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Role of the 5-lipoxygenase-activating protein (FLAP) in murine acute inflammatory responses.

Byrum RS, Goulet JL, Griffiths RJ, Koller BH - J. Exp. Med. (1997)

Bottom Line: Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin.The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP).Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill 27599-7248, USA.

ABSTRACT
Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor-induced shock compared to controls. Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO-deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.

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Ca2+ ionophore–induced eicosanoid production in Flap (+/+)  and Flap (−/−) mice. Peritoneal macrophages from three Flap (+/+)  and four Flap (−/−) mice were cultured in 1.5 ml tissue culture medium  for 30 min. Medium was collected and replaced with medium containing  Ca2+ ionophore A23187 (5 μg/ml). Supernatant was again collected 30  min after stimulation. LTC4 (A), PGE2 (B) and TXB2 (C) levels in the  prestimulation (solid bars) and poststimulation (hatched bars) supernatants  were determined by EIA. After ionophore stimulation and supernatant  collection, the number of adherent macrophages was determined by using  a hexosaminidase assay. Eicosanoid levels were then normalized to reflect  106 macrophages. Error bars indicate SEM.
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Figure 2: Ca2+ ionophore–induced eicosanoid production in Flap (+/+) and Flap (−/−) mice. Peritoneal macrophages from three Flap (+/+) and four Flap (−/−) mice were cultured in 1.5 ml tissue culture medium for 30 min. Medium was collected and replaced with medium containing Ca2+ ionophore A23187 (5 μg/ml). Supernatant was again collected 30 min after stimulation. LTC4 (A), PGE2 (B) and TXB2 (C) levels in the prestimulation (solid bars) and poststimulation (hatched bars) supernatants were determined by EIA. After ionophore stimulation and supernatant collection, the number of adherent macrophages was determined by using a hexosaminidase assay. Eicosanoid levels were then normalized to reflect 106 macrophages. Error bars indicate SEM.

Mentions: Peritoneal macrophages collected from four Flap (−/−) mice and from three (+/+) littermates were stimulated with calcium ionophore to determine the ability of these cells to release prostaglandins, thromboxanes, and leukotrienes. After A23187 activation, a >200-fold increase in LTC4 was detectable in supernatant from macrophages of wild-type mice, while no LTC4 was detected in cells from Flap (−/−) animals (Fig. 2 A). The ability of the FLAPdeficient mice to produce prostaglandins and thromboxanes by the cyclooxygenase-dependent pathway remained intact (Fig. 2, B and C). Similar to results obtained with 5-LO–deficient mice (42), levels of PGE2 and TXB2 that were released from the Flap (−/−) macrophages after stimulation were greater than those released from their (+/+) counterparts. While both PGE2 and TXB2 production by FLAP-deficient mice was increased compared to controls, only the difference in prostaglandin production reached statistical significance (P = 0.028 and 0.078, respectively).


Role of the 5-lipoxygenase-activating protein (FLAP) in murine acute inflammatory responses.

Byrum RS, Goulet JL, Griffiths RJ, Koller BH - J. Exp. Med. (1997)

Ca2+ ionophore–induced eicosanoid production in Flap (+/+)  and Flap (−/−) mice. Peritoneal macrophages from three Flap (+/+)  and four Flap (−/−) mice were cultured in 1.5 ml tissue culture medium  for 30 min. Medium was collected and replaced with medium containing  Ca2+ ionophore A23187 (5 μg/ml). Supernatant was again collected 30  min after stimulation. LTC4 (A), PGE2 (B) and TXB2 (C) levels in the  prestimulation (solid bars) and poststimulation (hatched bars) supernatants  were determined by EIA. After ionophore stimulation and supernatant  collection, the number of adherent macrophages was determined by using  a hexosaminidase assay. Eicosanoid levels were then normalized to reflect  106 macrophages. Error bars indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196234&req=5

Figure 2: Ca2+ ionophore–induced eicosanoid production in Flap (+/+) and Flap (−/−) mice. Peritoneal macrophages from three Flap (+/+) and four Flap (−/−) mice were cultured in 1.5 ml tissue culture medium for 30 min. Medium was collected and replaced with medium containing Ca2+ ionophore A23187 (5 μg/ml). Supernatant was again collected 30 min after stimulation. LTC4 (A), PGE2 (B) and TXB2 (C) levels in the prestimulation (solid bars) and poststimulation (hatched bars) supernatants were determined by EIA. After ionophore stimulation and supernatant collection, the number of adherent macrophages was determined by using a hexosaminidase assay. Eicosanoid levels were then normalized to reflect 106 macrophages. Error bars indicate SEM.
Mentions: Peritoneal macrophages collected from four Flap (−/−) mice and from three (+/+) littermates were stimulated with calcium ionophore to determine the ability of these cells to release prostaglandins, thromboxanes, and leukotrienes. After A23187 activation, a >200-fold increase in LTC4 was detectable in supernatant from macrophages of wild-type mice, while no LTC4 was detected in cells from Flap (−/−) animals (Fig. 2 A). The ability of the FLAPdeficient mice to produce prostaglandins and thromboxanes by the cyclooxygenase-dependent pathway remained intact (Fig. 2, B and C). Similar to results obtained with 5-LO–deficient mice (42), levels of PGE2 and TXB2 that were released from the Flap (−/−) macrophages after stimulation were greater than those released from their (+/+) counterparts. While both PGE2 and TXB2 production by FLAP-deficient mice was increased compared to controls, only the difference in prostaglandin production reached statistical significance (P = 0.028 and 0.078, respectively).

Bottom Line: Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin.The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP).Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill 27599-7248, USA.

ABSTRACT
Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO-activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor-induced shock compared to controls. Also, edema associated with Zymosan A-induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO-deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.

Show MeSH
Related in: MedlinePlus