An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells

E. Razin, L. C. Romeo, S. Krilis, Fu-Tong Liu, R. A. Lewis, E. J. Corey, K. F. Austen

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The quantitative relationships between the secretion of a granule-associated mediator, β-hexosaminidase, and the oxidative metabolism of arachidonic acid by the 5-lipoxygenase pathway were analyzed for a homogeneous population of T cell-dependent, bone marrow-derived, murine mast cells. The mast cells were either sensitized with a monoclonal IgE and challenged with specific antigen, or to bypass a transmembrane signal, were stimulated with calcium ionophore A23187. The released products of the 5-lipoxygenase pathway were quantitated by integrated ultraviolet absorbance after resolution by reverse phase-high performance liquid chromatography in the case of 5-hydroxy-eicosatetraenoic acid (5-HETE), and by separate radioimmunoassays for leukotriene C4 (LTC4) and leukotriene B4 (LTB4). The activation-release response of the cells was perturbated by the introduction of three pharmacologic agents, each directed to different steps in the 5-lipoxygenase pathway of arachidonic acid metabolism, and the action of each agent was determined for separate cell samples while present and after its removal by washing. 5,6-Dehydroarachidonic acid (5,6-DHA), an irreversible inhibitor of 5-lipoxygenase, prevented formation of 5-HETE from exogenous [14C]arachidonic acid and from membrane-derived arachidonic acid in a dose-related fashion when sensitized mast cells, preincubated with drug, were washed before antigen activation. Release of 5-HETE, LTC4, and LTB4 was inhibited by 5,6-DHA in a corresponding dose-related fashion, with a minimal preincubation period of 1 to 5 min before the cells were washed and subjected to antigen-dependent activation. In contrast, the inhibitory effect of 5,6-DHA on β-hexosaminidase release was lost after three washes and was not evident after one wash unless the preincubation period was extended to 15 min. The capacity of 5,6-DHA to prevent leukotriene generation without altering β-hexosaminidase release was also observed with ionophore-activated mast cells. Preincubation of sensitized cells with diethylcarbamazine (DEC), followed by a wash before antigen-dependent activation, produced inhibition of leukotriene generation, no effect on β-hexosaminidase release, and augmentation of 5-HETE release at the maximum dose studied; thus, DEC interrupts the pathway distal to the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) from arachidonic acid by 5-lipoxygenase. Preincubation of sensitized cells with incremental amounts of the prostacyclin analog U-60,257, followed by washing and antigen challenge, inhibited LTC4 release without altering the release of LTB4 or β-hexosaminidase. Selective inhibition of LTC4 release was also obtained after unsensitized cells were preincubated with U-60,257, washed, and activated with ionophore. The interposition of one or more wash step(s) before antigen activation of sensitized cells revealed a specificity of product inhibition that was not observed in the continued presence of the agents, and eliminated the inhibitory actions of 5,6-DHA and of DEC on β-hexosaminidase release and of U-60,257 on LTB4 release. That 5-lipoxygenase of arachidonic acid is not mandatory for the secretion of granule markers in IgE-Fc receptor-activated bone marrow-derived mast cells is indicated by the capacity of these agents to block 5-lipoxygenase, conversion of 5-HPETE to leukotrienes, and conversion of LTA4 to LTC4, without suppressing β-hexosaminidase release.

Original languageEnglish (US)
Pages (from-to)938-945
Number of pages8
JournalJournal of Immunology
Volume133
Issue number2
StatePublished - 1984
Externally publishedYes

Fingerprint

Hexosaminidases
Arachidonate 5-Lipoxygenase
Arachidonic Acids
Mast Cells
Leukotriene C4
Bone Marrow
Hydroxy Acids
Leukotriene B4
Diethylcarbamazine
Antigens
Leukotrienes
Acids
Ionophores
Arachidonic Acid
Leukotriene A4
IgE Receptors
Cohort Effect
Lipoxygenase Inhibitors
Fc Receptors
Calcium Ionophores

ASJC Scopus subject areas

  • Immunology

Cite this

Razin, E., Romeo, L. C., Krilis, S., Liu, F-T., Lewis, R. A., Corey, E. J., & Austen, K. F. (1984). An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells. Journal of Immunology, 133(2), 938-945.

