Introduction

Abstract Introduction Materials and Methods Results Discussion References

Airway epithelium has metabolic and immunologic properties that play an important role in the regulation of inflammatory processes and tissue repair in the airways. It also exerts the important function of a dynamic barrier that regulates the penetration and traffic of several molecules between the body and the environment (1). Many of these functions seem to be altered during inflammatory diseases of the airways such as asthma and chronic bronchitis.

Airway epithelial cells can be the target of several inflammatory mediators that are responsible for the epithelial damage and desquamation observed in autopsy and bronchial biopsy specimens from asthmatic individuals (2- 4). However, airway epithelial cells are not simply passive targets of inflammation, since they can respond to inflammatory stimuli by releasing several mediators that are in turn able to play an active role in the development of airway inflammation. During inflammatory responses of the airways, these cells express high levels of nitric oxide synthase (NOS) (5) and of membrane markers such as adhesion molecules (6), and release an increased amount of fibronectin and endothelin (7), cytokines (10), or chemokines (11). In addition, bronchial epithelial cells can release several arachidonic acid (AA) metabolites (12) that are capable of playing a crucial role in the development of airway inflammation in asthma.

Among these AA metabolites, an important role is played by 15(S)-hydroxyeicosatetraenoic acid (15[S]- HETE), which has been shown to be the major AA metabolite produced by human bronchi (15), eosinophils (16), and airway epithelial cells (17). In this respect, the release of 15(S)-HETE by bronchial epithelial cells obtained from asthmatic subjects has been shown to be greater than in normal subjects, and to be significantly correlated with the clinical severity of the disease (7). Furthermore, it has been shown that exogenously or endogenously generated 15(S)-HETE not only can exert its biologic activities on the surrounding cells in the bronchi, but also can be rapidly metabolized and/or incorporated into phosphatidylinositol (PI) of the cell membrane of human tracheal epithelial cells (18, 19). Recently, it was shown that the exposure of human tracheal epithelial cells to ozone, the major oxidant in photochemical air pollution, increases 15(S)-HETE production and 15(S)-HETE esterification into phospholipids (20).

The release of 15(S)-HETE can be modulated by several cytokines released in the pulmonary microenvironment. In particular, interleukin-4 (IL-4) has been shown to induce 15-lipoxygenase (15-LO) enzymatic activity in monocytes and tracheal epithelial cells (21, 22), and IL-4 receptors have been reported to be expressed along the airway epithelium in a distribution identical to that of 15-LO (23). Because IL-4 may play an important role in airway inflammation in asthma, as shown by increased amounts of IL-4 proteins recovered in bronchoalveolar lavage fluid (BALF) from allergic asthmatic individuals (24), or by increased numbers of IL-4-immunoreactive cells in the bronchial epithelium of these subjects (25), we undertook the present study to evaluate the effect of IL-4 on the expression of 15-LO and the incorporation of 15(S)-HETE into cellular phospholipids in a human pulmonary epithelial cell line (WI-26 VA4).

	Materials and Methods

Abstract Introduction Materials and Methods Results Discussion References

Reagents

Human recombinant IL-4 and polyclonal sheep antihuman IL-4 were obtained from Genzyme (Cambridge, UK). 15(S)-HETE, AA (tested for cell culture), prostaglandin B₂ (PGB₂), and phospholipid (PL) standards were obtained from Sigma Chemical Co. (St. Louis, MO). [³H]- 15(S)-HETE (182 Ci/mmol) was obtained from Dupont de Nemour NEN Products (Cologno, Milan, Italy). All solvents were of high-pressure liquid chromatography (HPLC) grade and were obtained from Merck (Darmstadt, Germany). Nutritive medium came from Whittaker (Veviers, Belgium), and fetal calf serum (FCS) from Hyclone (Logan, UT). Penicillin-streptomycin solution and L-glutamine were from GIBCO (Paisley, UK). A 15(S)-HETE radioimmunoassay (RIA) kit was purchased from Advanced Magnetics Inc. (Framingham, MA). 15-LO primer was obtained from PRIMM (Milan, Italy).

