The Airway Epithelium and Arachidonic Acid 15-Lipoxygenase1 - 3 E. SIGAL4 and J. A. NADEL

Introduction Airway inflammation is a prominent feature of many lung diseases such as asthma, bronchitis, and cystic fibrosis. Our hypothesis is that the airway epithelial cells is actively involved in the development of airway inflammation and its subsequent pathophysiologic effects (1). Furthermore, we believethat lipoxygenase metabolites of arachidonic acid are involved in this process. The background for this belief is the seriesof experiments on ozone inhalation, which elucidated the interrelationships between the epithelial cell, airway inflammation, and the development of airway hyperresponsiveness. We found that the inhalation of ozone increased bronchomotor responsiveness in human subjects (2) and in dogs (3). The level of responsiveness was correlated with the number of neutrophils recovered in lavage fluid and it was prevented entirely when circulating neutrophils were depleted (4). Furthermore, the inhalation of neutrophil chemoattractant leukotriene (LT) B4 induced both inflammation and hyperresponsiveness (5). Finally, we incubated canine tracheal epithelial cells with arachidonic acid and found that they released LTB4 as well as I2-hydroxyeicosatetraenoicacid (12-HETE) and I5-HETE (6). These studies led us to examine the metabolism of arachidonic acid by human tracheal epithelial cells, and we found that these cells generate large quantities of I5-lipoxygenase metabolites (7). The mammalian 5-, 12-, and I5-lipoxygenasesare named according to their ability to insert molecular oxygen at carbon number 5, 12, or 15 of arachidonic acid (8). Metabolites arising from the I5-lipoxygenase pathway are shown in figure 1. Human tracheal epithelial cells metabolize arachidonic acid predominantly via this pathway (7). This is consistent with the finding by Hamberg and coworkers (9) that I5-HETE is the major metabolite of whole-lung homogenates incubated with arachidonic acid. Interestingly, I5-lipoxygenaseactivity may even be increased in asthmatic lung (9), and local antigen challenge has been reported to increase I5-HETE in bronchoalveolar lavage fluid in four of five atopic subjects (10). In this report we discuss the discovery of I5-lipoxygenaseactivity in human tracheal epithelial cells, we review some of the proposed actions of I5-lipoxygenase metabolites, and we describe our current research on the purification and characterization of the human 15lipoxygenase. Because the biologic roles of the I5-lipoxygenase pathway remain unclear, we believe it is important to isolate the enzyme and characterize its biochemical and biologic functions. The availability of purified enzyme and clones of its cDNA will allow the development of antibodies useful in

SUMMARY Pulmonary epithelial cells may be primarily responsible for Initiating or regulating Inflammatory responses In the airways, in part by releasing chemical mediators. Among the most potent mediators of Inflammation are the Iipoxygenase metabolites of arachidonic acid, Including the leukotrienes and other mono and dihydroxyelcosatetraenoic acids (HETES). The human airway epithelium contains significant 15-lIpoxygenase activity. Although some biologic functions of 15Iipoxygenase metabolites are known, further understanding of the role of this enzyme In the airway requires localization In tissue and studies of expression, regulation, and biologic activity. Towards these aims, we purified and characterized 15-lipoxygenase from eosinophil-enriched leukocytes. First, we studied cofactors that may be involved in regulating enzymatic activity. Second, we Isolated to homogeneity, for the first time, human 1S-llpoxygenase. This led to the determination of the N-terminal amino acid sequence and the discovery of homology among various mammalian Iipoxygenases. Finally, we utilized this structural information to Isolate a eDNA that encodes for human 15-lIpoxygenase. The availability of a clone will permit studies of expression and the development of antibodies for tissue localization. Further research using molecular and antibody probes is expected to increase our understanding of the biologic roles of 1S-llpoxygenase In airway epithelium. AM REV RESPIR DIS 1991; 143:571-574

the immunocytochemical localization of the enzyme within tissue, thereby giving clues to its biologic functions. Purified enzyme from either native or recombinant sources will permit investigations of these biologic actions and their modulation. In addition, protein structural analysis of homogeneous protein will make possible a molecular understanding of lipoxygenation. For example, a comparison and analysis of the amino acid sequences of the various lipoxygenaseswill help identify the active site of lipoxygenation. Finally, the availability of molecular probes will allow us to study the regulation of 15lipoxygenase at the transcriptional level. Until now, cellular 15-lipoxygenase could only be studied at the level of its enzymatic product 15-HETE.

