Introduction

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Human mononuclear phagocytes migrate from bone marrow to inflamed tissue via the peripheral blood system, and they differentiate into mature macrophages, these being the phagocytic cell of the lineage. Activated monocytes can promote the bronchial inflammatory response by releasing numerous mediators, including arachidonic acid metabolites (Ferreri et al., 1986; Demoly et al., 1994) that contribute to the development of inflammatory processes such as asthma (Rankin, 1989). Histological studies on asthmatic subjects have shown that in airways, macrophages are in close contact with inflammatory cells, particularly mast cells, basophils, and Th2 cells (Bradley et al., 1991); these cells are able to release several immunomodulatory cytokines that may affect macrophage functions and activities.

Among the cytokines potentially playing a role in the inflammatory response IL-4 is known to regulate the expression of 15-lipoxygenase as well as 15(S)-HETE production in human monocytes (Conrad et al., 1992). Bronchial allergen challenge in atopic asthmatic subjects has been found to yield a 30-fold increase in the concentrations of 15(S)-HETE recovered in bronchoalveolar lavage fluid (Murray et al., 1986); interestingly, 15(S)-HETE exerts several immunoregulatory functions that may be relevant in asthma and chronic bronchitis (Samuelsson et al., 1987).

Thus, 15(S)-HETE is a potent mucosecretagogue in the human airway (Marom et al., 1982) and has been reported to possess chemotactic activity for neutrophils, contributing to the recruitment of these cells in the airways (Johnson et al., 1985; Kirsch et al., 1988). Furthermore, it can prolong the duration of airway obstruction during the early response to allergen challenge, augmenting the release of mediators from mast cells and their effects on airway smooth muscle (Lai et al., 1990). Bronchial epithelial cells from asthmatic subjects express more 15-LO immunoreactivity than cells obtained from normal subjects, and this enhanced expression appears to correlate with the clinical severity of the disease (Bradding et al., 1995).

Aside from its proinflammatory actions, 15(S)-HETE may also modulate the inflammatory response through the inhibition of leukotriene production in a variety of cell types (Vanderhoek et al., 1982; Profita et al., 2000a). Different studies have shown that steroid-dependent asthmatic patients generate 5(S),15(S)-diHETE and lipoxins from 15(S)-HETE, with a concomitant reduction of LTB₄ production (Chavis et al., 1998); recently it has been shown that FLAP, in addition to its role in leukotriene biosynthesis, may also function as a more general lipid-binding protein that significantly increases the metabolism of 15(S)-HETE by 5-lipoxygenase (Mancini et al., 1998).

The ability of IL-4 to induce the expression of 15-LO, and the potential effect of 15(S)-HETE on leukotriene biosynthesis prompted us to study the effect of IL-4 treatment of human monocytes on their ability to synthesize leukotrienes. In this study we provide evidence for a potential anti-inflammatory role of IL-4 in human monocytes through the enhanced expression of 15-LO and production of 15(S)-HETE, resulting in inhibition of the synthesis of the potent chemotactic factor LTB₄.

	Materials and Methods

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Reagents. Human recombinant interleukin-4 (IL-4) and polyclonal sheep anti-human IL-4 were obtained from Genzyme (Cambridge, MA). All solvents were HPLC grade and were obtained from Merck (Darmstadt, Germany). Cell culture media were from Invitrogen (Carlsbad, CA). Nutritive medium came from Whittaker (Veviers, Belgium) and fetal calf serum from Hyclone Laboratories (Logan, UT). 15(S)-HETE RIA kit was purchased from Advanced Magnetics (Cambridge, MA). LTB₄ RIA kit was purchased from Amersham-Pharmacia Biotec (Milan, Italy). Eicosanoid standards were from Cayman Chemical (Ann Arbor, MI). Reagents for PCR were from Promega (Madison, WI). Compound MK-886 was kindly provided by Dr. A.W. Ford-Hutchinson (Merck Frosst, Pointe Claire, PQ, Canada).

Purification and Culture of Monocytes. Mononuclear cells were isolated from buffy coats by density gradient centrifugation using Ficoll-Hypaque cushions (Conrad et al., 1992). The mononuclear cell band was removed and washed three times by centrifugation with PBS containing CaCl₂ and MgCl₂ at final concentrations of 0.5 and 1 mM, respectively (PBS buffer). After resuspension (4-20 × 10⁶ cells/ml) in RPMI 1640 + 10% heat-inactivated (56°C, 30 min) fetal calf serum + 1% penicillin-streptomycin solution + 1 mM l-glutamine (RPMI buffer), cells were allowed to adhere to 150-mm polystyrene tissue culture dishes for 2 h at 37°C. More than 96% of the adherent cells stained positive for nonspecific esterase, and the viability was >95%, as assessed by trypan blue exclusion.

