Arachidonate 5-lipoxygenase of porcine pancreas: its localization in acinar cells

Arachidonate 5-lipoxygenase has been found so far in various types of leukocyte. When a homogenate of porcine pancreas was incubated with arachidonic acid, 5-hydroxy-6,8,11,14-eicosatetraenoic acid was predominantly produced concomitant with small amounts of compounds derived from leukotriene A4. After differential centrifugation of the homogenate, the 5-lipoxygenase activity was found predominantly in the 1000 x g pellet and 105,000 x g supernatant. When porcine pancreas was investigated immunohistochemically with anti-5-lipoxygenase antibody, Langerhans islets were unstained, and infiltration of 5-lipoxygenase-positive leukocytes was hardly observed. In contrast, acinar cells were positively stained. Immunoelectron microscopy demonstrated the localization of the enzyme along the nuclear membranes of the acinar cells.     

Immunohistochemical study of arachidonate 12-lipoxygenase in porcine tissues

12-Lipoxygenase oxygenates the 12 position of arachidonic acid and produces its 12-hydroperoxy derivative. The enzyme is found in greatest amounts in porcine leukocytes and is distributed widely in various other tissues. An anti-12-lipoxygenase antibody was raised in rabbits with the immunoaffinity-purified enzyme as an antigen and was used in immunohisto- and cytochemical studies on the enzyme, the physiological significance of which remains to be clarified. When peripheral blood cells were examined by immunoelectron microscopy, the enzyme was found in neutrophils and monocytes but was not detected in lymphocytes, platelets, and erythrocytes. In immunostained neutrophils and monocytes the enzyme was localized in the cytosol but was not clearly detected in the plasma membrane, nuclear membrane, endoplasmic reticulum, and other organelles. Several other organs known to contain considerable amounts of 12-lipoxygenase were also investigated immunohistochemically, i.e., alimentary tract (ileum and jejunum), lymphatic organs (spleen, lymph node, and thymus), ovary, lung, liver, and others. In these organs, resident mast cells and granulocytes infiltrating the interstitial tissues were positively immunostained. The enzyme was not detected in parenchymal cells of these organs under our experimental conditions.     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

间接免疫过氧化物酶技术鉴定猪和牛的肥大细胞

用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体ＡＡ１,ＡＡ３及ＡＡ５的间接免疫过氧化物酶技术对经Ｃａｒｎｏｙ液或中性缓冲福尔马林固定的猪和犊牛空肠,舌及胸腺的石蜡切片进行了免疫染色。对猪和牛的肥大细胞特异性免疫染色与常规的组织化学染色的结果进行了比较。     

Arachidonate 5-lipoxygenase of porcine leukocytes studied by enzyme immunoassay using monoclonal antibodies

The cytosol fraction of porcine leukocytes contained 5-lipoxygenase, the activity of which was masked by a predominant activity of 12-lipoxygenase. The 5-lipoxygenase was partially purified to a specific activity of about 10 nmol of arachidonic acid oxygenated/min/mg of protein and given to mice as an antigen to prepare monoclonal antibodies against the enzyme. Two species of antibodies recognized separate sites of the 5-lipoxygenase protein and did not cross-react with 12-lipoxygenase. They were utilized to develop a peroxidase-linked immunoassay of sandwich-type, which allowed a quantitative determination of the 5-lipoxygenase protein. The assay was applied to a screening of the 5-lipoxygenase content in various porcine tissues. By far the highest content of 5-lipoxygenase was found in leukocytes. About one-tenth the amount of the enzyme was found in lung, pancreas, ileum, and thymus, which could not be attributed to the contaminating leukocytes in these tissues.     

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes

15-Hydroperoxy[1-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-diHEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 15-HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and ultraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin A5) and 5,14,15-trihydroxy-6,8,10,12,17-eicosapentaenoic acid (lipoxin B5). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 15-lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes.     

