Attenuated Zymosan-induced Peritoneal Vascular Permeability and IgE-dependent Passive Cutaneous Anaphylaxis in Mice Lacking Leukotriene C4 Synthase*

Yoshihide Kanaoka, Akiko Maekawa, John F. Penrose, K. Frank Austen, and Bing K. LamDagger

From the Department of Medicine, Harvard Medical School and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115

Received for publication, April 20, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Leukotriene C4 synthase (LTC4S), the terminal 5-lipoxygenase pathway enzyme that is responsible for the biosynthesis of cysteinyl leukotrienes, has been deleted by targeted gene disruption to define its tissue distribution and integrated pathway function in vitro and in vivo. The LTC4S -/- mice developed normally and were fertile. LTC4S activity, assessed by conjugation of leukotriene (LT) A4 methyl ester with glutathione, was absent from tongue, spleen, and brain and >=  90% reduced in lung, stomach, and colon of the LTC4S -/- mice. Bone marrow-derived mast cells (BMMC) from the LTC4S -/- mice provided no LTC4 in response to IgE-dependent activation. Exocytosis and the generation of prostaglandin D2, LTB4, and 5-hydroxyeicosatetraenoic acid by BMMC from LTC4S -/- mice and LTC4S +/+ mice were similar, whereas the degraded product of LTA4, 6-trans-LTB4, was doubled in BMMC from LTC4S -/- mice because of lack of utilization. The zymosan-elicited intraperitoneal extravasation of plasma protein and the IgE-mediated passive cutaneous anaphylaxis in the ear were significantly diminished in the LTC4S -/- mice. These observations indicate that LTC4S, but not microsomal or cytosolic glutathione S-transferases, is the major LTC4-producing enzyme in tissues and that its integrated function includes mediation of increased vascular permeability in either innate or adaptive immune host inflammatory responses.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The cysteinyl leukotrienes (cysLTs),1 leukotriene (LT) C4, and its metabolites, LTD4 and LTE4, are potent lipid mediators of tissue inflammation, particularly implicated in allergic and asthmatic diseases (1, 2). In humans, inhalation of cysLTs constricts bronchial smooth muscle and attracts eosinophils, and intradermal injection elicits an increase in vascular permeability (3-6). The contribution of the cysLTs to the pathophysiology of bronchial asthma is established by the therapeutic efficacy of inhibitors of their biosynthesis (7) and antagonists of their receptor-mediated action (8). The cellular generation of LTC4 requires activation with Ca2+-dependent translocation of cytosolic phospholipase A2 and 5-lipoxygenase (5-LO) to the perinuclear and endoplasmic reticular membranes. There, in the presence of 5-LO-activating protein (FLAP), the arachidonic acid released by cytosolic phospholipase A2 is converted to 5-hydroperoxyeicosatetraenoic acid and then to LTA4 (9-11). LTA4 is processed either to the dihydroxy leukotriene, LTB4, by LTA4 hydrolase (12) or to LTC4 through conjugation with reduced glutathione by LTC4 synthase (LTC4S) (13-15). After carrier-mediated export of LTC4 (16), glutamic acid and glycine are sequentially cleaved by gamma -glutamyl transpeptidase and dipeptidase to form LTD4 and LTE4, respectively (17, 18). Two cysLT receptors, termed CysLT1 and CysLT2 receptors, are presently known. Whereas the CysLT1 receptor has a marked preference for signal activation by LTD4, the CysLT2 receptor has a similar recognition of LTC4 and LTD4 with a higher Kd value relative to that of the CysLT1 receptor (19, 20). Mast cells also metabolize the released arachidonic acid to prostaglandin (PG) D2 by the successive action of PG endoperoxide synthase-1 or PG endoperoxide synthase-2 (21, 22) and hematopoietic PGD synthase (23, 24). Whereas a PGD2 receptor, termed DP, is prominent on smooth muscle such as airways and microvasculature, a recently identified PGD2 receptor, termed CRTH2, is localized to hematopoietic cells such as T helper type 2 (Th2) cells, basophils, and eosinophils (25, 26).

