Impaired polymorphonuclear leukocyte 15-lipoxygenase activity in juvenile and rapidly progressive periodontitis

15-lipoxygenase activity was investigated in sonicated polymorphonuclear leukocytes (PMNLs) from patients with juvenile and rapidly progressive periodontitis and adult periodontitis. The group with juvenile and rapidly progressive periodontitis had 17 patients (6 male, 11 female, mean age 27.4 years), and the age matched control group had 18 normal individuals (11 male, 7 female, mean age 26.3 years). The group with adult periodontitis had 14 patients with 9 male, 5 female, mean age 45.2 years and the age-matched control group had 6 normal subjects with 5 male, 1 female, mean age 43.7 years. 15-hydroxyeicosa-tetraenoic acid (15-HETE) synthesized in the group with juvenile and rapidly progressive periodontitis was 0.219 +/- 0.102 ng/mg protein (mean +/- S.D.), while it was 0.410 +/- 0.138 ng/mg protein in the age-matched control group. There was a significant difference between the two groups. The group with adult periodontitis produced 0.358 +/- 0.124 ng/mg protein and the age matched control group produced 0.448 +/- 0.176 ng/mg protein (no significant difference). These results are relevant to reports that PMNLs of patients with juvenile and rapidly progressive periodontitis have abnormal functions, while those of patients with adult periodontitis are normal.     

Activation of a 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen

Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.     

Inhibition of human leukocyte 5-lipoxygenase by 15-HPETE and related eicosanoids

The inhibition of human leukocyte 5-lipoxygenase by 15-hydroperoxyeicosatetraenoic acid and its chemical or enzymatic rearrangement products was investigated. 15-Hydroperoxyeicosatetraenoic acid was the most potent inhibitor tested. The inhibition was found to be time dependent and is not due to chemical or enzymatic decomposition products nor metabolism of 15-hydroperoxyeicosatetraenoic acid to 5,15-dihydroperoxyeicosatetraenoic acid.     

The effect of free radical derived hydroxyicosatetraenoic acids on hexose transport in the human polymorphonuclear leukocyte

The following racemic hydroxyicosatetraenoic acids were prepared and assayed for their ability to stimulate hexose transport in human polymorphonuclear leukocytes: 15-, 12-, 11-, 9-, 8-, and 5-hydroxyicosatetraenoic acids. The compounds were isolated from reduced, autoxidized arachidonic. The results demonstrate that only the 12-, and 5-hydroxyicosatetraenoic acids are biologically active, inducing half-maximal responses at 820 and 176 nM, respectively. Thus, the bioactions of hydroxicosatetraenoates are crucially dependent upon the position of the hydroxy residue. Response to both hydroxyicosatetraenoates was effectively blocked by two inhibitors of arachidonic acid metabolism: nordihydroguaiaretic acid and indomethacin. A third arachidonic acid antimetabolite, 5,8,11,14-eicosatetraynoic acid, completely inhibited the response to 12-HETE but caused only partial inhibition of the response to 5-HETE.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Metabolic response of polymorphonuclear leukocyte to arachidonic and linoleic acids

Various stimuli act on polymorphonuclear leukocytes (PMN), activating membrane-bound phospholipase A2 and C, and diglyceride lipase and then liberating unsaturated fatty acids (USFAs). These liberated USFAs are immediately metabolized through various metabolic pathways such as cyclooxygenase, lipoxygenase, phosphatidylinositol metabolism etc. It is possible that the metabolic intermediates of these pathways reveal various physiological actions. This work was undertaken to clarify whether stimuli on PMN depend on these USFAs themselves or on their oxidation products. The following results were obtained: 1. USFAs such as arachidonate and linoleate stimulate PMN, accelerating superoxide (O2) generation, depolarization of membrane potential and increase in [Ca2+]i. 2. Oxidation products of USFAs have no stimulative effect on PMN. The decrease in the stimulative effect of these USFAs following their oxidation is proportional to the quantitative decrease in non-oxidized linoleate. 3. USFAs accelerate membrane permeability of Ca2+, and their oxidation products enhance non-specific membrane permeability in proportion to the formation of monohydroxy compound. These results suggest that stimulative effects of USFAs on PMN do not depend on their oxidation products but on unoxidized fatty acids. Furthermore, among the oxidation products of the USFAs, monohydroxy compound acts as a strong perturber of membrane and accelerates membrane permeability.     

