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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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Targeting of the Flap gene. (A) Partial restriction enzyme  maps of the targeting construct, the endogenous genomic clone, and the  recombinant locus. Coding sequences replaced by homologous recombination are indicated by the filled black box. The hsv-tk and NEO selection cassettes are indicated as hatched boxes (hsv, herpes simplex virus; tk,  thymidine kinase). Restriction enzyme sites are abbreviated: N, NotI; B,  BamHI; Nh, NheI; X, XhoI; R5, EcoRV; R, RmaI; H, HindIII. The  probe used to screen for homologous recombination by Southern blot  analysis is indicated by the filled gray box. (B) Southern blot analysis of  EcoRV-digested tail DNA from Flap (+/+), Flap (+/−), and Flap (−/−)  mice hybridized with a 1.2-kb BamHI/NotI fragment downstream of the  targeting construct's region of homology. (C) Northern blot analysis of  total RNA isolated from peritoneal macrophages of Flap (+/+), Flap (−/−),  and 5-lo (−/−) mice hybridized with the rat FLAP cDNA. A rat β-actin  cDNA probe was used as a positive control.
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Figure 1: Targeting of the Flap gene. (A) Partial restriction enzyme maps of the targeting construct, the endogenous genomic clone, and the recombinant locus. Coding sequences replaced by homologous recombination are indicated by the filled black box. The hsv-tk and NEO selection cassettes are indicated as hatched boxes (hsv, herpes simplex virus; tk, thymidine kinase). Restriction enzyme sites are abbreviated: N, NotI; B, BamHI; Nh, NheI; X, XhoI; R5, EcoRV; R, RmaI; H, HindIII. The probe used to screen for homologous recombination by Southern blot analysis is indicated by the filled gray box. (B) Southern blot analysis of EcoRV-digested tail DNA from Flap (+/+), Flap (+/−), and Flap (−/−) mice hybridized with a 1.2-kb BamHI/NotI fragment downstream of the targeting construct's region of homology. (C) Northern blot analysis of total RNA isolated from peritoneal macrophages of Flap (+/+), Flap (−/−), and 5-lo (−/−) mice hybridized with the rat FLAP cDNA. A rat β-actin cDNA probe was used as a positive control.

Mentions: A targeting vector for the Flap gene was constructed from the murine genomic clone. Fragments identical to their corresponding regions in the gene were inserted 5′ and 3′ of the neomycin resistance gene (neo) of pJNS2 (36) such that coding material would be replaced by neo after homologous recombination of the plasmid with the endogenous locus. This construct was electroporated into E14TG2a embryonic stem (ES) cells established from the 129/Ola mouse line (37), and colonies resistant to neomycin and ganciclovir were identified as described previously (36). Genomic DNA was isolated from doubly resistant ES cell lines, digested with EcoRV and analyzed by Southern blot using a 1.2-kb BamHI/NotI fragment downstream of the targeting construct as a probe (Fig. 1 A). Verified recombinant ES cell lines were microinjected into mouse blastocysts, and chimeric mice were generated. Chimeras were bred to either B6D2 or 129/SvEv mice, and offspring were genotyped by Southern blot analysis of tail DNA digested with EcoRV using the 1.2-kb probe described above. All animals used in our studies of inflammation, with the exception of those assayed for platelet-activating factor (PAF) survival, were offspring of chimeras crossed with 129/SvEv mice. Likewise, the 5-LO–deficient mice used as controls for these experiments were on the 129 genetic background.


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)

Targeting of the Flap gene. (A) Partial restriction enzyme  maps of the targeting construct, the endogenous genomic clone, and the  recombinant locus. Coding sequences replaced by homologous recombination are indicated by the filled black box. The hsv-tk and NEO selection cassettes are indicated as hatched boxes (hsv, herpes simplex virus; tk,  thymidine kinase). Restriction enzyme sites are abbreviated: N, NotI; B,  BamHI; Nh, NheI; X, XhoI; R5, EcoRV; R, RmaI; H, HindIII. The  probe used to screen for homologous recombination by Southern blot  analysis is indicated by the filled gray box. (B) Southern blot analysis of  EcoRV-digested tail DNA from Flap (+/+), Flap (+/−), and Flap (−/−)  mice hybridized with a 1.2-kb BamHI/NotI fragment downstream of the  targeting construct's region of homology. (C) Northern blot analysis of  total RNA isolated from peritoneal macrophages of Flap (+/+), Flap (−/−),  and 5-lo (−/−) mice hybridized with the rat FLAP cDNA. A rat β-actin  cDNA probe was used as a positive control.
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Related In: Results  -  Collection

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Figure 1: Targeting of the Flap gene. (A) Partial restriction enzyme maps of the targeting construct, the endogenous genomic clone, and the recombinant locus. Coding sequences replaced by homologous recombination are indicated by the filled black box. The hsv-tk and NEO selection cassettes are indicated as hatched boxes (hsv, herpes simplex virus; tk, thymidine kinase). Restriction enzyme sites are abbreviated: N, NotI; B, BamHI; Nh, NheI; X, XhoI; R5, EcoRV; R, RmaI; H, HindIII. The probe used to screen for homologous recombination by Southern blot analysis is indicated by the filled gray box. (B) Southern blot analysis of EcoRV-digested tail DNA from Flap (+/+), Flap (+/−), and Flap (−/−) mice hybridized with a 1.2-kb BamHI/NotI fragment downstream of the targeting construct's region of homology. (C) Northern blot analysis of total RNA isolated from peritoneal macrophages of Flap (+/+), Flap (−/−), and 5-lo (−/−) mice hybridized with the rat FLAP cDNA. A rat β-actin cDNA probe was used as a positive control.
Mentions: A targeting vector for the Flap gene was constructed from the murine genomic clone. Fragments identical to their corresponding regions in the gene were inserted 5′ and 3′ of the neomycin resistance gene (neo) of pJNS2 (36) such that coding material would be replaced by neo after homologous recombination of the plasmid with the endogenous locus. This construct was electroporated into E14TG2a embryonic stem (ES) cells established from the 129/Ola mouse line (37), and colonies resistant to neomycin and ganciclovir were identified as described previously (36). Genomic DNA was isolated from doubly resistant ES cell lines, digested with EcoRV and analyzed by Southern blot using a 1.2-kb BamHI/NotI fragment downstream of the targeting construct as a probe (Fig. 1 A). Verified recombinant ES cell lines were microinjected into mouse blastocysts, and chimeric mice were generated. Chimeras were bred to either B6D2 or 129/SvEv mice, and offspring were genotyped by Southern blot analysis of tail DNA digested with EcoRV using the 1.2-kb probe described above. All animals used in our studies of inflammation, with the exception of those assayed for platelet-activating factor (PAF) survival, were offspring of chimeras crossed with 129/SvEv mice. Likewise, the 5-LO–deficient mice used as controls for these experiments were on the 129 genetic background.

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