Culture and Stimulation of WI-26 Epithelial Cells with IL-4

WI-26 (VA4) epithelial cells were purchased from the American Type Culture Collection (Rockville, MD) and cultured as adherent monolayers in complete medium (CM) composed of RPMI 1640 + 10% heat-inactivated (56°C, 30 min) FCS + 1% penicillin-streptomycin solution + 1 mM L-glutamine. WI-26 cells are a clonal diploid (2n = 6) cell line isolated from human embryonal lung, and have previously been used to study the functional properties of epithelial cells in inflammatory and repair processes; electron-microscopic analysis of these cells revealed type I pneumocytelike morphology, with tight junctions and no lamellar bodies (26). The cells were incubated in RPMI medium-10% FCS in the presence or absence of IL-4 (10 ng/ml) for 24, 48, and 72 h. Adherent WI-26 cells were recovered after a brief trypsinization (2 min) to standardize the number of cells used in each sample, were washed twice with RPMI 1640 with 10% FCS, and were resuspended at a final concentration of 2 × 10⁶ cells/ ml in RPMI supplemented with 1% FCS, L-glutamine, and antibiotics. Cell viability was assessed by trypan blue exclusion, and was > 98% throughout all experiments.

Assay of 15-LO Activity in Intact WI-26 Cells

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 100 µM AA dissolved in ethanol (0.5% final concentration) for 2, 5, 10, and 20 min in RPMI-1% FCS. To test the specific effects exerted by IL-4, the cells were also incubated in the presence of a polyclonal anti-IL-4 antibody. At the end of the incubation with AA, the cell supernatants were harvested and, after the addition of PGB₂ as an internal standard, were stored under an argon atmosphere at 80°C until analysis with reverse-phase (RP)-HPLC, as described subsequently. The fractions corresponding to the elution time of 15(S)- HETE were collected (LKB 2112 Redirac fraction collector), dried under a stream of N₂, redissolved in 400 µl of RIA buffer, and quantitated with a specific RIA (Advanced Magnetics) performed according to the manufacturer's protocol. The antibody used was raised against 15(S)-HETE, and cross-reactivities for 5(S)-HETE, 12(S)- HETE, 5,15-di-HETEs, and 8,15-di-HETEs were 0.1%, 0.5%, 1%, and 1%, respectively. All measurements were made in duplicate, and the results were expressed in nanograms per 2 × 10⁶ cells.

