Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Numerous studies conducted in recent years indicate that the epithelium of the conducting airways can produce a variety of inflammatory mediators, particularly when injured by microbial agents, toxic air contaminants, and allergens (1, 2). One group of inflammatory mediators are the eicosanoids, which are lipid metabolites formed from arachidonic acid (AA) by prostaglandin H synthase (PGHS) or lipoxygenase enzymes. Eicosanoids are believed to play an important role in pathogenetic mechanisms of airway inflammation, such as leukocyte trafficking, edema formation, and airway hypersecretion.

Two major groups of eicosanoid enzymes are responsible for the formation of these lipid mediators. The PGHS enzymes are responsible for the conversion of AA to prostaglandins, whereas lipoxygenases (LO) are responsible for the metabolism of AA to leukotrienes and other hydroperoxy- and epoxy fatty acids. The expression of these enzymes in tracheobronchial epithelium tends to be highly species specific (3). Human tracheal cells are reported to generate mainly 15-LO products (4, 5), whereas tracheal cells of rats secrete primarily prostaglandin E₂ (PGE₂), a product of PGHS (6). Two forms of PGHS exist, a constitutively expressed PGHS-1, and PGHS-2. In many cells, expression of the latter is regulated by many growth factors and inflammatory agents; however, recent work has shown that PGHS-2 is constitutively expressed in human lung epithelial cells (7). The substrate for these enzymes, AA, is stored in membrane phospholipids and must be converted to free AA by the phospholipase A₂ (PLA₂) enzymes. The PLA₂ family consists of a high molecular weight, 85-kD cytosolic form (cPLA₂) and a group of 13 to 15-kD secreted enzymes (sPLA₂) with a requirement for millimolar Ca²⁺ concentrations for activity.

We have been interested in studying changes in eicosanoid enzyme expression during differentiation of airway epithelium (6). Using rat tracheal epithelial cells grown in air/liquid interface cultures, which support mucociliary differentiation, we have shown that the undifferentiated epithelium typically seen during the logarithmic growth phase of these cultures did not express significant amounts of cytosolic PLA₂ (cPLA₂), PGHS-1, or PGHS-2. However, as mucous differentiation began in plateau-phase cultures, cPLA₂ and PGHS-2, but not PGHS-1, were expressed, and PGE₂ was secreted. We also reported that the expression of these enzymes hinged on the development of the vitamin A-dependent mucous phenotype, since vitamin A-deficient cultures, which differentiate into squamous instead of mucociliary epithelium, expressed neither cPLA2 nor PGHS-2, and secreted only very small amounts of PGE₂. However, these squamous cultures did express PGHS-1.

Human tracheobronchial epithelial cells have been reported to predominantly synthesize 15-LO products such as 15-hydroxyeicosatetraenoic acid (15-HETE) and smaller amounts of PGE₂ and PGF₂ (4, 5, 7). The physiologic and pathophysiologic roles of 15-LO products (e.g., 15-HETE and its derivatives) are still largely unclear, and may depend in part on the cells and tissues by which they are generated. They have been implicated as playing a role in inflammatory processes (8). 15-LO is also believed to play an important role in the pathogenesis of atherosclerosis (9). At the cellular level it has been shown to be involved in erythrocyte maturation (10, 11) and in programmed cell death (12). In monocytes, 15-LO expression is regulated by corticosteroids, interferon- (IFN-), interleukin (IL)-4, and IL-13 (13, 14). However, the regulation of 15-LO expression and activity in airway epithelial cells is poorly understood. The main purpose of the studies reported here was to examine the expression of key enzymes of AA metabolism during the differentiation of normal human tracheobronchial epithelial (NHTBE) cells. We simultaneously examined the expression of cPLA₂, PGHS isoforms, and 15-LO during different phases of differentiation, in order to compare the expression of these enzymes in human airway cells with that in rat airway cells in a previously reported study (6).

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials

Linoleic acid (LA) was obtained from Nucheck, Elysian, MN; [I-¹⁴C] carboxyl-labeled AA and LA (60 to 100 Ci/ mmol) were from NEN-DuPont, Boston, MA; trypsin was from Hyclone Labs, Logan, UT. Leupeptin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), calcium ionophore (A23187), and all trans-retinoic acid (RA) were purchased from Sigma Chemical Co., St. Louis, MO. Enhanced chemiluminescence (ECL) reagents were from Amersham, Arlington Heights, IL.