LTB₄ and 15(S)-HETE Production in Monocytes. IL-4-treated and untreated cells (2 × 10⁶ cells/ml) were allowed to adhere to tissue culture plates for 2 h at 37°C, and were then incubated with A23187 to a final concentration of 5 × 10⁶ M for 15 min. Alternatively monocytes were challenged with opsonized zymosan (100 particles per cell, 1 h at 37°C). Zymosan was opsonized by incubation with fresh human serum; after boiling 100 mg in 2 ml of PBS for 1 h, and washing twice with PBS, zymosan was resuspended in PBS (2 ml) and incubated with 6 ml of fresh human serum at 37°C for 20 min. After centrifugation and washing twice with PBS, opsonized zymosan was resuspended at a concentration of 10 mg/ml. Zymosan particle count was performed using an hemocytometer.

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Analysis of AA Metabolites by Electrospray Ionization-Tandem Mass Spectrometry. Arachidonic acid metabolites generated by monocytes stimulated with opsonized zymosan, and from selected experiments carried out with A23187 and exogenous 15(S)-HETE, were extracted on LC18 solid phase cartridges after addition of d₄-LTB₄ as an internal standard, and analyzed by electrospray ionization-tandem mass spectrometry. Aliquots of extracted samples (50 µl) were injected into a RP-HPLC column (Columbus 3 µm, 1 × 125 mm, Phenomenex, Torrance, CA) directly interfaced into the electrospray source of a triple quadrupole mass spectrometer (Sciex API-3000; Perkin Elmer Instruments, Norwalk, CT). The column was eluted at a flow rate of 50 µl min¹ using a linear gradient from 15 to 100% solvent B (A: water, 0.05% acetic acid, pH 5.7 with NH₃; B: AcCN/MeOH, 65:35) over 30 min, and arachidonic acid metabolites were detected by selected reaction monitoring, using collision-induced dissociation and specific transitions for the different metabolites (m/z 339 to 198 for d₄-LTB₄, 335 to 195 for LTB₄, 319 to 219 for 15(S)-HETE, and 335 to 201 for 5(S),15(S)-diHETE).

15-LO Activity and reverse transcription (RT)-polymerase chain reaction (PCR). To assess 15-LO activity in IL-4-treated and untreated samples, cells (2 × 10⁶ cells/ml) were allowed to adhere to tissue culture plates for 2 h at 37°C, and were then incubated with 100 µM AA dissolved in ethanol (0.5% final concentration) for 20 min in PBS buffer. At the end of the incubation with AA, the cell supernatants were harvested and, after the addition of prostaglandin B₂ as an internal standard, were stored under an argon atmosphere at 80°C, and 15(S)-HETE analyzed as described above.

Purification of Neutrophils from Peripheral Blood and Neutrophil Chemotaxis Assay. Peripheral blood was obtained from normal subjects and neutrophils were prepared by dextran sedimentation and centrifugation over Ficoll cushions. Neutrophils were resuspended at a concentration of 1 ×10⁶/ml in PBS buffer in the presence and absence of an LTB₄ receptor antagonist (LY 223982 10 µM; Eli Lilly, Basingstoke, UK), or 15(S)-HETE (1-100 µM), at 37°C. Chemotaxis was performed using a 48-well microchemotaxis chamber (Costar; Neuro Probe Inc., Cabin John, MD) as previously described (Profita et al., 2000a). Neutrophils were loaded into the upper well and the monocyte supernatant, or synthetic compound, were placed in the bottom chamber. The two wells were separated by a polycarbonate membrane filter with a pore size of 3 µm. Chamber was incubated at 37°C for 1 h. At the end of incubation, the filter was fixed, stained, and mounted on a glass microscope slide (observed at 400×). Migration was assessed by counting the number of cells that had migrated beyond a certain depth into the filter. Each experimental condition was performed in duplicate, and three to four fields were assessed for cell migration. Chemotactic activity of standard 15(S)-HETE, 5(S),15(S)-diHETE, and LTB₄ dissolved in PBS buffer was also evaluated.

