps (6000A and 510) coupled to a Model 680 gradient controller. UV absorbance of the eluate was monitored using two serial detectors. Radioactivity was determined by mixing 1 ml of the effluent with 5-10 ml of Atomlight (NEN) and counting in a liquid scintillating counter.
The procedure used for the precolumn extraction/ RP-HPLC of the supernatant was similar to that described previously, 19 with the six-port valve in the 'load' position. The sample, diluted to a total volume of 10 ml, was applied to a C8 reverse-phase guard column (Nucleosil C, 10 l.tm, 10 mm, Macherey Nagal) located in the sample loop of the six-port switching valve (Rheodyne) which had been equilibrated with solvent A (2.5 mH H3PO in 15% methanol). A 3 btm filter was placed between the outlet of the pump and the six-port valve. The precolumn was then washed with 8 ml of solvent A. The AA metabolites, remaining on the precolumn, were injected by turning the six-port valve to the 'inject' position onto a Spherisorb ODS2 (5 btm, 4.6 x 150 mm) column (phase separation). RP-HPLC was carried out using a mobile phase consisting of a nonlinear gradient starting with 30% solvent B (water/ acetic acid 0.05, v/v, buffered to pH 5.7 with ammonium hydroxide) leading to 100% solvent C (65% acetonitrile-35% methanol) with the following program: 0-10 min, linear to 65%; 10   was significantly greater than that induced by TNF0t (Fig. 2). The response to IL-1 or TNF was also time-dependent having a lag phase of 8 h before significant generation of HETEs in both cases (Fig. 2).
The simultaneous addition of IL-1I] and TNF0t stimulated HETE release to a greater extent than did either agent alone (Fig. 2). The effect was additive and the sum of the HETEs released by both IL-1 I] and TNF always exceeded the amount of HETEs generated after separate addition of IL-11] or TNF0 to SMC. In addition, preincubation of SMC with NDGA (10 -5 M) inhibited recovery of HETEs induced by IL-li] and TNF0t (90% and 95% inhibition for IL-1 and TNF, respectively). Metyrapone (10 to 10 -4 M), a cytochrome P450 inhibitor, did not modify the recovery of monohydroxylated compounds. The protein synthesis inhibitors cycloheximide (10 -5 M) and actinomycin (10 -s M) also abolished HETEs production induced by IL-1 and TNF. In contrast, pretreatment of cells with aspirin (10 to 10 -5 M) inhibited the synthesis of cyclooxygenase metabolites (20 to 90%) and slightly increased HETEs recovery (  capable of converting exogenous supplies of LTA4 into LTB4. The ability of macrophages to utilize exogenous LTA was examined by incubating macrophages (lx 106 cells) with exogenously added LTA4. As shown in Fig. 3, macrophages were able to transform, in dose-dependent manner, extremely low concentrations of LTA into LTB4, but the amount of LTB4 formed from the exogenous LTA4 (1 to 10 000 pmol) in the absence of AM but in the presence of HBSS/BAS remained very low (5.6 to 10 pmol, as shown in Fig. 3). This finding suggested that transcellular synthesis of LTB4 could occur when very limited amounts of LTA4 were available to these cells. were 14.7_+ 2.5, 34.9-+ 6.7, 17.8_+ 4.3, 12.6_+ 2.9, 6.7 +_ 2.7 and 2.4 + 0.8 pmol/106 cells respectively. In addition, the preincubation of SMC with NDGA (10 I.tM), cycloheximide (10 btM), and actinomycin (10 l.tM) abolished LWB production induced by IL-1 and TNF, when co-incubated with macrophages (Table 2). However, no LTB was found when IL-1and TNF-treated SMC were co-incubated with NDGA pretreated macrophages. In this study, the transcellular synthesis of LTB4 during cell-cell interaction between IL-I[ and TNF(x activated vascular SMC and macrophages has been Adherent smooth muscle cells (5x 106 cells) were incubated with 5x 10 macrophages in the presence of IL-1 (5 units/ml) and TNF (10 units/ml) for 24 h after preincubation with aspirin (10 -s M), NDGA (10 -s M), cycloheximide (10 -s M) and actinomycin (10 - The synthesis of LTB involves a complex series of reactions within macrophages. A limiting factor for LTB biosynthesis was found to be the availability of the unstable intermediate, LTA4. However, the generation of LTA4 depends upon activation of 5lipoxygenase as well as the presence of its substrate, arachidonic acid. '5 The production of LTs by human polymorphonuclear leukocytes has been known to be significantly influenced by adjacent cells possessing distinctly different metabolic properties. '-5 In this study, macrophages transformed very low concentrations of exogenous LTA4 into LTB4 suggesting that macrophages could metabolize LTA4 into biologically active LTB 4. The co-culture of SMC with various ratios of macrophages greatly increased LTB synthesis when compared with very low levels of gwg produced by individual cells. The highest augmentation in gWB was obtained when the ratio of SMC to macrophages was 10:1. Because IL-ll and TNFo: did not induce a significant LTB production either in SMC alone or in macrophages alone, and because the pretreatment of SMC cells and macrophages with the lipoxygenase inhibitor NDGA and protein synthesis inhibitors, blocked LTB formation, it could be concluded that cell-cell interaction could be responsible for this augmentation, i.e. macrophages utilizing SMC-derived LTA or its precursor, 5-HPETE for its syn-thesis of LTB during co-incubation of SMC and macrophages. According to the cell cooperation classification of Marcus, LTB biosynthesis could come from the interaction of type IA (cells can share a common precursor synthesized by different cells). 24 However, the mechanism of LTB4 transcellular synthesis is certainly more complex. Irvine 25 has demonstrated that arachidonic acid availability is a major limiting factor for LTs synthesis. Furthermore, several studies have suggested that a transfer of arachidonic acid takes place in in vitro cell-cell cooperation such as during platelet-neutrophil interaction. 8,m,5,26 For example, Palmentier and Borgeat have presented evidence that thrombin activated platelets offer free arachidonic acid to neutrophils that are utilized for LTB4 synthesis. Antoine et al. have also found that neutrophils can utilize plateletderived arachidonic acid for the formation of the 5lipoxygenase product. Therefore, the possibility that other mechanisms are responsible for LTB synthesis cannot be excluded from the present data.
The transcellular synthesis of l,WB between vascular SMC and macrophages may have an important physiological and pathophysiological significance. The lipoxygenase products of AA metabolism are involved in various aspects of inflammation and atherosclerotic lesions. '7 For example, the intimal accumulation of smooth muscle cells in rabbit carotid arteries, an early stage of atherosclerosis, is inhibited by dexamethasone, which prevents formation of both cyclooxygenase and lipoxygenase metabolites, but is not inhibited by nonsteroidal anti-inflammatory drugs such as indomethacin. ', '9 The transcellular LTB synthesis could be involved in the genesis of atherosclerosis and other vascular diseases because of its action of initiating vascular inflammation, promoting vascular constriction and proliferation as well as platelet aggregation.