Antibodies

Rabbit antiserum raised against the C terminus of rabbit cornifin  was a gift from Dr. A. M. Jetten, National Institute of Environmental Health and Science (NIEHS), Research Triangle Park, NC. Antiserum to human 15-LO was a gift from Dr. E. Sigal, Syntex Inc., Palo Alto, CA. Human PGHS-2 antibody was purchased from Cayman Chemical Co., Ann Arbor, MI. Antiserum to a 38-kD trypsin fragment of the COOH terminus of PGHS-1 was a gift from Dr. L. Marnett, Vanderbilt University, Nashville, TN. Antiserum against human denatured recombinant cPLA₂ was a gift from the Genetics Institute, Cambridge, MA. Antihuman Group II sPLA₂ was purchased from Upstate Biotechnology Incorporated, Lake Placid, NY. 17Q2 antibody against rhesus monkey tracheal secretions was a gift from Dr. J. St. George, Genzyme, Framingham, MA. Human 15-LO, PGHS-1, and PGHS-2 complementary DNAs (cDNA) were purchased from Oxford Biomedical Research Inc., Oxford, MI. Glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA was obtained from Oncor Inc., Gaithersburg, MD.

Cell Culture

Frozen passage-1 stocks of NHTBE cells (strain 2002; Clonetics Corp., San Diego, CA) were subcultured as previously described (15). Passage-2 NHTBE cells were seeded (5 × 10⁵/culture) on Transwell-clear (Costar Co., Cambridge, MA) culture inserts. Cells were cultured in a 1:1 mixture of bronchial epithelial growth medium (BEGM; Clonetics Corp) and Dulbecco's modified Eagle's medium (DMEM) supplemented as previously described (15), but with the addition of LA (1 µM) and the omission of hydrocortisone after 3 d of culture. In some experiments, RA, (5 × 10⁸ M) was omitted from the medium after 1 d of culture. Cells were fed both apically (0.5 ml) and basally (2.5 ml) until day 7 of culture, and thereafter were fed only basally, to establish air/liquid interface cultures.

Cell Counting

Cells were treated with 0.25% trypsin (1 ml apical compartment) at 37°C for 20 min. Cells were recovered by centrifugation and resuspended in culture medium, and triplicate samples were counted in a hemocytometer. Cell viability (95 to 97%) was monitored through trypan blue exclusion.

Mucin Measurement

Apical secretions produced over 24 h from individual inserts were collected in 3 × 500 µl washes of phosphate-buffered saline (PBS). Aliquots of samples were serially diluted 2-fold and transferred to nitrocellulose (Schleicher and Schuell, Keene, NH). Mucin was quantified against purified human mucin by dot-blot assay (15), using a 17Q2 antibody raised against rhesus monkey tracheal secretions. The blots were incubated with peroxidase-labeled antimouse antibody using ECL reagent. Exposed films were scanned and density readings converted to mucin standard equivalents.

Reverse-phase High-pressure Liquid Chromatographic Analysis

NHTBE cultures were incubated basally (2.5 ml) and apically (0.5 ml) with or without A23187 and with either ¹⁴C-labeled LA or ³H-AA (10 µM, 0.6 µCi/ml), in 1:1 BEGM:DMEM at 37°C for 30 min. The medium was removed and the insert containing the culture was extracted with methanol (0.5 ml). The methanol and medium were combined and diluted 2-fold with 1% acetic acid. PGB₂ (200 ng) internal standard was added, and the sample was subjected to solid-phase extraction on a C₁₈ column (Waters, Milford, MA). Eicosanoids were separated on an ODS 5-µm column (Ultrasphere, Beckman, CA), using a stepwise solvent gradient of water (solvent A consisting of 10% methanol-0.1% acetic acid, pH 5.05, with ammonium hydroxide) and methanol (solvent B), and changing from 53% solvent B to 60% solvent B at 27 min, to 73% solvent B at 52 min, and to 100% solvent B at 74 min. Radioactive metabolites were detected with a Radiomatic Flow-One beta detector (Packard Instruments, Downer Grove, IL).

