Localization of leukotriene D4-metabolizing metalloenzyme on the cell surface of human neutrophils

The localization of leukotriene D4-metabolizing enzyme on the cell surface was examined using human neutrophils. Intact neutrophils rapidly converted leukotriene D4 to leukotriene E4. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell surface enzymes selectively, the leukotriene D4-metabolizing activity of neutrophils decreased significantly without any inhibition of the cell viability or marker enzymes of cytosol, granules, microsome and mitochondria. The leukotriene D4-metabolizing enzyme activity of the membrane fraction was inhibited by modification to the same extent as that of Mg2+-dependent ATPase, a cell-surface marker enzyme. Among various enzyme inhibitors examined, a metal chelator, o-phenanthroline, strongly suppressed the leukotriene D4-metabolizing activity of intact neutrophils and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+, Mn2+ and Zn2+. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of leukotriene D4 to leukotriene E4 by neutrophils.     

Conversion of leukotriene C4 to leukotriene D4 by a cell-surface enzyme of rat macrophages

Leukotriene (LT) C4-metabolizing enzyme was studied using rat leukocytes. Neutrophils and lymphocytes hardly metabolized LTC4, whereas macrophages rapidly converted LTC4 to LTD4. The LTC4-metabolizing enzyme of macrophages was present in the membrane fraction but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the LTC4-metabolizing activity of macrophages decreased significantly (greater than 90%). These findings suggest that rat macrophages possess the LTC4-metabolizing enzyme which converts LTC4 to LTD4, on the cell surface membrane.     

Comparative studies on the leukotriene D4-metabolizing enzyme of different types of leukocytes

1. The leukotriene (LT) D4-metabolizing enzyme which catalyzes the conversion of LTD4 to LTE4, was investigated in various types of leukocytes from guinea pigs and humans. 2. In guinea pigs, the enzyme activity was present in macrophages but was hardly present in neutrophils, lymphocytes and eosinophils. 3. In humans, neutrophils, lymphocytes and macrophages all possessed the enzyme activity. However, enzyme activity varied with cell types and macrophages showed the highest enzyme activity among the leukocytes. 4. The subcellular localization of the LTD4-metabolizing enzyme was studied and leukocytes were divided into two groups: one which has the enzyme activity exclusively on the cell surface and the other which has the activity both on the cell surface and in the granules of leukocytes. 5. The enzyme activity was remarkably inhibited by o-phenanthroline and dithiothreitol and the inactivated enzyme was considerably reactivated by Co2+ and Zn2+, suggesting that the LTD4-metabolizing enzyme of leukocytes is a metalloenzyme.     

Leucine aminopeptidase as an echo-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5'-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5'-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Leucine aminopeptidase as an ecto-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5′-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5′-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Inactivation of phagocytosis-stimulating activity of tuftsin by polymorphonuclear neutrophils: A possible role of leucine aminopeptidase as an ecto-enzyme

The subcellular localization of the tuftsin-inactivating activity was studied using guinea-pig polymorphonuclear neutrophils and the following results were obtained. 1. The tuftsin-inactivating activity was present in the membrane function but not in the cytosol and the granular fractions. 2. Intact neutrophils inactivated tuftsin rapidly. However, when neutrophils were modified chemically by a poorly permeant reagent, diazotized sulfanilic acid, the tuftsin-inactivating activity decreased sifnificantly without any inhibition of marker enzymes of cytosol, microsome, granulesa and mitochondria, suggesting that the tuftsin-inactivating activity is located on the plasma membrane as an ecto-enzyme. 3. When neutrophils were modified by diazotized sulfanilic acid at different concentrations, the tuftsin-inactivating activity of neutrophils was inhibited in proportion to the degree of inhibition of the activity of leucine aminopeptidase, an ecto-enzyme. 4, Hydrolysis of L-leucyl-β-napthylamide, a synthetic substrate of leucine aminopeptidase, was inhibited competitively by tuftsin. 5. Treatmetn of neutrophils with serine protease inhibitors affected neither tuftsin-inactivating nor leucine aminopeptidase activity at all, indicating no involvement of serine proteases, which is said to be located on the cell surface membrane, in the tuftsin-inactivating activity of neutrophils. The possibility was deduced from the above results that leucine aminopeptidase may act as a tuftsin-inactivating enzyme.     