Primed In Situ Labeling of 15-LO mRNA

To evaluate whether the effect of IL-4 on 15-LO activity in WI-26 cells resulted from changes in specific activity of the enzyme rather than from its transcriptional modification, we performed primed in situ (PRINS) labeling for 15-LO mRNA on slides of cytospin cells (Shandon Southern Products Ltd., Runcorn, Cheshire, UK) obtained from three consecutive experiments with WI-26 incubated in the presence or absence of IL-4 (24, 48, and 72 h). The experiments were performed according to Koch and colleagues (27), with minor modifications. Briefly, slides were immediately placed in 4% paraformaldehyde/phosphate-buffered saline (PBS) for 5 min, treated with 0.3% Triton X-100 in PBS for 10 min, and then hydrated with Tris-glycine buffer (0.2 M Tris-HCl, 0.1 M glycine, pH 7.4) for 10 min. Slides were then treated with 2× standard saline citrate (SSC) (1× SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0) mixed with 1 volume of deionized formamide (10 min at room temperature). The temperature was subsequently raised to 65°C for 10 min, and slides were transferred to ice-cold nick-translation (NT) buffer (50 mM Tris-HCl, 10 mM MgSO₄, 100 mM dithiothreitol [DTT], 150 µg bovine serum albumin [BSA]). After incubation for 30 min at 4°C in NT buffer, slides were spotted with the reaction mixture (2 µg 15-LO primer: 5'-AGT GTC CAT TAT CTG CTC GAA AAT-3' [28]; 5 nmol each of deoxyadenosine triphosphate [dATP], deoxycytosine triphosphate [dCTP], and deoxyguanosine triphosphate [dGTP]; 0.5 nM deoxythymidine triphosphate [dTTP]; 0.078 nM digoxigenin-deoxyuridine triphosphate [dUTP]; 0.25 U ribonuclease [RNase] inhibitor; and 0.5 U avian myeloblastosis virus [AMV] reverse transcriptase in a total volume of 10 µl of NT buffer); and were incubated at 42°C for 90 min in a humidified chamber. After washing in SSC (10 min, 42°C) and in Tris-buffered saline (TBS) (150 mM NaCl, 50 mM Tris-HCl, pH 7.4), slides were incubated with 3% bovine serum albumin (BSA)/TBS (10 min) and with a sheep polyclonal antidigoxigenin antiserum conjugated with alkaline phosphatase (Dig-AP) in TBS (1 h). After incubation, slides were washed three times with TBS (15 min) and with equalization buffer (0.1 M HCl, 0.1 M NaCl, 50 mM MgCl₂, pH 9.5) (5 min). Color development was achieved by spotting the slides with a freshly prepared solution of substrate (0.175 mg 5-bromo-4-chloro-3-indolyl phosphate [BCIP] and 0.37 mg nitroblue tetrazolium [NBT] salt per milliliter of equalization buffer) for 20 to 40 min at room temperature. Slides were finally washed with TBS and tap water, counterstained with hematoxylin for 5 s, and mounted with glycerol gelatine. Control slides were prepared during the same experimental series either by: (1) pretreating the slides with RNase A before PRINS, (2) omitting the specific primer, or (3) omitting the Dig-AP conjugate.

At least four fields per slide were evaluated under light microscopy (×400 final magnification). Results were expressed as the percentage of 15-LO mRNA-positive cells over the total.

Uptake of Radioactive 15(S)-HETE and Cellular Lipid Analysis

To evaluate the incorporation of 15(S)-HETE into cellular phospholipids by WI-26 cells (2 × 10⁶ cells/ml), cells were grown for 24, 48, and 72 h with or without IL-4 (10 ng/ml), and were then incubated with [³H]15(S)-HETE (0.5 µCi/ ml) for 20 min at 37°C. At the end of the incubation period, cell-free medium was collected and radioactivity evaluated by scintillation counting (Model LS 1801; Beckman Instruments, Fullerton, CA) after addition of 10 ml of Ready Safe Scintillation Cocktail, with separate aliquots being saved and used for RP-HPLC analysis. After washing, the adhering cells were rapidly trypsinized, centrifuged, resuspended in 1 ml of PBS in siliconized glass centrifuge tubes, and stored at 80°C.

After one night at 80°C, cell suspensions were thawed and extracted with 2 volumes of 2-propanol containing 1.2% (vol/vol) acetic acid:chloroform (1:1) according to Conrad and coworkers (21). The resulting mixture was vortexed and centrifuged to yield separate phases. The lower phase, containing lipid extract, was dried under N₂ and resuspended in 400 µl of methanol. An aliquot (50%) was placed into siliconized glass tubes and stored at 80°C until normal-phase (NP)-HPLC analysis. A separate aliquot was used for base-catalyzed hydrolysis of phospholipids. Briefly, 100 µl of 2 N NaOH were added to samples (29) and heated at 60°C for 30 min under an argon atmosphere. The solution was neutralized by addition of 100 µl of 2 N acetic acid, and the final volume was adjusted to 800 µl with water. After centrifugation, supernatants were analyzed with RP-HPLC, as described subsequently, to identify cell-associated radioactivity. One-minute fractions were collected and the radioactivity of each fraction was evaluated by scintillation counting.

