John R. Brashler

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel, participate glutathione S-transferase

Abstract Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A 4 to a mixture of leukotrienes C 4 and D 4 in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S -transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein −1 min −1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S -transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.

On the structure of slow reacting substance of anaphylaxis: evidence of biosynthesis from arachidonic acid.

The mononuclear cells in peritoneal washings from normal rats can be induced to produce large amounts of slow reacting substance of anaphylaxis by incubation with 10 mM cysteine in the presence of the calcium ionophore A-23187. This production of slow reacting substance could be inhibited by the addition of non-steroidal anti-inflammatory drugs, e.g., indomethacin, ibuprofen and flurbiprofen, Furthermore, mediator production was inhibited by eicosatetraynoic acid, the substrate analog of arachidonic acid, and by 9,11-azoprosta-5, 13-dienoic acid (AZO analog 1), a structural analog of the prostaglandin endoperoxide, PGH2, which known to inhibit thromboxane synthesis. Relatively high concentrations of hydrocortisone acetate inhibited mediator production; this inhibition could be partly reversed by the addition of arachidonic acid or to a lesser extent by eicosatrienoic acid. Preliminary results suggest that a small fraction of the 3H-labled arachidonic acid which was taken up by these cells in vitro was associated with slow reacting substance. We postulate that slow reacting substance of anaphylaxis may be derived from a prostaglandin endoperoxide which is formed during the oxidation of arachidonic acid by the prostaglandin fatty acid cyclooxygenase.

Identification of a component of rat mononuclear cell SRS as leukotriene D

Abstract Slow reacting substance (SRS), produced by rat peritoneal mononuclear cells after stimulation with ionophore A23187, consists of two main components ( Bach, M.K. et al. (1979) J. Immunol. 122, 160–165). One of these components was recently identified as leukotriene C-1. The other component has now been identified as leukotriene D.

Leukotriene C synthetase, a special glutathione S-transferase: Properties of the enzyme and inhibitor studies with special reference to the mode of action of U-60,257, a selective inhibitor of leukotriene synthesis

The cytosolic glutathione S-transferases of rat liver have been fractionated by chromatofocusing into 10 distinct fractions based on their reactivity with 2,4-dinitrochlorobenzene. All these fractions were capable of generating leukotriene C4 (LTC4) from leukotriene A4 (LTA4) to some extent. An inhibitor of leukotriene synthesis, U-60,257, inhibited the activity of these enzymes. The cytosolic glutathione S-transferases of rat basophil leukemia (RBL) cells have been similarly fractionated. U-60,257 inhibited the activity of some of these fractions but not that of others. None of the fractions of the enzyme from RBL cells formed LTC4 from LTA4. The microsomal glutathione S-transferase from rat liver also produced LTC4 from LTA4. It differs from the microsomal LTC synthetase of RBL cells in at least two respects: (1) The enzyme from RBL cells did not react with chromophoric substrates like dinitrochlorobenzene while the enzyme from liver did react. (2) Triton X-100 potentiated the activity of the enzyme from basophil leukemia cells and solubilized it, while it inhibited the activity of the leukotriene-synthesizing enzyme in the rat liver preparation. These results, along with a distinctly different inhibitor profile, indicate that LTC synthetase is a new and distinct glutathione S-transferase.

Inhibition of the leukotriene synthetase of rat basophil leukemia cells by diethylcarbamazine, and synergism between diethylcarbamazine and piriprost, a 5-lipoxygenase inhibitor

Diethylcarbamazine inhibited the formation of sulfidopeptide leukotrienes in rat basophil leukemia (RBL) cells (50% inhibitory concentration, EC50, 3 mM). Similar concentrations also inhibited the formation of leukotriene C4 (LTC4) by LTC synthetase, a detergent-solubilized cell free particulate enzyme from RBL cells which is capable of coupling LTA4 to glutathione. By contrast, the conversion of LTA4 to LTC4 using enzymes from rat liver was at least ten times less sensitive to this inhibitor. The EC50 for inhibition of the leukotriene C synthetase of RBL cells was directly proportional to the LTA4 concentration in the incubations, ranging from 1.5 mM at 10 microM LTA4 to over 40 mM at 500 microM LTA4. Kinetic analysis revealed that the inhibition of the leukotriene C synthetase reaction by diethylcarbamazine was competitive with respect to LTA4. In contrast to diethylcarbamazine, piriprost (U-60,257; 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1), which inhibits the formation of sulfidopeptide leuktrienes in RBL cells at the 5-lipoxygenase step (EC50 5 microM), did not inhibit the leukotriene synthetase of these cells. On the other hand, low concentrations of piriprost, which had no demonstrable inhibitory activity on leukotriene formation by themselves, markedly synergized the inhibitory activity of diethylcarbamazine. These results are consistent with the interpretation that both piriprost and diethylcarbamazine inhibit leukotriene formation but that they act on sequential steps in the biosynthetic pathway in such a manner as to synergistically interfere with the availability or utilization of LTA4 in the leukotriene C synthetase reaction.

Activation of Human Eosinophils by Platelet-Derived Growth Factor

Activated eosinophils are believed to be major contributors to the chronic inflammatory sequelae of asthma, but the details of the mechanism of eosinophil activation in vivo are unknown. In our search for physiologically important modes of eosinophil activation, we studied the effects of recombinant human platelet-derived growth factor (PDGF) on human peripheral blood eosinophils. We compared two activation end-points: secretion of granule contents, exemplified by the release of eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and the generation of active oxygen metabolites (O2- production). PDGFc-sis dose dependently stimulated the secretion of large amounts of EPO and EDN from eosinophils. Higher concentrations of PDGF induced a dose-dependent O2- production, especially if the cells were first primed with low concentrations of phorbol ester. These activities were not seen with the AA homodimer of PDGF, suggesting that the activation was receptor dependent. However, several attempts to directly demonstrate the existence of such receptors were unsuccessful. The magnitude of the secretory response to PDGF, and the realization that eosinophils could be easily exposed to this substance as they travel towards the lung, suggests the possibility that this growth factor may be a physiologically important activator of eosinophils in the pulmonary inflammation which is associated with asthma.