Functional characteristics of leukotriene C4- and D4-metabolizing enzymes (gamma-glutamyl transpeptidase, dipeptidase) within human plasma

Several properties of the leukotriene C4- and leukotriene D4-metabolizing enzymes within human plasma were studied after fractionation of the plasma proteins using ammonium sulfate precipitation. Leukotriene D4-metabolizing enzymes were widely distributed among the fractions obtained. They showed different pH optima (pH 6.5, pH 7.0 and pH greater than or equal to 8.5) and revealed a different degree of thermal stability. The results indicate the presence of more than one enzyme in plasma which interacts with leukotriene D4. EDTA and L-cysteine inhibited the metabolism of leukotriene D4. Two leukotriene C4-metabolizing activities (gamma-glutamyl transpeptidases) differing in their molecular weights were detected after gel filtration. Their molecular weights were estimated to be Mr greater than or equal to 150 000 and Mr between 55 000 and 100 000.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25 +/- 0.27 mU/10(7) cells and 6.18 +/- 0.87 mU/10(7) cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10(7) cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10(7) cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10(7) cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10(7) cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Possible involvement of angiotensin I-converting enzyme in the inactivation of bradykinin by intact macrophages

As intact macrophages inactivated bradykinin, the subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig macrophages. The bradykinin-inactivating activity was found to be present in membrane and cytosol fractions but not in granular and nuclear fractions. The bradykinin-inactivating activity of the membrane fraction was inhibited by captopril, a specific inhibitor of angiotensin I-converting enzyme, whereas that of the cytosol fraction was hardly inhibited by various proteinase inhibitors used. Angiotensin I-converting enzyme activity was located predominantly in the membrane fraction and its activity was inhibited by captopril. Angiotensin I-converting enzyme activity measured with a synthetic substrate was competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for macrophage angiotensin I-converting enzyme. When macrophages were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent, both the bradykinin-inactivating activity and the angiotensin I-converting enzyme activity of macrophages decreased significantly without any inhibition of the cytosol bradykinin-inactivating activity. These findings seem to suggest that the angiotensin I-converting enzyme would be responsible for the inactivation of bradykinin in intact macrophages.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Metabolism of leukotriene D4 by rat peritoneal mast cells

Metabolism of sulfidopeptide leukotrienes, leukotrienes (LT) C4 and D4 by rat peritoneal mast cells was studied. Rat peritoneal mast cells converted LTD4 to LTE4 but not LTC4 to LTD4. The LTD4-metabolizing activity was equally distributed on the cell surface and inside cells, but not released to the extracellular milieu even when a considerable portion of histamine and the secretory granule enzymes were released. Among various enzyme inhibitors examined, o-phenanthroline, a metal chelator, and dithiothreitol significantly suppressed the LTD4-metabolizing activity of mast cell. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of LTD4 to LTE4 by rat peritoneal mast cells.     

Inhibition of leukotriene D4 catabolism by D-penicillamine

Inhibition of the catabolism of the most biologically potent cysteinyl leukotriene, LTD4, was studied in rat hepatoma cells in vitro and in the rat in vivo. LTD4 dipeptidase, an ectoenzyme on the surface of AS-30D hepatoma cells, exhibited an apparent Km value of 6.6 microM for LTD4. D-Penicillamine and L-penicillamine inhibited this enzyme activity with apparent Ki values of 0.46 mM and 0.21 mM respectively. Bestatin, an inhibitor of the aminopeptidase activity of hepatoma cells, did not affect LTD4 hydrolysis at concentrations as high as 5 mM, indicating that the aminopeptidase did not contribute to LTD4 catabolism. In the rat in vivo, D-penicillamine also inhibited LTD4 catabolism. After intravenous injection of [3H]LTC4 an accumulation of [3H]LTD4 and a retarded formation of [3H]LTE4 were observed in the circulating blood after D-penicillamine pretreatment. Within 1 h after intravenous [3H]LTC4 injection, about 80% of the administered radioactivity was recovered in bile. After D-penicillamine pretreatment [3H]LTD4 was the major biliary leukotriene metabolite, whereas in untreated controls leukotriene metabolites more polar than LTC4 predominated in bile. After stimulation of endogenous leukotriene production in vivo by platelet-activating factor, N-acetyl-LTE4 was the major cysteinyl leukotriene detected in bile. D-Penicillamine treatment prior to platelet-activating factor resulted in the accumulation of LTD4, which under these circumstances was the major endogenous leukotriene metabolite detected in bile.     