Cell-free Preparations

Cells in culture were detached from the inserts with trypsin, washed with DMEM (10 ml), and resuspended in 0.4 ml of 100 mM ice-cold Tris buffer (pH 8.0) containing 3 mM ethylenediamine tetraacetic acid (EDTA), 1 µg/ml leupeptin and pepstatin, and 0.5 mM PMSF. Cells were sonicated four times for 15 s each at 50% power for a total protein preparation for Western blot analysis. For some metabolism studies of cell-free preparations, the sonicate was further diluted to 1 ml with Tris-EDTA buffer and incubated with [¹⁴C]-AA (10 µM) and CaCl₂ (5 mM) for 30 min at 37°C before being analyzed as described earlier.

Western Blot Analysis

Protein preparations were quantified through the bicinchoninic acid method. Aliquots of the protein preparation were boiled in Laemmli sample buffer, separated by 8% polyacrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to HYBOND nitrocellulose (Amersham). Blots were blocked with 10% milk in 0.1% Tween in PBS before incubation with the appropriate specific antibodies. Immunoreactive protein was detected with the ECL Western blotting system.

Northern Blot Analysis

Cell cultures on inserts were lysed through the addition of 1 ml of guanidinium isothiocyanate lysis solution, and total RNA was prepared by phenol-chloroform extraction. Total RNA (10 µg) was separated on 0.9% agarose gels containing 1.2% formaldehyde and 1× MOPS running buffer. RNA was transferred to nylon membranes, (HYBOND-N; Amersham) by capillary blotting, using 10× standard saline citrate (SSC), and was crosslinked to the membrane by ultraviolet irradiation. cDNA probes were labeled with [-³²P] deoxycytosine triphosphate ([-³²P] dCTP) using the Prime-It 11 random prime kit (Stratagene, La Jolla, CA). Blots were prehybridized at 44°C for 2 h, followed by hybridization overnight. Blots were washed at 44°C with 1× saline-sodium phosphate-EDTA (SSPE), 0.1% SDS for 15 min followed by 0.1× SSPE, 0.1% SDS for 10 min.

Immunoassays

Eicosanoids were quantified from medium aliquots by immunoassay according to the manufacturer's protocol. 13(S)-hydroxy-9,11-octadienoic acid [13(S)-HODE] was measured with an enzyme-linked immunosorbent assay (ELISA) (Oxford Biomedical Research Inc.). 15(S)-HETE was measured with a radioimmunoassay (RIA) (PerSeptive Diagnostics, Cambridge, MA), and PGE₂ was determined by ELISA (Cayman Chemical Co., Ann Arbor, MI).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Effect of Retinoid-induced Mucous Differentiation on 15-LO and PGHS-2 Expression

NHTBE cells grown in the presence of 5 × 10⁸ M RA for 14 d produced significant amounts of mucinous secretions (99.0 ± 12.0 µg/10⁶ cells), indicating the development of a secretory phenotype. In contrast, cells grown in the absence of RA secreted negligible amounts of mucin (0.1 ± 0.1 µg/10⁶ cells). We measured the expression of cornifin , a component of the crosslinked envelope, which has been shown to be highly expressed in late stages of squamous differentiation (16). Western blot analysis showed (Figure 1) that cornifin  levels in cell extracts from RA-sufficient cultures was very low. RA-deficient cultures showed significant amounts of cornifin  protein, consistent with differentiation to the squamous phenotype.

View larger version (6K): [in this window] [in a new window]   Figure 1.   Cornifin  protein expression in NHTBE cells grown in the absence or presence of RA for 14 d. Total cell lysates (5 µg) were analyzed with SDS-PAGE on a 16% gel, and immunoblotted with a polyclonal antibody to cornifin .