Contribution of macrophages to immediate hypersensitivity reaction

The interaction of mast cells with other leukocytes during immediate hypersensitivity reactions was tested by in vivo and in vitro experiments. Intraperitoneal challenge of passively sensitized rats with antigen caused the production of peptidoleukotrienes, leukotriene (LT)B4, thromboxane (TX)B2, and 6-keto-prostaglandin (PG) F1 alpha in the peritoneal cavity. Pretreatment of the rats with thioglycollate i.p. markedly changed the amount of eicosanoids formed. When polymorphonuclear leukocytes were the predominant cell type in the peritoneal exudate, both LTC4 and 6-keto-PGF1 alpha were decreased by 75% each and TXB2 by 50%. When elicited macrophages were predominant, there was an additional reduction in LTC4 by 68% as compared with 18 hr after thioglycollate treatment, but no additional change in the other arachidonic acid metabolites. In vitro antigen challenge of passively sensitized mouse bone marrow-derived mast cells caused the release of LTC4, LTB4, 6-trans-LTB4, 5-hydroxyeicosatetraenoic (5-HETE), and TXB2. Exposure to antigen of these mast cells in the presence of resident peritoneal macrophages markedly altered eicosanoid formation. Early in the time course (2 to 15 min), macrophages markedly enhanced all 5-lipoxygenase products. However, later in the time course (30 to 120 min), these products were decreased. This decrease was reversed by catalase and superoxide dismutase, which suggests the involvement of oxygen radicals. These active oxygen species also seemed to be generated by mast cells, because these enzymes caused an increase in 5-lipoxygenase products when mast cells were challenged alone. RIA of cyclooxygenase products showed that mast cells released only TXB2 when stimulated with antigen. When they were stimulated in the presence of macrophages, TXB2 and also PGE2 and 6-keto-PGF1 alpha were synthesized. Therefore, macrophages probably contribute the PGE2 and 6-keto-PGF1 alpha. Because the same amount of TXB2 was generated whether macrophages were present or not, the mast cells seem to be the major source of this compound. These data indicate that macrophages and possibly polymorphonuclear leukocytes participate in immediate hypersensitivity reactions.     

Biochemistry of the lipoxygenase pathways in neutrophils

Three mammalian lipoxygenases have been reported to date. They catalyze the insertion of oxygen at positions 5, 12, and 15 of various 20-carbon polyunsaturated fatty acids. In the case of arachidonic acid, the immediate products are hydroperoxyeicosatetraenoic acids (HPETEs). HPETEs can undergo different transformations. One reaction is a reduction of the hydroperoxy group yielding the corresponding hydroxyeicosatetraenoic acids (HETEs). In the neutrophils, the major pathway of arachidonic acid metabolism is the 5-lipoxygenase. In these cells the 5-HPETE undergoes a cyclization reaction leading to a 5(6)-epoxy(oxido)eicosatetraenoic acid or leukotriene A4. The 5(6)-epoxy fatty acid can undergo three additional transformations: (a) a nonenzymatic hydrolysis to epimeric dihydroxyeicosatetraenoic acids (diHETEs); (b) stereospecific enzymatic hydrolysis to a specific diHETE, leukotriene B4; or (c) ring opening by reduced glutathione (GSH) to yield a peptidolipid, named leukotriene C4, in which GSH is attached via a sulfoether linkage. The leukotrienes constitute a group of biologically active substances probably involved in allergic and inflammatory reactions. The 5(6)-epoxy-eicosatetraenoic acid and the products derived from it contain a conjugated triene unit; the term leukotriene also denotes the cells (leukocytes) recognized to form these products, mainly the neutrophils, eosinophils, basophils, monocytes, mast cells, and macrophages. In the present article various aspects of the biochemistry of the lipoxygenase pathways of neutrophils are reviewed.     