An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells. / Razin, E.; Romeo, L. C.; Krilis, S.; Liu, Fu-Tong; Lewis, R. A.; Corey, E. J.; Austen, K. F.

In: Journal of Immunology, Vol. 133, No. 2, 1984, p. 938-945.

Research output: Contribution to journalArticle

Razin, E. ; Romeo, L. C. ; Krilis, S. ; Liu, Fu-Tong ; Lewis, R. A. ; Corey, E. J. ; Austen, K. F. / An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells. In: Journal of Immunology. 1984 ; Vol. 133, No. 2. pp. 938-945.
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T1 - An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells

AU - Razin, E.

AU - Romeo, L. C.

AU - Krilis, S.

AU - Liu, Fu-Tong

AU - Lewis, R. A.

AU - Corey, E. J.

AU - Austen, K. F.

PY - 1984

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N2 - The quantitative relationships between the secretion of a granule-associated mediator, β-hexosaminidase, and the oxidative metabolism of arachidonic acid by the 5-lipoxygenase pathway were analyzed for a homogeneous population of T cell-dependent, bone marrow-derived, murine mast cells. The mast cells were either sensitized with a monoclonal IgE and challenged with specific antigen, or to bypass a transmembrane signal, were stimulated with calcium ionophore A23187. The released products of the 5-lipoxygenase pathway were quantitated by integrated ultraviolet absorbance after resolution by reverse phase-high performance liquid chromatography in the case of 5-hydroxy-eicosatetraenoic acid (5-HETE), and by separate radioimmunoassays for leukotriene C4 (LTC4) and leukotriene B4 (LTB4). The activation-release response of the cells was perturbated by the introduction of three pharmacologic agents, each directed to different steps in the 5-lipoxygenase pathway of arachidonic acid metabolism, and the action of each agent was determined for separate cell samples while present and after its removal by washing. 5,6-Dehydroarachidonic acid (5,6-DHA), an irreversible inhibitor of 5-lipoxygenase, prevented formation of 5-HETE from exogenous [14C]arachidonic acid and from membrane-derived arachidonic acid in a dose-related fashion when sensitized mast cells, preincubated with drug, were washed before antigen activation. Release of 5-HETE, LTC4, and LTB4 was inhibited by 5,6-DHA in a corresponding dose-related fashion, with a minimal preincubation period of 1 to 5 min before the cells were washed and subjected to antigen-dependent activation. In contrast, the inhibitory effect of 5,6-DHA on β-hexosaminidase release was lost after three washes and was not evident after one wash unless the preincubation period was extended to 15 min. The capacity of 5,6-DHA to prevent leukotriene generation without altering β-hexosaminidase release was also observed with ionophore-activated mast cells. Preincubation of sensitized cells with diethylcarbamazine (DEC), followed by a wash before antigen-dependent activation, produced inhibition of leukotriene generation, no effect on β-hexosaminidase release, and augmentation of 5-HETE release at the maximum dose studied; thus, DEC interrupts the pathway distal to the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) from arachidonic acid by 5-lipoxygenase. Preincubation of sensitized cells with incremental amounts of the prostacyclin analog U-60,257, followed by washing and antigen challenge, inhibited LTC4 release without altering the release of LTB4 or β-hexosaminidase. Selective inhibition of LTC4 release was also obtained after unsensitized cells were preincubated with U-60,257, washed, and activated with ionophore. The interposition of one or more wash step(s) before antigen activation of sensitized cells revealed a specificity of product inhibition that was not observed in the continued presence of the agents, and eliminated the inhibitory actions of 5,6-DHA and of DEC on β-hexosaminidase release and of U-60,257 on LTB4 release. That 5-lipoxygenase of arachidonic acid is not mandatory for the secretion of granule markers in IgE-Fc receptor-activated bone marrow-derived mast cells is indicated by the capacity of these agents to block 5-lipoxygenase, conversion of 5-HPETE to leukotrienes, and conversion of LTA4 to LTC4, without suppressing β-hexosaminidase release.

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