Uptake and Metabolism of Radiolabeled 15(S)-HETE. Metabolism of 15(S)-HETE by monocytes (2 × 10⁶ cells), was studied in cells grown for 72 h with and without IL-4 (10 ng/ml). Cells were then incubated with [³H]15(S)-HETE (0.5 µCi/ml) for 20 min at 37°C, and stimulated with A23187 at a final concentration of 5 × 10⁶ M for 15 min 37°C, in the presence or absence of MK-886. At the end of the incubation the cell-free medium was analyzed by RP-HPLC as described above. One-minute fractions were collected and radioactivity analyzed by liquid scintillation counting.

Statistical Analysis. Results are given as mean ± S.E. of n observations. Statistical analysis was performed by one-way analysis of variance. Mean data for individual experiments were also compared using Student's t test for paired or unpaired samples, as appropriate. A p value < 0.05 was considered statistically significant.

	Results

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Treatment with IL-4 for 24, 48, and 72 h significantly increased the amount of 15(S)-HETE observed after stimulation with the Ca²⁺ ionophore A23187, resulting in over a 10-fold increase after 72 h of incubation (Fig. 1A). Concomitant measurement of LTB₄ production showed a progressive decrease over IL-4 incubation time, reaching a minimum after 72 h, where LTB₄ production was less than 15% of that observed in the absence of IL-4 pretreatment (Fig. 1B). The effect of IL-4 was abolished upon pretreatment with a specific anti-IL-4 polyclonal antibody, and the production of 15(S)-HETE and LTB₄ after 72 h of incubation with IL-4 resulted in 2.1 ± 1 and 3.5 ± 1.1 ng/ml, respectively, compared with values of 22.6 ± 2.5 and 0.6 ± 0.1 ng/ml observed in samples treated with IL-4 in the absence of the anti-IL-4 polyclonal antibody.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effects of IL-4 on 15(S)-HETE and LTB4 production in human monocytes. The effects of IL-4 treatment on the production of 15(S)-HETE (A) and LTB4 (B) were evaluated in normal human peripheral monocytes activated with A23187 (5 µM). Monocytes (2 × 106 cells/ml) were cultured in the presence or absence of IL-4 for 24, 48, and 72 h. 15(S)-HETE and LTB4 were quantified in supernatants by specific RIAs after RP-HPLC separation as described under Materials and Methods. Data are expressed as mean ± S. E. of eight consecutive experiments. Statistical analysis was performed using Student's t test for unpaired samples.

To test if the effect of IL-4 on LTB₄ could be observed in the presence of a more physiological stimulus, control and IL-4-treated (72 h) monocytes were challenged with opsonized zymosan. LC/MS/MS analysis of supernatants showed the presence of 15(S)-HETE in IL-4-treated cells only (Fig. 2). As observed in A23187-activated monocytes, LTB₄ production in IL-4-treated monocytes was also inhibited (45 ± 11%, n = 3, p = 0.05; LTB₄ production in controls was 0.44 ± 0.24 ng/2 × 10⁶ cells).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   LC/MS/MS analysis of supernatant from opsonized zymosan-activated human monocytes. Untreated (top panel) and IL-4 treated (bottom panel; 10 ng/ml, 72 h, 37°C) monocytes were challenged with opsonized zymosan (100 particles per cell, 1 h at 37°C). Supernatant was extracted and analyzed by LC/MS/MS as described under Materials and Methods. Negative ion chromatograms represent the specific transition from m/z 319 to m/z 219, resulting from the collision induced dissociation of the carboxylate anion of 15(S)-HETE.

In agreement with data previously published, 15-LO activity, evaluated upon addition of excess arachidonic acid (Table 1), as well as 15-LO mRNA (Fig. 3) showed a significant increase in IL-4 treated monocytes compared with untreated cells. On the contrary, the decrease in LTB₄ production observed in IL-4 treated monocytes did not reflect decreased expression of mRNA for enzymes involved in its synthesis, such as 5-LO and FLAP (Fig. 3).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Expression of lipoxygenases and FLAP mRNA in normal human peripheral monocytes cultured in the presence and absence of IL-4 for 72 h. Equal amounts of total RNA (2 ng) obtained from monocytes were reverse-transcribed into cDNA. 5-LO and FLAP were amplified with nested specific primers. Arrows indicate predicted sizes (bp) of amplification products. Number on the top of figure indicates the sample: lane 1, size marker (pBR322 DNA-Mps I Digest Biolabs, Beverly, MA); lane 2, monocytes without IL-4; lane 3, monocytes with IL-4 (10 ng/ml) for 72 h; lane 4, negative control (no template cDNA control).