LTC4S is an 18-kDa integral membrane protein that shows glutathione S-transferase (GST) activity that is strictly specific for LTA4 as a substrate (13-15). The human LTC4S cDNA encodes a protein of 150 amino acids and belongs to a recently recognized superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes FLAP and microsomal GSTs (MGSTs) (27). LTC4S shows 44% amino acid identity with MGST2 (28) and 31% identity with FLAP. MGST2 and MGST3 (29) conjugate glutathione not only to xenobiotics but also to LTA4 to form LTC4, and they are ubiquitously expressed even in cells lacking the capacity to provide LTA4. LTC4 can be formed through the transcellular metabolism of LTA4 by cells that express LTC4S, such as platelets (30), or MGST2, such as endothelial cells (31). With the exception of platelets, LTC4S has been identified only in hematopoietic cells that also express 5-LO.

We sought to establish that LTC4S was the dominant constitutive enzymatic source of LTC4 in situ in the mouse by targeted gene disruption of the LTC4S gene. The loss of function in the gene-disrupted mice relative to their controls was then used to quantitate the roles of LTC4S in models of innate and adaptive immune inflammation. Zymosan, a yeast cell wall polysaccharide, was used to elicit an intraperitoneal extravasation of plasma proteins, and hapten-specific, IgE-mediated passive cutaneous anaphylaxis provided a permeability increment at the skin. We also assessed for the catalytic function of LTC4S in various tissues relative to the bifunctional GSTs by the decrement in LTC4 biosynthesis in the LTC4S -/- mice. Finally, we determined whether the absence of LTC4S in bone marrow-derived mast cells (BMMC) subjected to IgE-dependent activation would lead to increased non-enzymatic hydrolysis of the substrate LTA4 to 6-trans-LTB4 diastereoisomers or to the shunting of LTA4 to LTA4 hydrolase to form more LTB4.

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EXPERIMENTAL PROCEDURES
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Generation of LTC4S -/- Mice-- A 5.7-kb 129/Ola mouse genomic fragment containing the LTC4S gene (32) was subcloned into a pcDNA3 vector (Invitrogen). After the endogenous neomycin resistance gene (neo) of the pcDNA3 and ~400 base pairs of the 5' region of the 5.7-kb genomic fragment were removed, a neo gene cassette from pMC1Neo Poly(A) (Stratagene) was inserted to replace 289 nucleotides of intron 1, exon II to exon IV, and 32 nucleotides of intron 4 of the mouse LTC4S gene. The herpes simplex virus thymidine kinase (TK) gene was inserted at the 3'-end of the gene. The resultant targeting vector was linearized and electroporated into the embryonic stem cell line, AB2.2 (Stratagene). The embryonic stem cells were selected with G418 (200 µg/ml; Life Technologies, Inc.) and ganciclovir (2 µM), and homologous recombination was confirmed by Southern blot analysis of EcoRI-digested genomic DNA from each embryonic stem cell clone with the ~400-base pair 5' fragment as a probe (see Fig. 1A). The verified embryonic stem cell clones were microinjected into blastocysts from C57BL/6 and BALB/c mice, and chimeric mice were obtained. Chimeras were bred to C57BL/6 and BALB/c mice, and offspring were genotyped by Southern blot analysis of tail DNA as described above. Heterozygotes were backcrossed to a C57BL/6 or BALB/c genetic background, and heterozygotes in the N2 or N3 generation were intercrossed to obtain homozygotes. All the experiments were performed with LTC4S -/- mice derived from a BALB/c background except for the zymosan-induced peritonitis model in which mice from the C57BL/6 background were utilized. All procedures were approved by the Harvard Medical Area Standing Committee on Animals.

Culture of BMMC and LTC4S mRNA Analysis-- Bone marrow cells were collected from femurs and tibiae of mice and cultured for 4-6 weeks in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1% culture supernatant from Chinese hamster ovary cells expressing mouse interleukin-3 (33, 34). The culture medium for the BMMC was changed every week, and the cell density was adjusted to 3 × 105/ml at every passage. After 4 weeks, more than 97% of the cells were BMMC as assessed by staining with Wright-Giemsa and toluidine blue.