Pitfalls in the quantitative estimation of the secretion of granule proteins by eosinophils

The secretion of preformed granule proteins by eosinophils is an important correlate of eosinophil activation. However, a review of the literature reveals large disparities in the amounts of these substances which were reportedly secreted when eosinophils were activated. In the present study we report that our attempts to quantitate the secretion of eosinophil peroxidase and eosinophil-derived neurotoxin from activated eosinophils by measuring these substances in the incubation supernatants were uniformly unsuccessful. We found that, once they were secreted, both eosinophil peroxidase and eosinophil-derived neurotoxin were promptly lost to assay and presumably destroyed. Thus the measurement of the difference in the concentration of these substances in eosinophils prior to and after activation, revealed that as much as 65% of the eosinophil-derived neurotoxin and 62% of the peroxidase in the eosinophils were lost to assay during activation of the cells whereas the largest amount of these substances which could be measured in the incubation supernatants never exceeded 2%. Evidence is presented that the destruction of eosinophil-derived neurotoxin must occur prior to the release of this substance into the medium. Attempts to inhibit the destruction of eosinophil peroxidase and of eosinophil-derived neurotoxin by incorporating various inhibitors into the incubations were unsuccessful. These results emphasize the need to monitor the overall recoveries of secreted products from activated eosinophils and suggest that meaningful estimates of the secretion of these granule proteins from activated eosinophils can only be obtained by measuring the residual content of these substances in eosinophils after they have been activated and comparing these values to the contents of eosinophils prior to activation.

Stimulated production of slow reacting substances by mercaptans from ionophore A 23187-induced mononuclear cells: mercaptan structure-activity studies.

Abstract When isolated mononuclear cells from the peritoneal cavity of the rat are challenged with the ionophore, A 23187, slow reacting substance (SRS) is produced. SRS production is markedly enhanced by the addition of mercaptans to the incubations. In confirmation of previously published reports, this enhancement is dependent on the duration of a preincubation of the cells with the mercaptans before addition of the ionophore, a two minute preincubation being optimal. Quantitative structure-activity studies revealed that a variety of mercapto carboxylic acids, where the mercapto group was one or two carbon atoms removed from the carboxyl group, were all active in enhancing SRS production and the enhancement followed parallel dose response curves. A 4-mercapto acid, while weakly active, had a distinctly different dose response curve and cysteamine, which lacks a carboxyl function, was inactive. Preliminary characterization of the products of the incubations produced in the presence of different mercaptans has revealed that, while they all qualitatively meet the criteria of stability to alkali, lability in acid and destruction by arylsulfatase which are associated with slow reacting substance of anaphylaxis, there are quantitative differences in stability which suggest that the products may not be identical.

Evidence that Granulocyte/Macrophage-Colony-Stimulating Factor and Interferon-γ Maintain the Viability of Human Peripheral Blood Monocytes in Part by Their Suppression of IL-10 Production

Prolonged culture of human peripheral blood monocytes hPBMs requires the addition of both granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interferon (IFN)-gamma. Cultured hPMBs challenged with lipopolysaccharide produced large amounts of several cytokines but very little interleukin (IL)-10. However, when GM-CSF and IFN-gamma were omitted from the cultures, IL-10 production was readily demonstrated. Addition of IL-10 to the cultures potently inhibited the production of several cytokines and, in the presence of GM-CSF and IFN-gamma, there was no loss in cell number. In contrast, when IL-10 was added to cultures in the absence of GM-CSF and IFN-gamma, there was an accelerated loss of viable cells. A monoclonal antibody to IL-10, which had no effect on cell survival in the presence of GM-CSF and IFN-gamma, partly prevented the loss of cells which occurred in the absence of IL-10 and these additives. Preliminary studies suggest that inclusion of anti-IL-10 can partly prevent the apoptosis which occurs when GM-CSF and IFN-gamma are omitted from the cultures. These observations suggest that there is a cause and effect relationship between the failure of hPBMs to produce IL-10 when they are cultured in the presence of GM-CSF and IFN-gamma and protection from apoptosis by these additives.

A comparison of the leukotriene synthesizing ability of subfractions of rat liver glutathione s-transferases

The cytosolic glutathione S-transferases of rat liver have been partially purified by affinity adsorption to glutathionyl Sepharose and elution with glutathione, and have been fractionated by chromatofocusing. The column eluates were pooled into ten fractions, although evaluation of the enzymatic activities of the column eluates with different chromogenic substrates indicated that some of the fractions were heterogeneous. The pooled fractions were characterized with respect to their substrate specificity, their susceptibility to inhibition by several inhibitors, and their ability to catalyze the conjugation of glutathione to leukotriene A4. It was found that all the fractions were able to catalyze leukotriene C formation. The fraction having the highest specific activity with three different chromogenic substrates also had the highest specific activity when LTA was used as the substrate while, in general, there were marked differences in the relative activities of the different pooled fractions. The most active fraction represented approximately 50% of the total glutathione S-transferase activity in the whole preparation and had an apparent isoelectric point of 9.05. There was no apparent relationship between the ability of the different fractions to utilize LTA and any of the other substrates which were tested.