Leukotriene A3. A poor substrate but a potent inhibitor of rat and human neutrophil leukotriene A4 hydrolase

Analysis of leukotriene B4 production by purified rat and human neutrophil leukotriene (LT) A4 hydrolases in the presence of 5(S)-trans-5,6-oxido-7,9-trans-11-cis-eicosatrienoic acid (leukotriene A3) demonstrated that this epoxide is a potent inhibitor of LTA4 hydrolase. Insignificant amounts of 5(S), 12(R)-dihydroxy-6-cis-8,10-trans-eicosatrienoic acid (leukotriene B3) were formed by incubation of rat neutrophils with leukotriene A3 or by the purified rat and human LTA4 hydrolases incubated with leukotriene A3. Leukotriene A3 was shown to be a potent inhibitor of leukotriene B4 production by rat neutrophils and also by purified rat and human LTA4 hydrolases. Covalent coupling of [3H]leukotriene A4 to both rat and human neutrophil LTA4 hydrolases was shown, and this coupling was inhibited by preincubation of the enzymes with leukotriene A4. Preincubation of rat neutrophils with leukotriene A3 also prevented labeling of LTA4 hydrolase by [3H]leukotriene A4. This result indicates that leukotriene A3 prevents covalent coupling of the substrate leukotriene A4 and inhibits the production of leukotriene B4 by blocking the binding of leukotriene A4 to the enzyme.     

Bioconversion of leukotriene D4 by lung dipeptidase

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.     

Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils

The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin.     

Co-localization of leukotriene a4 hydrolase with 5-lipoxygenase in nuclei of alveolar macrophages and rat basophilic leukemia cells but not neutrophils.

The synthesis of leukotriene B(4) from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A(4) hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A(4) hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A(4) hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A(4) hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A(4) hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situ and ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A(4) hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A(4) hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A(4) hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A(4) hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A(4) hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B(4).     

Identification of leukotriene D4 receptor binding sites in guinea pig lung homogenates using [3H]leukotriene D4

We have characterized [3H]leukotriene D4 binding to guinea pig lung homogenates. Both biphasic dissociation kinetics and curvilinear Scatchard plots indicated the presence of [3H]leukotriene high and low affinity states of the binding sites. The rank order of potency for the competition study was leukotriene C4 = leukotriene D4 greater than leukotriene E4 much greater than arachidonic acid, and for their contractile effect on lung strips was leukotriene C4 = leukotriene D4 = leukotriene E4 much greater than arachidonic acid. FPL-55712 was the only other agent tested that inhibited binding. These results suggest that binding of [3H]leukotriene D4 to the homogenate is consistent with its binding to specific leukotriene D4 receptor sites.     

Cilastatin-sensitive dehydropeptidase I enzymes from three sources all catalyze carbapenem hydrolysis and conversion of leukotriene D4 to leukotriene E4

The dipeptidase, dehydropeptidase I (EC 3.4.13.11), was purified to homogeneity from rat lung, rat kidney, and hog kidney. Analysis of physical parameters (subunit molecular weights, Km values for glycyldehydrophenylalanine, Ki values for dehydropeptidase I inhibitors, and immunoreactivity) showed the rat dipeptidases to be similar to each other but different from the hog dipeptidase. However, all three enzymes hydrolyzed imipenem and converted leukotriene D4 to leukotriene E4, and these activities were inhibited by cilastatin.     

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.     

Transformation of leukotriene D4 catalyzed by lysosomal cathepsin H of rat liver

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.