Arachidonate 15-lipoxygenase from human eosinophil-enriched leukocytes: partial purification and properties

Arachidonate 15-lipoxygenase was purified from human eosinophil-enriched leukocytes after showing that 15-lipoxygenase activity was 100-fold greater in eosinophils than in neutrophils. Partial purification was achieved using ammonium sulfate precipitation, cation-exchange and hydrophobic-interaction chromatography. New evidence is presented suggesting that 15-lipoxygenase has electrostatic and hydrophobic properties distinct from 5-lipoxygenase. In addition, ATP is shown to inhibit, and phosphatidylcholine is shown to stimulate, 15-lipoxygenase, suggesting a regulatory role for these compounds in the lipoxygenation of arachidonic acid.     

Histochemistry and morphology of porcine mast cells

Summary Mast cells have been described extensively in rodents and humans but not in pigs, and the objective of this study was to characterize porcine mast cells by histochemistry and electron microscopy. Carnoy's fluid proved to be a good fixative but fixation with neutral buffered formalin blocked staining of most mast cells. Alcian Blue stained more mast cells than did Toluidine Blue (pH 0.5), although Alcian Blue also stained goblet cells. In pigs, unlike rodents, the Alcian Blue method did not distinguish between mast cells in the intestinal mucosa and those in the connective tissue of the intestinal submucosa, tongue and skin. Mast cells were significantly larger in adult pigs than in piglets; in adult pigs and piglets, mast cells in the intestinal mucosa were significantly larger than those in submucosal connective tissue, and they were more varied in shape in piglets and adults. Granules in mast cells in the intestinal mucosa stained less intensely than those in mast cells in connective tissue of tongue, skin and intestinal submucosa. Mast cells in the connective tissue of the tongue, skin and intestinal submucosa fluoresced strongly when stained with berberine sulphate or with a mixture of berberine sulphate and Acridine Orange, but mast cells in the intestinal mucosa did not. All mast cells reacted positively in an enzyme-histochemical method previously used to detect human tryptase but not in a method previously used to detect human chymase. Mast cells in the medulla of thymus stained similarly to mast cells in the intestinal mucosa. Ultrastructural differences between mast cells were not detected.     

Inhibitory effect of linoleyl-hydroxamic acid on the oxidation of linoleic acid by 12-lipoxygenase from porcine leukocytes]

Linoleic acid oxidation by 12-lipoxygenase from porcine leukocytes has been studied as affected by linoleyl-hydroxamic acid. Linoleyl-hydroxamic acid has been found to be an effective inhibitor of porcine leucocyte 12-lipoxygenase. Aerobic preincubation of 12-lipoxygenase with 0.1-6 microM of linoleyl-hydroxamic acid led to a time- and dose-dependent inhibition of the enzyme. The inhibitor's concentration able to induce a 50% loss of the enzyme activity with and without 15-min preincubation were 3.5 and 0.65 microM, respectively. Experimental results obeyed a kinetic scheme, which supposed 2 extra substrate molecules bounding with the enzyme-substrate complex in the presence of linoleyl-hydroxamic acid.     

Metabolic profile of linoleic acid in porcine leukocytes through the lipoxygenase pathway

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.     

Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells

Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Demonstration of a 12-lipoxygenase activity in bovine polymorphonuclear leukocytes

In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.     

Assessment of leukotriene B4 synthesis in human lymphocytes by using high performance liquid chromatography and radioimmunoassay methods