Lipoxygenase Metabolites from Human Trachea Our group has reported the initial evidence that human tracheal epithelium metabolizes arachidonic acid predominantly via the 15lipoxygenase pathway (7). Epithelial cells of 99010 purity and 92% viability were isolated from human tracheas obtained post-mortem and incubated with arachidonic acid. The lipoxygenase metabolites were identified using reverse-phase and straight-phase highpressure liquid chromatography, ultraviolet spectroscopy, and gas chromatography/mass spectrometry. Epithelial cells incubated without arachidonic acid failed to generate metabolites, whereas cells incubated with arachidonic acid at 1 to 15 ug/ml for 1 to 30 min invariably generated predominantly 15lipoxygenase products, including 15-HETE, four isomers of 8,15-diHETE, I4,15-diHETE, and small amounts of 12- and 8-HETE, but no detectable 5-HETE. These cells are capable of generating greater than 7,079 ± 674

pmol of 15-HETE per 106 cells (mean ± SE, n = 5) (11), which is significantly greater than the amount of 5-lipoxygenaseproducts generated by inflammatory cells. For example, the amount of LTB4 generated from human alveolar macrophages (12)and neutrophils (13) is 183 ± 96 (± SE) and 143 ± 63 pmol per 106 cells, respectively. The amount of LTB4 released from mast cells (14)and eosinophils (13) are both approximately 60 pmol per 106 cells. Whereas human tracheal epithelial cells express predominantly 15-lipoxygenase activity, dog cells and sheep cells exhibit predominantly 5-lipoxygenase and cyclooxygenaseactivities, respectively (15).

Biologic Activities of 1S-Lipoxygenase Metabolites A few biologic functions of the 15-lipoxygenase metabolites are becoming clarified. Of particular interest is the chemotactic activity of 8S,I5S-diHETE, which may serveas an epithelial cell signal to recruit inflammatory cells. This activity was originally described by Shak and coworkers (16) who studied human neutrophil chemotaxis in vitro. Because 1 From the Cardiovascular Research Institute and the Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California. 2 Supported in part by Program Project Grant HL-24136 from the National Institutes of Health and by the Cystic Fibrosis Foundation RDP Center. 3 Correspondence and requests for reprints should be addressed to E. Sigal, M.D., Cardiovascular Research Institute, Box 0911, University of California, San Francisco, San Francisco, CA

94143-0911. 4 Recipient of Clinical Investigator Award HL02047 from the National Institutes of Health and an Arthritis Investigator Award from the Arthritis Foundation.

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cxxx: ~--1=--~ 15-HPETE

Fig. 1. The metabolites formed from arachidonic acid via the 15-lipoxygenase pathway.

, ... 'SdiHETE

Lipo.in B

this action was questioned by Evans and colleagues (17), we studied the effect of 8S,15SdiHETE in vivo in the dog trachea (18). We developed an in vivo chemotaxis assay in the canine trachea using a double balloon endotracheal catheter that permits mechanical ventilation of dogs while an isolated tracheal segment is perfused with a chemotactic agent (18). The chemotacticeffect of 8S,15S-diHETE is significant at a concentration of 10-7 M and dose-dependent up to 10-6 M (18), at which point it appears equipotent to LTB4 • Weconclude from this study that 8S,15S-diHETE is a potent signal that recruits neutrophils to the airways. Once recruited to the airway, neutrophils may release LTB4 and platelet-activating factor, which can attract eosinophils, platelets, and other neutrophils. Hence, a wide array of inflammatory cells and mediators may be mobilized. In addition, the 15-lipoxygenase pathway may have other important inflammatory effects during this process. For example, 15-HETE has been shown to stimulate the releaseof the slow-reactingsubstance L1C4 from mastocytoma cells (19). If this finding is applicable to the mast cells of the human airway, 15-HETE production could lead to airway inflammation and smooth muscle hyperresponsiveness via effects of mast cells. 15-HETE has also been reported to stimulate mucous glycoprotein release from cultured human airways (20), and, therefore, it may playa role in the development of airway hypersecretion. A potential epithelial-neutrophil interaction has been suggested by the work of Serhan and coworkers (21) on the generation of lipoxin A. The lipoxins are trihydroxy acids derived from the sequential action of 15-and 5-lipoxygenase. Serhan and coworkers showed that neutrophils generate lipoxin A when incubated with 15-HETE (21). Macrophages