NP-HPLC Separation of Phospholipid Classes

An aliquot of the cellular lipid extract obtained after incubation of WI-26 cells with [³H]15(S)-HETE was dried under a stream of N₂, resuspended in 200 µl of mobile phase A, and analyzed with NP-HPLC, using a gradient liquid-chromatography (System Gold apparatus, Model 126; Beckman Instruments), connected to a diode-array UV detector (Model 168; Beckman). The column (Lichrospher Si-100 5 µm, 4 mm × 25 cm column; Merck) was eluted isocratically at a flow rate of 0.5 ml/min, with a mobile phase of hexane/2-propanol/ethanol/25 mM potassium phosphate (pH 7)/acetic acid, 367:490:100:62:0.6 (vol/vol), according to Patton and colleagues (30). The mobile phase was initially saturated with potassium phosphate and filtered to remove any precipitated potassium phosphate. One-minute fractions were collected, and the radioactivity of each fraction was evaluated by scintillation counting. Phospholipid standards were detected by monitoring UV absorbance at 205 nm.

RP-HPLC of AA Metabolites

AA metabolites were analyzed with RP-HPLC, using the System Gold apparatus (Beckman). The column (Ultrasphere octadecylsilyl [ODS] 5 µm, 4.6 × 250 mm; Beckman) was eluted at a flow rate of 1 ml/min, using a gradient from 20% B (acetonitrile/acetic acid, 100/0.1 [vol/vol]) to 100% B over a period of 18 min, with A represented by water/acetic acid (100/0.1 [vol/vol]). The retention time of the 15(S)-HETE standard was 21 ± 0.6 min, and was checked daily with [³H]15(S)-HETE standard.

Data Analysis

Analysis for statistical significance was done with one-way analysis of variance (ANOVA) followed by post hoc analysis with the Tukey-Kramer test for multiple comparison, or with Dunnett's test for multiple comparison versus a control, as appropriate. A value of P < 0.05 was accepted as significant. Results are expressed as means ± SEM of n observations.

	Results

Abstract Introduction Materials and Methods Results Discussion References

Effect of IL-4 on 15-LO Activity in WI-26 Cells

Addition of exogenous AA (100 µM) to WI-26 cells for different periods of time (2, 5, 10, and 20 min) released significant amounts of 15(S)-HETE into the extracellular medium, as assessed with RP-HPLC separation coupled to specific RIA. The maximal concentration of 15-HETE was observed at the shortest incubation time tested (1.85 ± 0.14 ng/2 × 10⁶ cells, P < 0.001 versus time 0 and P < 0.01 versus 5 min; Tukey-Kramer test), suggesting that significant reuptake or further metabolism were taking place at longer incubation times (Figure 1). The preincubation with IL-4 (10 ng/ml) revealed a significant increase in 15-LO activity, expressed as release of 15(S)- HETE upon addition of exogenous AA, which appeared maximal after 48 h of pretreatment (12.1 ± 1 ng/2 × 10⁶ cells, n = 3, P < 0.001 versus control; Dunnett's test) (Figure 2). Addition of a polyclonal anti-IL-4 antibody (20 mg/ ml) totally prevented the effect of IL-4 (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 1.   15-LO activity in WI-26 cells. WI-26 cells (2 × 106 cells/ ml) were allowed to adhere to tissue culture plates for 2 h at 37°C and were then incubated with exogenous AA (100 µM for 2, 5, 10, and 20 min) in RPMI-1% FCS. The samples were analyzed with RP-HPLC, and fractions corresponding to 15(S)-HETE were collected and quantitated by specific RIA. The results are expressed as means ± SEM (n = 9). **P < 0.01; ***P < 0.001.