To verify if newly expressed 15-LO, which utilizes AA, may reduce LTB₄ biosynthesis simply by decreasing substrate availability, monocytes were challenged in the presence of excess substrate (30 µM AA). The results showed a significant decrease of LTB₄ in IL-4-treated monocytes compared with untreated cells (10 ± 3 and 98 ± 3 ng/ml, respectively; n = 3, p < 0.01).

We have previously shown that 15(S)-HETE is able to inhibit the synthesis of 5-LO metabolites in human neutrophils. To test if the product of arachidonic acid metabolism by 15-LO may be responsible for the observed inhibition of LTB₄ production, we tested the effect of 15(S)-HETE on A23187-induced formation of LTB₄. Preincubation of isolated monocytes with exogenous 15(S)-HETE prior to A23187 challenge resulted in a concentration-dependent inhibition of LTB₄ production, with an IC₅₀ of 1 µM (Fig. 4). The observed inhibitory effect did not reflect a decreased availability of the substrate, as AA release was not reduced by pretreatment with 15(S)-HETE (31.5 ± 7.5 and 50.6 ± 11.5 ng/2 × 10⁶ cells; control and 15(S)-HETE 10⁵ M, respectively).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effect of exogenous 15(S)-HETE on LTB4 production by human monocytes challenged with A23187. Monocytes (2 × 106 cells/ml) were preincubated with increasing concentrations of 15(S)-HETE and challenged with A23187 (5 µM) for 15 min. LTB4 was quantified in supernatants by specific RIA after RP-HPLC separation as described under Materials and Methods. Results are expressed as mean ± S.E. of six consecutive experiments. Statistical analysis was performed using Student's t test for paired samples.

Incubation of radiolabeled 15(S)-HETE with human monocytes showed significant production of the double lipoxygenation product 5(S),15(S)-diHETE when cells were activated with A23187 (Fig. 5). Formation of 5(S),15(S)-diHETE required 5-LO activation and translocation as shown by complete inhibition of its synthesis by the FLAP inhibitor MK-886. LC/MS/MS analysis of monocytes preincubated with 15(S)-HETE, also showed the presence of the 5(S),15(S)-diHETE, as detected by the specific transition from m/z 335 to 201 (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Metabolism of radiolabeled 15(S)-HETE by human monocytes. Monocytes (2 × 106 cells/ml) were preincubated with radiolabeled 15(S)-HETE and challenged with A23187 (5 µM) for 15 min in the presence or absence of the FLAP inhibitor compound MK-886 (1 µM). Supernatants were extracted and analyzed by RP-HPLC as described under Materials and Methods. One-minute aliquots were collected and radioactivity was evaluated by liquid scintillation counting. Retention time of synthetic standard compounds are shown by arrows.

Given the potent neutrophil chemotactic activity of LTB₄, it was of interest to see if the treatment with IL-4 was able to significantly affect the biological activity of the substances released by activated monocytes. We therefore evaluated the neutrophil chemotactic activity of supernatants obtained from control and IL-4-treated (72 h) monocytes after challenge with A23187. Neutrophil chemotaxis induced by the supernatants of A23187-activated monocytes was significantly lower in IL-4-treated monocytes (Fig. 6). To evaluate the specific contribution of LTB₄ to the overall chemotactic activity released by activated monocytes, we also tested their activity in the presence of the specific LTB₄ receptor antagonist LY 223982. Pretreatment with LY 223982 significantly inhibited the chemotactic activity of supernatants from control monocytes, pointing out that about 50% of the chemotactic activity released by activated monocytes was the result of the biological activity of LTB₄. A similar pretreatment did not have significant effects against the chemotactic activity of supernatants obtained from IL-4 treated cells (Fig. 6), suggesting that the reduced biological activity released by IL-4-treated monocytes upon A23187 activation well correlated with the decreased amounts of LTB₄ observed under these experimental conditions.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effect of IL-4 treatment on neutrophil chemotactic activity of supernatants from A23187-challenged monocytes. Chemotaxis was performed using a 48-well microchemotaxis chamber as described under Materials and Methods. Neutrophils were preincubated either in the absence or the presence of the LTB4 receptor antagonist LY 223982 at the concentration of 10 µM. Results are expressed as mean ± S.E. of eight consecutive experiments. Statistical analysis was performed using Student's t test for unpaired samples.