and mast cells may also generate lipoxin A under similar conditions (22, 23). Lipoxin A has a variety of biologic actions, including the ability to cause neutrophils to degranulate and to generate superoxide radicals (21), to contract guinea-pig lung strips (24), and to activate protein kinase C (25), a potential mechanism involved in regulating the chloride channel in airway epithelium (26). Furthermore, Dahlen (27) has recently reported that lipoxin A contracts human bronchi. Hence, the 15-lipoxygenase pathway in the human airway epithelium has the capacity to incite an inflammatory response, to initiate a cascade of inflammatory mediators, to activate mast cells and neutrophils directly, to cause mucus release, and finally, through cellto-cell interactions, to generate slow-reacting substances such as L1C4 and lipoxins that may cause bronchoconstriction. Curiously, the substances that activate other cells to release lipoxygenase products do not cause their release from epithelial cells. For example, the ionophore A23187releases LTB4 from neutrophils (13) and macrophages (12) and LTC4 and lipoxins from eosinophils (13, 28), but it has little effect on epithelial cells. This suggests that the epithelial cell has a different, perhaps non-receptor-based, mechanism for release of these mediators. Because the epithelial cell functions as a sentinel cell and because many of its signals come from outside the body (irritants, viruses, etc.), it seems reasonable for this cell to respond to products of cell damage. Because damaged or senescent cells are capable of releasing arachidonic acid, and because epithelial cells are relatively resistant to the cytotoxic effects of arachidonic acid in concentrations of less than 75 ~M, but generate large quantities of I5-HETE at these concentrations (11), this may be the mechanism by which I5-lipoxygenase metabolites are released.

Purification of Human 1S-Lipoxygenase Advances in understanding the regulation and biologic role of 15-lipoxygenase depend on its purification and characterization. Therefore, after the initial description of the release of 15-lipoxygenase metabolites from intact epithelial cells, we concentrated on enzyme purification. We used human eosinophils as a cell source for purification because of the significant 15-lipoxygenase activity in these cells(29, 30) and because of a convenient supply of eosinophil-enriched leukocytes from patients with malignancies undergoing leukophoresis during interleukin-2 therapy (30). Wedisrupted these eosinophil-enriched granulocytes via sonication and recovered 70 to 95010 of the enzymatic activity in the 100,000 x g supernatant, which suggests a cytosolic localization for the 15-lipoxygenase. During the development of purification procedures, we explored the effect of cofactors known to enhance 5-lipoxygenase activity on enriched preparations of 15-lipoxygenase. Our intent was twofold: first, we hoped to discover factors that would increase our yield of I5-lipoxygenase during purification; second, we were curious about factors that might regulate 15-lipoxygenase activity in biologic systems (30). We found that the 15-lipoxygenase reaction was approximately linear at 330 C for approximately 7 to 10min. Activity was markedly decreased at pH less than 7, optimum in the range of pH 7 to 8.5, and, linearly dependent on protein concentration in the range 100 to 500 ug/ml, The enzyme lipoxygenated both arachidonic and linoleic acid and exhibited a calcium dependence in crude extracts, an effect that disappeared during purification. Phosphatidylcholine stimulated I5-lipoxygenase activity, whereas ATP inhibited it (30). Because the eosinophil contains both 5- and 15-lipoxygenase activity, the modulation of lipoxygenase activity within this cell may be regulated by ATP. Purification of 15-lipoxygenase was achieved by using a combination of ammonium sulfate precipitation, hydrophobicinteraction, hydroxyapatite, and cationexchange chromatography (31).A single protein peak, coeluting precisely with a peak of lipoxygenase activity, was obtained in the final chromatographic step. Specific activity measurements documented a 1,8oo-fold enrichment. The results from SDS/polyacrylamide gel electrophoresis(stainedwith Coomassieblue) of samples obtained from each purification step are shown in figure 2. In the purified preparation (lane 4), one major protein band was observed with an estimated molecular weight of 70,000 D. In addition, one major amino acid sequence. was obtained from this final fraction. N-Terminal Amino Acid Sequence and Homology to Other Mammalian Lipoxygenases We analyzed the N-terminal amino acid sequence of the protein shown in figure 2, lane

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AIRWAY EPITHELIUM AND ARACHIDONIC ACID 15-L1POXYGENASE

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Fig. 3. N-terminal amino acid sequences for human leukocyte 15-lipoxygenase (Jane 4), rabbit reticulocyte lipoxygenase, human 5-lipoxygenase, and soybean 15-lipoxygenase. Sequence identity is indicated by the bold lines. (Reproduced with permission from reference 31.)