Previous studies have shown that the expression of PGHS isozymes is regulated by retinoid-induced differentiation in rat tracheal epithelium (6). Human tracheal epithelium is a known source of prostaglandins (7) as well as products of 15-LO metabolism (4, 5). We investigated the expression of PGHS isozymes and 15-LO in differentiated mucous (⁺RA) or squamous (RA) NHTBE plateau-phase cultures. Fourteen-day-old mucous cultures strongly expressed 15-LO protein, which was detected as a single band at 66 to 72 kD on SDS-PAGE gels (Figure 2). Mucous cultures also weakly expressed PGHS-2 protein, which was detected at 70 to 72 kD. In contrast, neither 15-LO nor PGHS-2 protein could be detected in squamous cultures. PGHS-1 protein could not be detected in either phenotype (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 2.   The effect of retinoid-induced differentiation on expression of eicosanoid enzymes. NHTBE cells were cultured for 12 to 14 d to either a mucociliary (+RA) or squamous (RA) phenotype. Ten micrograms of total cell lysate were separated on 10% SDS-PAGE gels and immunoblotted with polyclonal antibody to either 15-LO or PGHS-2. Data are representative of three experiments.

Expression of Eicosanoid Enzymes during the Development of Mucociliary Differentiation

Previous studies of rat tracheal epithelial cell cultures have shown that expression of PGHS-2 and cPLA₂ occurred in a time-dependent manner coordinately with development of the mucous phenotype. We investigated the expression of enzymes of the AA cascade during mucociliary differentiation of RA-sufficient NHTBE cultures. Mucin secretion was used as an indicator of mucous differentiation. From Day 12 onwards the epithelium began to produce significant amounts of mucin, which reached a maximum at Day 14 (Figure 3). By Day 21, 1% of the cells were ciliated (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 3.   Mucin secretion of NHTBE cells as a function of time in culture. Cells were grown in the presence of RA for 21 d. Mucin production was quantified as described in MATERIALS AND METHODS; n = 3 cultures, values expressed as mean ± SD.

PGHS-1 protein was not detected in either early or late NHTBE cultures (data not shown), but both PGHS-2 message and protein were strongly expressed in early undifferentiated cultures (Figures 4 and 5A). As the cultures differentiated, expression of PGHS-2 message and protein declined to barely detectable levels by Day 17. In contrast, 15-LO message and protein were not expressed in undifferentiated cultures but were strongly expressed from day 14 onwards in the differentiated cultures (Figures 4 and 5B). Eicosanoid synthesis is dependent on release of the substrate, AA, from phospholipids by PLA₂. The high-molecular-weight cPLA₂ protein was detected as a 100-kD band on 10% SDS-PAGE gels, and was expressed throughout the course of culture in both undifferentiated and differentiated cultures (Figure 4). The low-molecular-weight Group II sPLA₂ protein was not detected in either early or late NHTBE cultures (data not shown). These results indicate that the mRNA levels of both PGHS-2 and 15-LO are downregulated and upregulated, respectively, during mucociliary differentiation of NHTBE cells.

View larger version (27K): [in this window] [in a new window]   Figure 4.   Expression of cPLA2, 15-LO, and PGHS-2 protein during mucociliary differentiation of NHBTE cells. Cells were grown in the presence of RA for 21 d. Total cell lysates (10 µg) were separated on 10% SDS-PAGE gels and immunoblotted with polyclonal antibody to cPLA2, 15-LO, or PGHS-2.

View larger version (23K): [in this window] [in a new window]   Figure 5.   Expression of PGHS-2 and 15-LO mRNA during mucociliary differentiation of NHTBE cells. Cells were grown in the presence of RA for 21 d; 10 µg of total RNA were separated on 0.9% agarose gels and hybridized to either (A) human PGHS-2 cDNA or (B) human 15-LO cDNA, followed by GAPDH (C and D).

Characterization of Eicosanoid Metabolism during the Development of Mucociliary Differentiation

Eicosanoid synthesis from exogenous substrate. Eicosanoid products were characterized from early and late RA-sufficient NHTBE cultures. Day 10 (early stage of differentiation) and Day 21 (late stage of differentiation) cultures were incubated with [³H]AA (10 µM, 30 min) and calcium ionophore (5 µM). One major metabolite (39 ± 12 ng/10⁶ cells) was formed by 10-d-old cultures (cell extracts and medium combined), and coeluted with authentic PGE₂ standard in methanol/water RP-HPLC (Figure 6A).