View larger version (42K): [in this window] [in a new window]   Figure 2.   15-LO activity in WI-26 cells: effect of IL-4. WI-26 cells (2 × 106 cells/ml) treated with IL-4 (10 ng/ml) (24, 48, 72 h) were incubated with exogenous AA (100 µM for 2 min). The samples were analyzed with RP-HPLC, and fractions corresponding to 15(S)-HETE were collected and quantitated by specific RIA. The results are expressed as means ± SEM (n = 3). **P < 0.01; ***P < 0.001.

Effect of IL-4 on 15-LO mRNA Expression

In control incubations, WI-26 cells expressing 15-LO mRNA, as assessed by PRINS labeling, represented 31.7 ± 1.7% of the cells. In the presence of IL-4, 15-LO mRNA-expressing cells increased to 55 ± 2.9%, 90 ± 5.8%, and 78 ± 1.5% after 24, 48, and 72 h, respectively (Figure 3). Control slides, prepared by pretreating slides with RNase A, or by omitting the specific primer or the Dig-AP conjugate, gave no cellular hybridization signal.

View larger version (117K): [in this window] [in a new window]   Figure 3.   Effect of IL-4 on expression of 15-LO mRNA in WI-26 cells. Expression of 15-LO mRNA in cytospin preparations of WI-26 cells was assessed by PRINS labeling in the absence (a) or presence of IL-4 (10 ng/ml) for 24 h (b), 48 h (c), and 72 h (d).

Effect of IL-4 on the Uptake and Incorporation into Cellular Phospholipid of [³H]15(S)-HETE

In the absence of pretreatment with IL-4, the radioactivity recovered in the cell-free medium of WI-26 cells exposed to [³H]15(S)-HETE (0.5 µCi/ml, 20 min at 37°C) accounted for approximately 90% of total [³H]15(S)-HETE added (Figure 4). This value was significantly decreased upon pretreatment with IL-4 for 48 and 72 h, suggesting that IL-4 was able to stimulate the uptake of 15(S)-HETE (Figure 4). RP-HPLC analysis showed that the radioactivity remaining in the supernatant was represented by intact 15(S)-HETE, and confirmed the decrease in 15(S)-HETE remaining in the cell-free medium after incubation with IL-4 for 48 h (a decrease of 22.2 ± 5% versus control incubations) and 72 h (a decrease of 34 ± 4.1% versus control incubations).

View larger version (16K): [in this window] [in a new window]   Figure 4.   Uptake of [3H]15(S)-HETE by WI-26 cells. Cells were incubated with [3H]15(S)-HETE (0.5 µCi/ml) for 20 min at 37°C after 24, 48, and 72 h, with or without IL-4 (10 ng/ml). Aliquots of incubation medium were taken for scintillation counting, and 15(S)-HETE remaining in the medium was expressed as percent of added radioactivity. The results are expressed as means ± SEM (n = 3). **P < 0.01; ***P < 0.001.

RP-HPLC analysis of base-catalyzed hydrolysates from the cellular lipid extract obtained after incubation with [³H]15(S)-HETE showed that the increased uptake of 15(S)-HETE observed upon incubation with IL-4 was accompanied by increased amounts of 15(S)-HETE incorporated into cellular lipids (Figure 5). The profile of radioactivity from RP-HPLC analysis indicated that unmetabolized 15(S)-HETE represented more than 80% of the radioactivity released from cellular lipids upon basic hydrolysis (Figure 6).

View larger version (15K): [in this window] [in a new window]   Figure 5.   [3H]15(S)-HETE incorporation into cellular lipids. Cells were incubated with [3H]15(S)-HETE (0.5 µCi/ml) for 20 min at 37°C after 24, 48, and 72 h with or without IL-4 (10 ng/ml). The cells were washed free of supernatant, and phospholipid fatty acids were obtained by base-catalyzed hydrolysis (see MATERIALS AND METHODS). Samples were analyzed with RP- HPLC, and radioactivity in 1-min fractions was evaluated by scintillation counting. 15(S)-HETE in phospholipid hydrolysates was expressed as percent of added radioactivity. The results are expressed as means ± SEM (n = 3). **P < 0.01; ***P < 0.001.