Leukotrienes (LT), mainly LTB4, have been shown recently to affect several functions of human lymphocytes in vitro, and they are regarded as putative modulators of the immune response. Although it is recognized that human neutrophils, eosinophils, monocyte-macrophages, and mast cells can generate LTs, the synthesis of 5-lipoxygenase products by lymphocytes is still the subject of a controversy. Human peripheral blood mononuclear leukocytes, nylon wool-purified lymphocytes, CD4+, CD4- T cells, large granular lymphocytes, and various fractions of pure lymphocyte preparations obtained by counter flow centrifugal elutriation were stimulated for 10 min to 24 hr with ionophore A23187, phytohemagglutinin, concanavalin A, or lipopolysaccharide with or without exogenous arachidonic acid (AA); supernatants were analyzed by reverse-phase high performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) methods for the presence of LTB4. Pure human lymphocyte preparations, which were shown to be free of monocytes, did not release any detectable amount of LTB4. Increasing percentage of contaminating monocytes was clearly paralleled by increasing amounts of LTB4. Murine thymocytes, interleukin 2-dependent CTLL2 cytotoxic lymphocytes, EL4 thymoma cells, and human Jurkatt cells were also found to be unable to generate detectable amounts of LTB4 after stimulation with ionophore A23187, phytohemagglutinin, phorbol myristate acetate, recombinant interleukin 1, or interleukin 2 with or without exogenous AA. The addition of increasing numbers of adherence-purified monocytes to Jurkatt cells was followed by increased synthesis of LTB4. In conclusion, the present study indicates that the synthesis of LTB4 by pure human lymphocyte preparations or some human and animal lymphoid cell lines is not detectable by combined HPLC-RIA methods in any of the conditions used.     

Lipoxin synthesis by arachidonate 5-lipoxygenase purified from porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes produced several more polar compounds from 5,15-dihydroperoxy-eicosatetraenoic acid added as such or generated from 15-hydroperoxy acid. These polar products with absorption maxima at 301-302 nm and shoulders at 289 nm and 316-317 nm were identified as 5S,6R,15S-11-cis-lipoxin A and its 6-epimer, all-trans-lipoxin A isomers, and all-trans-lipoxin B isomers. Most of these lipoxins were presumably degradation products of a 5,6-epoxy intermediate formed by the catalysis of leukotriene A synthase, an integral part of 5-lipoxygenase. The rate of the enzymatic lipoxin synthesis from 15-hydroperoxy acid was about 6% of arachidonate 5-oxygenation.     

Molecular cloning and expression of human arachidonate 12-lipoxygenase

The cDNA for a 12-lipoxygenase was isolated from cDNA library of human erythroleukemia cells. The cDNA had an open reading frame encoding 663 amino acids with a calculated molecular weight of 75,513. The deduced amino acid sequence of human 12-lipoxygenase exhibited 41.5%, 65.3% and 65.4% identity with human 5-lipoxygenase, human 15-lipoxygenase and porcine 12-lipoxygenase, respectively. Blot hybridization analysis of RNA from human erythroleukemia cells demonstrated a single species (3.1 kb) of mRNA with the cDNA probe for 12-lipoxygenase of these cells, but not with the cDNA for porcine leukocyte enzyme. The cytosol of Escherichia coli transformed with a recombinant pUC19 plasmid oxygenated the position 12 of arachidonic acid.     

BW755C inhibits the 5-lipoxygenase in E-mast cells without affecting degranulation

The inhibitory effect of the drug BW755C on the 5-lipoxygenase pathway was analyzed for bone marrow-derived murine mast cells, termed E-mast cells. The drug prevented the formation of 5-HETE from exogenous [14C]arachidonic acid when IgE-sensitized cells were challenged by the antigen. BW755C also prevented formation of leukotriene C4 in a dose-dependent fashion when IgE-sensitized mast cells, preincubated with the drug, were activated with either the specific antigen or the ionophore. Leukotriene C4 inhibition occurred with a minimal drug preincubation period of 1 min before the cells were subjected to antigen-dependent activation. BW755C did not affect the degranulation response of these cells. Thus in an intact cell system BW755C prevents 5-lipoxygenation of arachidonic acid. Furthermore, this study reveals that even with transmembrane activation of E-mast cells through their IgE-Fc receptors, granule secretion is not dependent upon corresponding metabolites from the 5-lipoxygenase pathway.     

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB₄ is initiated by the enzyme 5-lipoxygenase and completed by LTA₄ hydrolase. Epithelial cells constitutively express LTA₄ hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A₄ hydrolase, indicating a capacity to produce LTB₄. Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB₄. This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.