View larger version (16K): [in this window] [in a new window]   Figure 6.   Eicosanoid production during mucociliary differentiation. NHTBE cells were cultured with RA for 10 and 21 d and were incubated with [3H]-AA (10 µM, 30 min). The medium and cell extract were combined and analyzed with RP-HPLC, and the chromatograms were corrected for recovery. (A) Products formed from undifferentiated Day 10 cultures incubated with A23187 (5 µM, 30 min). (B) Products formed from differentiated Day 21 cultures incubated with A23187 (5 µM, 30 min). (C) Products formed by incubation of [3H]-AA with cell-free lysate of Day 21 cultures.

View larger version (20K): [in this window] [in a new window]   Figure 7.   LA metabolism in mucociliary NHTB cultures. Day 21 cultures were incubated with [14C]- LA (10 µM, 30 min). The medium and cell extract were combined with the products and analyzed by RP-HPLC. (A) Incubation without ionophore. (B) Incubation with A23187 (5 µM, 30 min).

Eicosanoid synthesis from endogenous substrate. We investigated the production of eicosanoids from endogenous substrate during mucociliary differentiation of NHTBE cultures. Every 24 h, the medium was removed from some of the cultures for analysis of PGE₂, 15-HETE, and 13-HODE by immunoassay. Early, poorly differentiated cultures (Day 7) produced > 80 ng PGE₂/10⁶ cells (Figure 8). As the cultures differentiated, the production of PGE₂ rapidly decreased to undetectable levels by Day 21. The culture medium was also analyzed for 15-HETE by RIA (detection limit, 25 pg/ml), and for 13-HODE with an ELISA (detection limit, 2 ng/ml). Neither eicosanoid could be detected in media throughout the time course of culture, or in media from Day 14 and Day 21 cultures treated with calcium ionophore for 30 min (data not shown). These data suggest that undifferentiated NHTBE cultures secrete PGE₂ but that differentiated cultures do not secrete detectable levels of eicosanoids, including 15-LO metabolites, without the addition of exogenous substrates.

View larger version (10K): [in this window] [in a new window]   Figure 8.   PGE2 production during differentiation of NHTBE cells to mucociliary epithelium. NHTBE cells were grown for 21 d in the presence of RA. Twenty-four-hour production of PGE2 in the culture medium was quantified by ELISA; n = 3 cultures, values are mean ± SD.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The differentiation state of the airway epithelium is frequently altered as a result of different types of injury and inflammation. In severe asthma and during viral and bacterial infections, large portions of the mucociliary epithelium can become necrotic. Subsequently, during the period of healing and regeneration, the wound is covered by rapidly proliferating, undifferentiated epithelium, which in most cases redifferentiates into a normal mucociliary epithelium. Other types of injuries caused by toxic or carcinogenic air contaminants (e.g., tobacco smoke) can result in transient or long-lasting metaplastic squamous differentiation of the epithelium. Abnormal squamous differentiation is also seen during severe vitamin A deficiency, and is reversible upon reintroduction of vitamin A to the diet (18).

Our studies have been aimed at elucidating biochemical changes accompanying normal and abnormal differentiation of the tracheobronchial epithelium. In this study and a previous study with rat epithelium (6), we focused our attention on the metabolism of AA and the expression of enzymes responsible for the formation of prostaglandins and other metabolites, because of their importance as proinflammatory as well as anti-inflammatory agents. Some of the eicosanoids have also been shown to regulate airway secretions. NHTBE cell cultures, similar to the rat tracheal epithelial (RTE) cell cultures used in our earlier study (6), when grown at an air/liquid interface, go through phases of differentiation. During the first 7 to 10 d the cultures rapidly proliferate and are morphologically undifferentiated. Regardless of their vitamin A status, they produce little mucin during this time. Between days 10 and 12 cell growth rapidly diminishes and increasing amounts of mucin are secreted as a sign of mucous differentiation, provided the medium contains RA or some other type of retinoid. In the absence of RA the cultures become squamous metaplastic, and increasingly express markers of squamous differentiation such as cornifin (16).

In our previous studies with RTE cells (6), RA-induced mucociliary differentiation was accompanied by increased expression of cPLA₂ and PGHS-2 protein. PGE₂ formation from endogenous and exogenous AA increased simultaneously. In contrast, in human tracheobronchial cells, as shown here, cPLA₂ protein expression was constant throughout the development of the mucous phenotype. PGHS-2 message and protein were highly expressed in the undifferentiated cultures and decreased markedly as the cultures differentiated. In contrast, 15-LO message and protein were not expressed in the early cultures but were expressed in the late, fully differentiated cultures. Thus, there appears to be an inverse relationship between PGHS-2 and 15-LO expression.