View larger version (13K): [in this window] [in a new window]   Figure 6.   RP-HPLC analysis of cell lipids after base-catalyzed hydrolysis. Cellular lipid extracts from WI-26 cells pretreated with IL-4 (10 ng/ml, 72 h) and exposed to [3H]15(S)-HETE (0.5 µCi/ml) for 20 min at 37°C were hydrolyzed and analyzed with RP-HPLC, as described in MATERIALS AND METHODS. One-minute fractions were collected, and radioactivity was evaluated by scintillation counting.

NP-HPLC class analysis of cellular phospholipids showed that 15(S)-HETE was preferentially incorporated into PI. Pretreatment with IL-4 caused a progressive increase in [³H]15(S)-HETE incorporation into PI, peaking at 72 h (Figure 7). Taken together, the results shown in Figures 6 and 7 suggest that intact 15(S)-HETE was preferentially incorporated into PI without further metabolism, and that preincubation with IL-4 induced a threefold increase in the amount of 15(S)-HETE incorporated into PI.

View larger version (13K): [in this window] [in a new window]   Figure 7.   Separation of phospholipid classes by NP-HPLC. Cellular lipid extracts from WI-26 cells cultured in the absence (a) or presence (b) of IL-4 for 72 h, and exposed to [3H]15(S)-HETE (0.5 µCi/ml) for 20 min at 37°C, were analyzed with NP-HPLC as described in MATERIALS AND METHODS. One-minute fractions were collected, and radioactivity evaluated by scintillation counting. NL = neutral lipid; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine; PC = phosphatidylcholine. Tracings are representative of three different experiments.

	Discussion

Abstract Introduction Materials and Methods Results Discussion References

The data presented indicate that IL-4 was able to increase significantly 15-LO activity, measured as production of 15(S)-HETE upon exposure to exogenous AA, in WI-26 cells, a human lung epithelial cell line. This change was associated with increased expression of 15-LO mRNA as assessed by PRINS labeling. In addition, we showed that IL-4 was able to modulate the incorporation of radioactive 15(S)-HETE into cellular lipid, significantly increasing the esterification of this metabolite into inositol-containing phospholipids.

IL-4 is a cytokine released by Th2 cells and mast cells (8), and plays a major role in the pathogenesis of allergic (24, 25) and possibly also of nonallergic asthma (31). Besides its IgE-regulating effects (32, 33), IL-4 is responsible for a complex cascade of biologic events. In normal subjects, IL-4 has been found to be an inhibitor of mononuclear phagocyte functions in vitro and ex vivo (34, 35), significantly suppressing IL-6 production and expression of IL-6 mRNA in lipopolysaccharide (LPS)-stimulated alveolar macrophages (AM) (34). Although these effects suggest a possible role of IL-4 in termination of the inflammatory response, this cytokine is also involved in a variety of immunologic reactions that can maintain the inflammatory process. In fact, IL-4 stimulates the maturation of blood monocytes (36) and increases the expression of cell-membrane markers, such as CD23 and class II major histocompatibility complex (MHC) antigen on monocytes (37).

IL-4 appears to be unique among several different cytokines for its ability to induce 15-LO protein and mRNA in different cell types (21, 22); in accord with these reports, we found that preincubation of WI-26 cells with IL-4 resulted in a significant increase in 15-LO activity, defined as the cellular capacity for oxygenation of exogenous AA to 15(S)-HETE. Maximal 15-LO activity was observed after 48 h of incubation with IL-4, whereas a decrease in activity was observed after 72 h. Changes in 15-LO activity correlated well with the number of cells positive for specific 15-LO mRNA as assessed by PRINS labeling, suggesting that the increase in 15-LO activity is the result of increased expression of the enzyme, rather than of changes in the specific activity of 15-LO.