Characterization of monoclonal antibodies against human leukocyte 5-lipoxygenase

Four mouse monoclonal IgG1 antibody-producing cell lines (5LO-1, 5LO-2, 5LO-3, 5LO-4), produced against highly purified human leukocyte 5-lipoxygenase have been characterized. The monoclonal antibodies produced by these cell lines exhibited differential reactivity against 5-lipoxygenase as determined by ELISA and immunoprecipitation analyses. Monoclonal antibodies 5LO-2 and 5LO-3 inhibited the activity of recombinant human leukocyte 5-lipoxygenase in a dose-dependent manner. This inhibition was selective for 5-lipoxygenase activity since these monoclonal antibodies did not inhibit human leukocyte 15-lipoxygenase or porcine leukocyte 12-lipoxygenase.     

Calcium regulation of the human PMN cytosolic 15-lipoxygenase

Addition of tracer (pg) amounts of [3H]arachidonic acid to the 120,000 x g cytosolic fraction of human polymorphonuclear leukocytes (PMNs) produced [3H]-15-HETE, the product of the 15-lipoxygenase, as the major metabolite. In the presence of nanomolar and low micromolar amounts of calcium, [3H]-15-HETE formation was increased as much as 15-fold which corresponded to 17% conversion of added substrate. This enhancement of the cytosolic 15-lipoxygenase activity, which was reversible by EGTA, was inhibited by phosphatidyl serine and phosphatidyl choline. Millimolar levels of calcium inhibited the cytosolic 15-lipoxygenase and the 5-lipoxygenase product 5-HETE could reverse this inhibition. These results indicate that calcium is an important modulator of the PMN 15-lipoxygenase when the enzyme is in a cytosolic milieu.     

Anthrax toxin components stimulate chemotaxis of human polymorphonuclear neutrophils

Effects of the three-component toxin of Bacillus anthracis on chemotaxis of human polymorphonuclear leukocytes (PMN) were investigated in an effort to determine the basis of the reported antiphagocytic effect of the toxin. The three toxin components, edema factor (EF), protective antigen (PA), and lethal factor (LF), were tested alone and in various combinations for their effect on PMN chemotaxis under agarose to formyl peptides and zymosan-activated serum. No component was active alone; combinations of EF + PA, LF + PA, and EF + LF + PA markedly stimulated chemotaxis (directed migration), but had little or no effect on unstimulated random migration. The toxin components were not themselves chemoattractants. EF in combination with PA had previously been identified as an adenylate cyclase in Chinese hamster ovary (CHO) cells. We found that EF + PA produced detectable cyclic adenosine 3'-5'monophosphate (cAMP) in PMN, but the level of cAMP was less than 1% of that produced in CHO cells by EF + PA, and in PMN by other bacterial adenylate cyclases. LF + PA (which stimulated chemotaxis to an equivalent extent) had no effect on cAMP levels. Thus, the enhancement of chemotaxis by anthrax toxin (at least by LF + PA) does not seem to be related to adenylate cyclase activity.     

Kinetic studies of human polymorphonuclear leukocyte phosphofructokinase.

Phosphofructokinase from human polymorphonuclear leukocytes has low cooperativity and high affinity for its substrate, F-6-P. It is resistant to ATP inhibition at pH 8; however, at pH 7.1 it becomes sensitive to the effect of this compound. It is activated by F-1, 6-P2; it is not very sensitive to citrate inhibition and F-2, 6-P2 has no effect on its activity. With these kinetic characteristics we assume that perhaps the predominant L-type subunit is accompanied by an F-type component.     