Val Cys

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Fig 2. Polyacrylamide gel electrophoresis (Coomassie blue stain) of fractions obtained during the purification of 15-lipoxygenase. The samples are 30 to 60% precipitate (fane 1),phenyl-Sepharose (Jane 2), hydroxyapatite (fane 3), and Mono S cation exchange (Jane 4). (Reproduced with permission from reference 31.)

4, and we compared this with the first 15 amino acid residues of various lipoxygenases (figure 3). Only a partial sequence of the rabbit reticulocyte lipoxygenase (32) was available, but the entire sequences of the soybean lipoxygenase (33) and the human 5-lipoxygenase are known (34, 35). There is a 71070 sequence identity with the rabbit reticulocyte lipoxygenase and a 36% sequence identity with the human 5-lipoxygenase. Furthermore, when one considers the sequence similarity at positions 5, 8, 11, 13, and 14, there is 60070 sequence similarity among all three mammalian lipoxygenases. In contrast, a search of the entire sequence of the soybean lipoxygenase failed to locate any sequence identity to the N-terminus of the human 15-lipoxygenase. These results suggest that the mammalian lipoxygenases are members of a homologous family of proteins. Despite the fact that others have presented functional and immunologic data (36) suggesting that the reticulocyte lipoxygenase is unique to reticulocytes, we speculate, based on the strong sequence identity shown here, that these two lipoxygenases, from different speciesand different tissues,are closelyrelated in their structure. In view of this, the human 15-lipoxygenase of airway epithelial cells and eosinophils should be evaluated for the degradative functions of the reticulocyte lipoxygenase such as the ability to degrade mitochondrial membranes selectively, to inhibit cellular respiration by decreasing the synthesis of ATP, and to inactivate sulfhydryl enzymes. Such degradative properties may be important in the pathophysiology of inflammatory and allergic responses.

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Fig. 4. The isolated cDNA and partial restriction map for human 15-lipoxygenase. The locations of verified restriction sites are indicated by vertical bars, and the protein coding region is shown by the open bar. (Reproduced with permission from reference 37.)

Isolation of a cDNA that Encodes Human 15-Lipoxygenase Because the purification procedures yielded only microgram quantities of homogeneous protein, and because large numbers of eosinophils are difficult to obtain, it was clear that further biologic and biochemical studies would require a molecular biologic approach. Using the amino acid information obtained from the isolated protein and the resulting homologies, oligonucleotide probes were designed to regions of the human and rabbit 15-lipoxygenase. A cDNA library was prepared from human peripheral blood cells and screened with the oligonucleotide probes. A cDNA was isolated, sequenced, and found to contain a region coding for the N-terminal amino acid sequence of human leukocyte 15lipoxygenase, which is shown in figure 3 (37). This is the first mammalian 15-lipoxygenase to be cloned and fully sequenced. The verified restriction sites of the isolated clone are fully sequenced. The verified restriction sites of the isolated clone are shown in figure 4. The predicted primary structure of the enzyme consists of 661 amino acid residues, it has a calculated molecular weight of 74.6 kD, and it exhibits a sequence similarity of 61 and 45% with human 5-lipoxygenase and the soybean lipoxygenase isoenzyme I, respectively. When all three lipoxygenasesare aligned, there are two distinct regions of significant sequence identity. These results extend our observation that the lipoxygenases are a family of enzymes, and they provide a basis for exploring functional domains of the lipoxygenase enzymes. Expression of recombinant 15lipoxygenase using this clone has permitted antibodies to be developed for studies of tissue localization (38, 39). From these studies, the relationship between the 15-lipoxygenases of epithelial cells and eosinophils will be better defined.

Conclusions We began investigating the metabolism of arachidonic acid by human airway epithelial cells because of studies that suggested that these cellssignal the recruitment of an inflammatory response and thereby contribute to the development of hyperresponsiveness. Because of the predominance of 15-lipoxygenase activity in airway epithelium and because of the known biologicactivities of 15-lipoxygenase metabolites, we have initiated detailed studies of this enzyme. The isolation and characterization of the enzyme from eosinophil-enriched leukocytes has led to insight into its structure and its regulation. The discovery of the homology between the eosinophillipoxygenase and the reticulocyte lipoxygenasesuggests that we should reexamine the degradative properties of the latter and their relevance to lung biology. The availability of a clone of 15-lipoxygenase will permit the study of this enzyme at the molecular level. Future efforts to study the 15-lipoxygenaseshould lead to further understanding of the biochemical features and biologic roles of this enzyme in health and in disease.

Acknowledgment The writers thank Beth Cost and Patty Snell for assisting in the preparation of the manuscript.

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