Our findings raise important questions about the causal relationship between the vitamin A status and differentiation status, respectively, of NHTBE cell cultures, and the expression of these enzymes. That cPLA₂ is not expressed in RA-deficient squamous metaplastic cultures but is expressed in RA-sufficient undifferentiated as well as in mucous differentiated cultures might suggest that in NHTBE cells this enzyme is simply RA dependent, rather than differentiation dependent. Alternatively, the lack of expression of PGHS-2 and 15-LO in RA-deficient squamous cultures, and the expression in RA-sufficient cultures of PGHS-2 only in undifferentiated proliferating cultures and of 15-LO only in mucous differentiated cultures, may suggest that in addition to vitamin A status, differentiation status also plays an important role in the regulation of expression of these two enzymes.

PGE₂ is a second messenger with pleiotropic effects: it can act as a mitogen in NIH 3T3 cells (19), inhibit proliferation of airway smooth-muscle cells in vitro (20), and inhibit mucous secretion in human lung explants (21, 22). In RTE cell cultures in which PGE₂ is the only eicosanoid produced, PGE₂ secretion was found to be increased during development of the mucous phenotype, and stimulation of mucin gene expression and secretion by tumor necrosis factor- (TNF-) was shown to be PGE₂ dependent (23). The studies presented here suggest that in NHTBE cells, PGE₂ probably has a very different function than in rat epithelium, since its secretion decreased as mucous differentiation and mucin secretion increased. Since PGE₂ secretion occurs during the proliferation phase of the NHTBE cultures, it may be related to cell growth or to regulation of extracellular matrix in the regenerating epithelium (24).

We examined the metabolism of AA by human tracheobronchial cell cultures in either the squamous or the mucocilliary state of differentiation. In undifferentiated RA-deficient cultures, PGHS-2 is expressed and exogenous AA is metabolized to PGE₂. Furthermore, these cultures were able to form PGE₂ from endogenous substrate, since they expressed cPLA₂. At late stages of mucociliary differentiation no prostaglandin was formed, because of lack of PGHS-2 expression. Surprisingly, no 15-LO metabolites were formed even when the cultures were stimulated with calcium ionophore, despite the cells content of ample 15-LO protein. However, cell lysates prepared from late, differentiated cultures did metabolize AA to 15-HETE, indicating that the cells contained active 15-LO. In subsequent experiments, LA metabolism was examined, since reports in the literature indicate that LA is the preferred substrate for human 15-LO (17). We observed that upon stimulation with calcium ionophore the cultures metabolized exogenous LA to 13-HODE at substrate concentrations as low as 10 µM.

The question thus arises why the NHTBE cultures in our studies failed to produce 15-HETE, since other investigators (4, 5) have reported the formation of 15-HETE by freshly isolated or cultured bronchial cells. One possible explanation is that freshly isolated bronchial-cell preparations may be contaminated with nonepithelial cells (5), which may produce 15-HETE. Furthermore, in most of the published studies, significant 15-HETE production was observed only at very high AA concentrations (> 50 µM) (4). It is conceivable that 15-HETE is formed by normal human bronchial cells only when the epithelium is injured or activated by inflammatory cytokines. Our studies suggest that under physiologic conditions, 13-HODE may be the more prevalent 15-LO metabolite in bronchial epithelium.

Studies with both rabbit and human 15-LO show that it can readily oxygenate cell-membrane phospholipids, and may be involved in the oxidation of mitochondrial membranes during reticulocyte maturation (10, 11). Immunofluorescence studies show that 15-LO is expressed in both the ciliated and basal cells of human tracheal epithelium (25). In our studies, 15-LO was expressed only during mucous differentiation, when the cultures contained secretory and (later) ciliated cells. It is conceivable that 15-LO is involved in membrane oxygenation of intracellular organelles during terminal differentiation to ciliated cells.

Additional studies are required to further explore conditions and stimuli that activate the formation of AA and LA metabolites by 15-LO, and the physiologic or pathophysiologic role of such metabolites in the conducting airways.