15(S)-HETE assayed in cellular supernatant upon addition of exogenous AA progressively decreased with time, suggesting that either metabolism or an active process of uptake by the WI-26 cells was taking place. Further experiments were performed to study the fate of radiolabeled 15(S)-HETE exposed to WI-26 cells, and results showed that IL-4 significantly increased incorporation of 15(S)- HETE into cellular lipids, with preferential incorporation into inositol-containing phospholipids.

Although different cell types, such as neutrophils (38), macrophages, and endothelial cells (39), are known specifically to incorporate 15(S)-HETE into the PI pool of cellular phospholipids, this is the first report of the modulation by IL-4 of the incorporation of 15(S)-HETE. RP-HPLC analysis of radioactivity obtained upon basic hydrolysis of cellular lipid after incorporation of [³H]15(S)-HETE confirmed the incorporation of intact 15(S)-HETE. Furthermore, analysis of radioactivity remaining in the supernatant upon exposure to [³H]15(S)-HETE showed that the radioactivity corresponded exclusively to intact 15(S)- HETE, ruling out significant catabolism of 15(S)-HETE by WI-26 cells.

The ability of IL-4 to increase 15-LO activity in pulmonary epithelial cells has several potential implications in the pathophysiology of airway inflammation. The increased potential for production of 15(S)-HETE by IL-4-treated cells may play an important role in development of the inflammatory process in the airways. 15(S)-HETE is indeed a potent mucosecretagogue in human airways (40), possesses chemotactic activity for neutrophils (41), and prolongs the duration of airway obstruction during the early response (42). Several studies have shown increased production of 15(S)-HETE in chronic inflammatory diseases of the airways, such as asthma (7, 43), and recently, ozone-treated airway epithelial cells showed increased intracellular levels of 15(S)-HETE with reduced synthesis of PGE₂, a cyclooxygenase metabolite of AA having several antiinflammatory activities in the airways (20).

On the other hand, the ability of IL-4 to increase 15(S)- HETE incorporation into PI by pulmonary epithelial cells may represent a mechanism regulating or altering intracellular signal transduction (19). The incorporation of 15(S)- HETE into PI causes reduction of its subsequent phosphorylation to PI phosphate (PIP) and diphosphate (PIP₂), altering the amount and/or production of second messengers such as diacylglycerol (DAG) and inositol trisphosphate (IP₃) (46). 15(S)-HETE esterified into cellular phospholipids has been shown to modulate the receptor-mediated activation of human neutrophils (47), to decrease the ability to generate leukotriene B₄, and to cause a marked reduction in agonist-induced cell aggregation (38).

The uptake and incorporation of 15(S)-HETE by epithelial cells may also contribute to decreasing the pericellular concentration of this proinflammatory mediator, promoting resolution of the inflammatory process. This hypothesis is supported by the finding that at sites of airway mucosal injury, airway epithelial cells can also incorporate 5-, 12-, or 15(S)-HETE released by inflammatory cells, such as macrophages, neutrophils, and eosinophils, potentially decreasing the extracellular concentrations of these proinflammatory mediators (19). Taken together, this evidence strongly suggests that autocrine or paracrine incorporation of 15(S)-HETE by pulmonary epithelial cells represents an important antiinflammatory mechanism.

Given the potential antiinflammatory activities of IL-4, it can be hypothesized that the IL-4-dependent increases in both 15-LO activity and the enzymatic activity(ies) resulting in 15(S)-HETE esterification into cellular phospholipids in pulmonary epithelial cells are important in downregulation of the proinflammatory process in the airways. However, further studies are necessary to test this hypothesis.

In conclusion, the results of this study support the concept that IL-4 is a specific and potent modulator of 15-LO activity, as well as of the uptake and incorporation of 15(S)-HETE into phospholipids. The selective incorporation of 15(S)-HETE into the PI pool of cellular phospholipid and its regulation by IL-4 suggest that this AA-metabolite may exert an autocrine function, possibly through the modification of intracellular signal-transduction pathways.