Given that significant neutrophil chemotactic activity has previously been reported for 15(S)-HETE in vivo (Johnson et al., 1985), we tested the in vitro chemotactic activity of 15(S)-HETE and 5(S),15(S)-diHETE. Both compounds, at the concentration of 1 µM, had an efficacy that was less than 50% of the effect observed for LTB₄ 10 nM, tapering off at a higher concentration (Fig. 7); the lower efficacy and potency for 15-LO metabolites as chemotactic factors is well in agreement with the observed reduction of chemotactic activity observed in supernatants from the IL-4-treated monocyte, where a shift of arachidonic acid metabolism from LTB₄ to 15(S)-HETE was observed.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Neutrophil chemotactic activity of LTB4 (A), and 15(S)-HETE and 5(S),15(S)-diHETE (B). Neutrophil chemotactic activities of LTB4 (1-50 nM), 15(S)-HETE, and 5(S),15(S)-diHETE (1-100 µM) were tested using a 48-well microchemotaxis chamber as described under Materials and Methods. Results are expressed as mean ± S.E. of six consecutive experiments.

	Discussion

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The results of this study showed that IL-4 induced expression of 15-LO in human monocytes is accompanied by a significant inhibition of LTB₄ production upon challenge with either the Ca²⁺ ionophore A23187 or a physiologically relevant stimulus such as opsonized zymosan. Inhibition of LTB₄ synthesis upon treatment with IL-4 and activation of monocytes in the presence of excess AA, showed that the effect of IL-4 did not simply reflect the presence of newly expressed 15-LO, and therefore a potential decrease in substrate availability to 5-LO.

Treatment with IL-4 did not change the expression of mRNA for key enzymes involved in leukotriene biosynthesis, such as 5-LO and FLAP. Although we did not measure actual proteins or enzymatic activity, the results of the mRNAs expression suggest that the observed inhibition of LTB₄ synthesis did not result from a direct transcriptional effect of IL-4 on 5-LO and FLAP. Concerning the potential effect of IL-4 on the LTA₄ hydrolase, a recent paper reported that IL-4 may indeed increase the expression of this enzyme (Zaitsu et al., 2000). Exogenous 15(S)-HETE concentration-dependently inhibited the formation of LTB₄ by monocytes, without affecting the availability of the substrate AA, as evaluated measuring the AA released after A23187 challenge; this result suggests that 15(S)-HETE formed by IL-4-treated monocytes may be responsible for the observed inhibition of LTB₄ production.

IL-4 is a cytokine released by Th2 and mast cells (Bradding et al., 1995) that may play a major role in the pathogenesis of inflammatory lung disease (Robinson et al., 1992; Walker et al., 1992; Humbert et al., 1996). Besides its IgE-regulating effects (Del Prete et al., 1988; Pene et al., 1988), IL-4 is involved in the maturation of blood monocytes (Mosmann et al., 1986), as well as in the expression of cell membrane markers such as CD23 and class II major histocompatibility complex antigen on monocytes (de Velde et al., 1988). On the other hand, together with its potential proinflammatory activity, in normal subjects IL-4 has been found to be an inhibitor of mononuclear phagocyte functions in vitro and ex vivo (Hart et al., 1989; Yanagawa et al., 1991; Wong et al., 1992). This inhibition appeared to be mediated by transcriptional effects of IL-4 (Yanagawa et al., 1991) and was not due to a cytotoxic effect.

