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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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Passive systemic IgE-induced anaphylaxis. Flap (−/−) and  (+/+) mice received 20 μg of a monoclonal mouse anti–mouse DNP  IgE in 200 μl PBS intravenously. 24 h later basal body temperatures were  recorded, and mice received an intravenous injection of 1 mg DNP-HSA  and 0.5% Evans blue dye in 200 μl PBS to induce mast cell degranulation  and anaphylaxis. Basal body temperature was again recorded 30 min after  this injection. (A) Animals of the indicated genotypes were killed and  8-mm-diam ear discs were isolated. Extravasation of plasma proteins into  tissue was quantitated by extraction of dye with formamide and spectrophotometric analysis of extracts at 610 nm. Dye leakage into both the left  and right ears was recorded in this systemic response. (B) Change in basal  body temperature for Flap (−/−) and (+/+) mice 30 min after treatment  was recorded as another measure of systemic anaphylaxis. n = five Flap  (−/−) and four (+/+) mice. Error bars indicate SEM.
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Figure 6: Passive systemic IgE-induced anaphylaxis. Flap (−/−) and (+/+) mice received 20 μg of a monoclonal mouse anti–mouse DNP IgE in 200 μl PBS intravenously. 24 h later basal body temperatures were recorded, and mice received an intravenous injection of 1 mg DNP-HSA and 0.5% Evans blue dye in 200 μl PBS to induce mast cell degranulation and anaphylaxis. Basal body temperature was again recorded 30 min after this injection. (A) Animals of the indicated genotypes were killed and 8-mm-diam ear discs were isolated. Extravasation of plasma proteins into tissue was quantitated by extraction of dye with formamide and spectrophotometric analysis of extracts at 610 nm. Dye leakage into both the left and right ears was recorded in this systemic response. (B) Change in basal body temperature for Flap (−/−) and (+/+) mice 30 min after treatment was recorded as another measure of systemic anaphylaxis. n = five Flap (−/−) and four (+/+) mice. Error bars indicate SEM.

Mentions: Systemic IgE anaphylaxis in the mouse is characterized by bronchconstriction, plasma extravasation, hypotension with resultant tachycardia, and drop in body temperature. In the passive IgE-induced anaphylaxis model, these changes are dependent on binding of IgE to the high affinity IgE receptor, followed by mast cell degranulation (36). Leukotrienes, which are not stored in granules but are synthesized when the cells are activated, are among several proinflammatory mediators released by stimulated mast cells that can contribute to these physiological effects. To test the role of FLAP in the anaphylactic response, IgE-dependent passive systemic anaphylaxis was induced in five FLAP-deficient and four control mice. Mast cells of the animals were loaded with a monoclonal mouse anti–mouse DNP IgE. 24 h later DNP-HSA and Evans blue were administered. Body temperature was taken using a rectal probe before and 30 min after the DNP-HSA treatment. Finally, mice were killed, and edema was quantitated by measuring the amount of Evans blue dye in 8-mm-diam ear biopsies. Flap (−/−) mice averaged a 3.76 ± 0.35°C drop in body temperature after the induction of passive anaphylaxis that was less extreme than the 4.98 ± 1.04°C drop seen in the (+/+) mice (Fig. 6 A). This reduced response, however, was not statistically significant (P = 0.174). Likewise, the A610 of ear extracts from each group showed similar levels of Evans blue dye extravasation (Fig. 6 B). The mean A610 of extracts from the ears of FLAP-deficient mice was 0.1006 ± 0.0057. Wild-type mice edema levels reached A610 = 0.0859 ± 0.0082, which again indicates no significant difference between Flap (−/−) and (+/+) animals (P = 0.075). Analogous results have been seen on examination of the 5-LO–deficient mice (data not shown).


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)

Passive systemic IgE-induced anaphylaxis. Flap (−/−) and  (+/+) mice received 20 μg of a monoclonal mouse anti–mouse DNP  IgE in 200 μl PBS intravenously. 24 h later basal body temperatures were  recorded, and mice received an intravenous injection of 1 mg DNP-HSA  and 0.5% Evans blue dye in 200 μl PBS to induce mast cell degranulation  and anaphylaxis. Basal body temperature was again recorded 30 min after  this injection. (A) Animals of the indicated genotypes were killed and  8-mm-diam ear discs were isolated. Extravasation of plasma proteins into  tissue was quantitated by extraction of dye with formamide and spectrophotometric analysis of extracts at 610 nm. Dye leakage into both the left  and right ears was recorded in this systemic response. (B) Change in basal  body temperature for Flap (−/−) and (+/+) mice 30 min after treatment  was recorded as another measure of systemic anaphylaxis. n = five Flap  (−/−) and four (+/+) mice. Error bars indicate SEM.
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Figure 6: Passive systemic IgE-induced anaphylaxis. Flap (−/−) and (+/+) mice received 20 μg of a monoclonal mouse anti–mouse DNP IgE in 200 μl PBS intravenously. 24 h later basal body temperatures were recorded, and mice received an intravenous injection of 1 mg DNP-HSA and 0.5% Evans blue dye in 200 μl PBS to induce mast cell degranulation and anaphylaxis. Basal body temperature was again recorded 30 min after this injection. (A) Animals of the indicated genotypes were killed and 8-mm-diam ear discs were isolated. Extravasation of plasma proteins into tissue was quantitated by extraction of dye with formamide and spectrophotometric analysis of extracts at 610 nm. Dye leakage into both the left and right ears was recorded in this systemic response. (B) Change in basal body temperature for Flap (−/−) and (+/+) mice 30 min after treatment was recorded as another measure of systemic anaphylaxis. n = five Flap (−/−) and four (+/+) mice. Error bars indicate SEM.
Mentions: Systemic IgE anaphylaxis in the mouse is characterized by bronchconstriction, plasma extravasation, hypotension with resultant tachycardia, and drop in body temperature. In the passive IgE-induced anaphylaxis model, these changes are dependent on binding of IgE to the high affinity IgE receptor, followed by mast cell degranulation (36). Leukotrienes, which are not stored in granules but are synthesized when the cells are activated, are among several proinflammatory mediators released by stimulated mast cells that can contribute to these physiological effects. To test the role of FLAP in the anaphylactic response, IgE-dependent passive systemic anaphylaxis was induced in five FLAP-deficient and four control mice. Mast cells of the animals were loaded with a monoclonal mouse anti–mouse DNP IgE. 24 h later DNP-HSA and Evans blue were administered. Body temperature was taken using a rectal probe before and 30 min after the DNP-HSA treatment. Finally, mice were killed, and edema was quantitated by measuring the amount of Evans blue dye in 8-mm-diam ear biopsies. Flap (−/−) mice averaged a 3.76 ± 0.35°C drop in body temperature after the induction of passive anaphylaxis that was less extreme than the 4.98 ± 1.04°C drop seen in the (+/+) mice (Fig. 6 A). This reduced response, however, was not statistically significant (P = 0.174). Likewise, the A610 of ear extracts from each group showed similar levels of Evans blue dye extravasation (Fig. 6 B). The mean A610 of extracts from the ears of FLAP-deficient mice was 0.1006 ± 0.0057. Wild-type mice edema levels reached A610 = 0.0859 ± 0.0082, which again indicates no significant difference between Flap (−/−) and (+/+) animals (P = 0.075). Analogous results have been seen on examination of the 5-LO–deficient mice (data not shown).

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