Appearance of an arachidonic acid 15-lipoxygenase pathway upon differentiation of the human promyelocytic cell-line HL-60

The metabolism of arachidonic acid and 15-HPETE was studied in a human promyelocytic cell line (HL-60). Upon exposure to DMSO, HL-60 cells undergo differentiation and acquire a 15-lipoxygenase activity while undifferentiated cells challenged with either arachidonic acid or 15-HPETE did not enzymatically transform these precursors. Products of the arachidonic acid 15-lipoxygenase pathway were identified by HPLC. UV-absorption and gas chromatography-mass spectrometry. Results indicate that upon differentiation HL-60 cells express a 15-lipoxygenase activity as well as the ability to transform 15-HPETE to 8,15-DHETEs and 14,15-DHETE. Moreover, these findings suggest that products of the 15-lipoxygenase cascade may be generated by a single cell system.     

Urate causes the human polymorphonuclear leukocyte to secrete superoxide.

The human polymorphonuclear leukocyte generates O2-. and H2O2 when it is treated with uric acid. A transition metal catalyzed reaction between O2-. and H2O2 can give the hydroxyl radical and myeloperoxidase forms hypochlorous acid from H2O2 and chloride. Therefore, the uric acid-induced secretion of oxidants may be responsible for a large part of the inflammation associated with gout.     

Arachidonic acid 15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Partial purification and properties

Arachidonic acid 15-lipoxygenase was purified from rabbit peritoneal polymorphonuclear leukocytes. The enzyme was recovered in the cytosol fraction after sonication and purified about 250-fold by acetone precipitation, column chromatography on CM52, Sephadex G-150, and hydroxyapatite. The enzyme catalyzed the conversion of arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which then decomposed to a mixture of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), 15-keto-5,8,11,13-eicosatetraenoic acid, 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid. The enzyme was specific for oxygenation at carbon 15 of arachidonic acid. The apparent molecular weight of the enzyme was about 61,000 as measured by Sephadex G-150 gel filtration chromatography. The enzyme was sensitive to sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid. The enzyme activity was inhibited by eicosatetraynoic acid (ETYA) or 3-amino-1-(m-(trifluoromethyl)-phenyl)2-pyrazoline (BW755C), but not by indomethacin up to 200 micrograms/ml.     

Chemoattractants stimulate phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes

Chemoattractant receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C is instrumental for leukocyte activation. Previous studies have demonstrated that chemoattractant treatment of intact polymorphonuclear leukocytes (PMN) causes a transient decrease in PIP2 due to phospholipase C activation, followed by an increase in cellular PIP2 levels. The present study determined whether chemoattractants altered the activities of the two enzymes responsible for the synthesis of PIP2, phosphatidylinositol kinase, and phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact PMN with the N-formylated peptide chemoattractant formyl-methionyl-leucyl-phenylalanine at 37 degrees C caused a rapid (3 min), 2-fold stimulation of PIP kinase activity isolated from a particulate membrane fraction. The increase in PIP kinase was dose-dependent for a variety of N-formylated chemoattractants as well as leukotriene B4. Lineweaver-Burk analysis showed that the Vmax of PIP kinase was increased 2-fold by formyl-methionyl-leucyl-phenylalanine, without a significant change in the apparent Km of the enzyme for ATP. Phosphatidylinositol kinase was, however, not altered by any chemoattractants tested. Nonchemotactic activators of the oxidative burst in leukocytes such as phorbol myristate acetate and ionophore A23187 did not significantly alter PIP kinase, suggesting a specificity for chemotactic agents. These findings demonstrate direct, chemoattractant-induced stimulation of PMN PIP kinase which may serve to replenish the important phospholipid, PIP2, in the membrane following its hydrolysis by phospholipase C.     

Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and -valerolactone were used, 2,5-hexanedione at 75 g/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of flourescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.Abbreviations 2,5-Hxdn 2,5-hexanedione - 2-Hxl 2-hexanol - 2-Hxn 2-hexanone - PMN polymorphonuclear leukocyte - TMA DPH trimethylammonium diphenylhexatriene - -V1 -valerolactone     

Regulation of 5-lipoxygenase activity by the glutathione status in human polymorphonuclear leukocytes

The influence of the glutathione status of human polymorphonuclear leukocytes (PMN) on 5-lipoxygenase activity was studied by treating cells with increasing concentrations of 1-chloro-2,4-dinitrobenzene (Dnp-Cl) or azodicarboxylic acid bis(dimethylamide) (Diamide). Subsequent incubation with arachidonate resulted in an up to tenfold-stimulated formation of 5-hydroxyeicosatetraenoic acid, leukotriene B4, leukotriene B4 isomers and omega-hydroxyleukotriene B4. Higher concentrations of the GSH reagents were inhibitory. At maximal stimulation by Dnp-Cl, 5-hydroperoxyeicosatetraenoic acid started to be built up at the expense of 5-HETE at glutathione levels which were diminished by about 50% compared to resting cells. No increase in cytosolic Ca2+ could be measured under these conditions by the fura-2 method. In PMN homogenates Dnp-Cl and Diamide were without effect and even caused inhibition when 5-lipoxygenase was stimulated by Ca2+ and ATP. 15-Lipoxygenase was either unchanged in the case of Diamide, or even increased after pretreatment with Dnp-Cl. The results allow us to conclude that 5-lipoxygenase activity in intact PMN is regulated not only by Ca2+ but in a complex manner also by the glutathione redox status. Conditions of oxidative stress increase the activity which may reflect the in vivo situation under phagocytosis and oxidative burst.     

Cell damage unmasks 15-lipoxygenase activity in human neutrophils

Metabolism of arachidonic acid (10 microM) into 15(S)-hydroxyl-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) was proportional to lactate dehydrogenase release from human neutrophils incubated with supratherapeutic concentrations of non-steroidal anti-inflammatory agents. In contrast to others (Vanderhoek, J., and Bailey, J. (1984) J. Biol. Chem. 259, 6752-6756), we report that increased 15-HETE formation was not uniquely attributable to 5 mM ibuprofen, and it did not originate from enzymatic activation. For instance, ibuprofen (1-5 mM) did not affect the isolated 15-lipoxygenase enzyme in the 100,000 X g supernatant from neutrophil lysates, and dose-dependent increases in 15-HETE biosynthesis, proportional to lactate dehydrogenase release, were evident with benoxaprofen, naproxen, flurbiprofen, or etodolac. At similar supratherapeutic concentrations (1-5 mM), aspirin and phenylbutazone did not influence lactate dehydrogenase release or 15-HETE production. In further contrast, neutrophils did not tolerate 1-5 mM ibuprofen. Biochemical, morphological, flow cytometric, and fluorochromatic analyses each indicated cytological damage. A correlation between lactate dehydrogenase release and increased 15-HETE formation was a dose-dependent property also exhibited by arachidonic acid alone (10-100 microM). We conclude that cytological damage, facilitating access of arachidonic acid to 15-lipoxygenase in a cytosolic compartment, accounts for this phenomenon.     

Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase

Purified polymorphonuclear leukocyte elastase degraded native human liver type III collagen at 27 degrees C by making a cleavage through the triple helix. The enzyme had no effect on human type I collagen. The reaction was inhibited by phenylmethanesulfonyl fluoride (PhCH2SO2F) but not by EDTA. The collagen reaction products were identical with those generated by human rheumatoid synovial collagenase when analyzed by polyacrylamide gel electrophoresis and gel filtration. NH2-trminal sequence analysis indicated that the enzyme cleaved at an isoleucyl-threonyl bond located 4 residues on the carboxyl side of the established cleavage site for animal collagenases. Therefore, it is likely that in pathologic states, type III collagen can be selectively depleted from the matrix by this enzyme.     

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.