The potential for IL-4 to significantly change the profile of arachidonic acid metabolites from the production of leukotrienes to that of 15(S)-HETE raises important questions on the biological relevance of such an effect. 15(S)-HETE represents the major AA metabolite produced in lung homogenates (Hamberg et al., 1980), as well as in cultured human lung tissue obtained from both asthmatic and normal donors. It has been shown that when lung tissue obtained from asthmatics undergoes in vitro allergen challenge, 15(S)-HETE production is approximately 100 times greater than that of LTC₄ (Dahlen, 1983). Bronchial allergen challenge in atopic asthmatic subjects leads to a 30-fold increase of the concentrations of 15(S)-HETE recovered in bronchoalveolar lavage fluid (Murray et al., 1986). Once released, 15(S)-HETE can exert several immunoregulatory functions that may be relevant to the pathogenesis of asthma. 15(S)-HETE has been shown to be a potent mucosecretagogue in the human airway (Marom et al., 1982), and to possess chemotactic activity for neutrophils directly, contributing to the recruitment of these cells in the airways (Johnson et al., 1985). It has also been found that 15(S)-HETE can prolong the duration of airway obstruction during the early response, suggesting that it either augments the release of mediators from mast cells or potentiates the effects of other mediators on airway smooth muscle (Lai et al., 1990). On the other hand, it has been reported that 15(S)-HETE inhibits 5-lipoxygenase (Vanderhoek et al., 1980; Borgeat et al., 1983; Profita et al., 2000a), and incorporation of 15(S)-HETE into membrane phospholipids impairs the response of human polymorphonuclear neutrophil to inflammatory stimuli such as the formylated tripeptide fMLP (Brezinski and Serhan, 1990).

Recently we showed that 15(S)-HETE is present in high concentration in induced sputum obtained from asthmatics (Profita et al., 2000b), as well as that of chronic bronchitis patients (Profita et al., 2000a). Furthermore we showed that its concentration inversely correlates with the percentage of neutrophils recovered in induced sputum, and that 15(S)-HETE inhibits LTB₄ production in A23187, as well as in fMLP-activated human neutrophils (Profita et al., 2000a). Hence, the ability of IL-4 to increase the release of 15(S)-HETE by human monocytes/macrophages, and to concomitantly inhibit the production of a potent chemotactic factor such as LTB₄, may play an important role in the evolution of pulmonary inflammatory responses. In agreement with the expected effect of reduced production of LTB₄, supernatants from IL-4-treated monocytes showed a significantly lower chemotactic activity toward neutrophils. Neutrophil chemotaxis evaluation in the presence of an LTB₄-receptor antagonist showed that over 50% of the chemotactic activity of activated monocytes depends on the presence of the 5-LO metabolite LTB₄. This result suggests that neutrophilic influx following monocyte activation may be significantly inhibited by the expression of 15-LO induced by IL-4.

A recent paper raised the possibility that inhibition of 5-LO product formation by 15(S)-HETE in human neutrophils (Petrich et al., 1996) may simply reflect that 15(S)-HETE is an alternative substrate to arachidonic acid for 5-LO. We tested the metabolism of radiolabeled 15(S)-HETE in monocytes and found that measurable formation of the dihydroxy-derivative 5(S),15(S)-diHETE could indeed be observed. Interestingly, as recently shown by Mancini and coworkers by selective expression of 5-LO and FLAP in Sf9 insect cells (Mancini et al., 1998), conversion of 15(S)-HETE by 5-LO was dependent on FLAP, and pretreatment with the FLAP inhibitor MK-886 completely abolished the conversion of radiolabeled 15(S)-HETE in monocytes. This evidence suggests that FLAP is responsible for appropriate handling of substrate, either AA of 15(S)-HETE, to 5-LO. Irrespective of the mechanism underlying the inhibition of LTB₄ synthesis (true inhibitor or alternative substrate) it is important to stress that IL-4-induced expression of 15-LO causes a significant shift in arachidonic acid metabolism from LTB₄ to 15(S)-HETE, and possibly minor amounts of 5(S),15(S)-diHETE. This shift appears to be biologically relevant as we found that 15(S)-HETE, as well as 5(S),15(S)-diHETE, both showed only a very modest neutrophil chemotactic activity (approximately 50% of the efficacy of LTB₄ at 100-fold higher concentration); furthermore this effect tapered off at higher concentrations. This modest chemotactic activity may be responsible for the results observed after in vivo administration of 15-HETE in dogs, in which enhanced influx of neutrophils as well as mast cells was observed (Johnson et al., 1985). On the other hand, the potential effect on the production of LTB₄ may contribute to the observed inhibitory effect of 15(S)-HETE on neutrophil responses in vitro (Brezinski and Serhan, 1990). These complex mechanisms may well play a role in the regulation of the resolution of the inflammatory response.

In conclusion, this study shows that in human monocytes IL-4 may directly regulate the production of arachidonic acid-derived lipid mediators, such as LTB₄ and 15(S)-HETE, therefore contributing to the evolution of inflammatory responses resulting in monocyte activation. The evidence provided in this study underscores a potential modulatory role for IL-4 in the inflammatory response, unveiling a novel anti-inflammatory activity of this cytokine.