Total RNA was isolated from BMMC with Tri-Reagent (Sigma). A 20-µg sample of the total RNA was resolved by electrophoresis on a formaldehyde-denatured gel and transferred to a nylon membrane (Gelman Sciences) with 20× SSC for 24 h. The membrane was baked at 80 °C for 2 h, prehybridized at 42 °C for 2 h in 5× SSC, 5 × Denhardt's solution, 50% formamide, 0.2% SDS, 100 µg/ml denatured salmon sperm DNA, and then hybridized at 42 °C for 16 h with a 32P-labeled mouse LTC4S cDNA fragment prepared with a Megaprime DNA labeling kit (Amersham Pharmacia Biotech). The blot was washed once in 0.5× SSC, 0.1% SDS at 60 °C for 30 min and twice in 0.2× SSC, 0.1% SDS at 60 °C for 30 min and was then exposed to an Eastman Kodak Co. AR film for 24 h at -80 °C with an intensifying screen. The probe was stripped, and the blot was hybridized with a 32P-labeled mouse glyceraldehyde-3-phosphate dehydrogenase cDNA probe.

Enzyme Assay-- Mice were euthanized with CO2, and various tissues were isolated and homogenated with a Tissue-Tearor homogenizer (Biospec Products) in five volumes of a buffer containing 50 mM HEPES, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, pH 7.9. BMMC were washed, suspended in phosphate-buffered saline (PBS), and sonicated for 2 min at 4 °C. The tissue homogenates and the BMMC lysates were centrifuged at 1,000 × g for 5 min at 4 °C, and the supernatants were collected for measurements of LTC4S activity and protein concentration. To assay LTC4S activity, the samples were incubated in 200 µl of HEPES buffer, pH 7.6, containing 10 mM MgCl2, 10 mM glutathione at room temperature. The reaction was started by the addition of 2 µl of LTA4-methyl ester (ME) (Dr. J. Rokach, Florida Institute of Technology, Melbourne, FL), which had been dried in nitrogen and dissolved in methanol containing 3% triethylamine, to give a final concentration in the reaction of 20 µM. After incubation for 10 min at room temperature, the reactions were terminated by the addition of three volumes of methanol containing 200 ng of PGB2. Samples were analyzed for LTC4-ME by reverse phase-high performance liquid chromatography (RP-HPLC) (14). Protein concentration was determined by the Bradford method (35) with bovine gamma -globulin as a standard. Enzyme activity was expressed as pmol of LTC4-ME/mg/10 min.

IgE-dependent Activation of BMMC-- BMMC were washed, suspended at a concentration of 1 × 107 cells/ml in Hanks' balanced salt solution containing 1 mM CaCl2, 1 mM MgCl2, and 0.1% bovine serum albumin (HBSA2+), and sensitized with 2 µg/ml monoclonal anti-dinitrophenyl (DNP) IgE (Sigma) for 1 h at 4 °C. After being washed with HBSA2+, the cells were resuspended at a concentration of 1 × 107 cells/ml in HBSA2+ and stimulated with 10 µg/ml goat anti-mouse immunoglobulin (Jackson ImmunoResearch). After 15 min, the reaction was stopped by centrifugation at 120 × g for 5 min at 4 °C, and the supernatants were retained for assays of beta -hexosaminidase (beta -HEX) and eicosanoids. The cell pellets were suspended in HBSA2+ and disrupted by repeated freeze-thawing. beta -HEX, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis of the hydrolysis of p-nitrophenyl-beta -D-2-acetamido-2-deoxyglucopyranoside (36). The percent release of beta -HEX was calculated by the formula [S/(S+P)] × 100, where S and P are the beta -HEX contents of equal portions of supernatant and cell pellet, respectively. PGD2 was measured by enzyme immunoassay according to the manufacturer's instructions (Cayman Chemical). Leukotrienes and 5-hydroxyeicosatetraenoic acid (5-HETE), the decay product of 5-hydroperoxyeicosatetraenoic acid, were measured by RP-HPLC as described (37). Briefly, samples were applied to a C18 Ultrasphere RP column (Beckman Instruments) equilibrated with a solvent of methanol/acetonitrile/water/acetic acid (10:15:100:0.2, v/v), pH 6.0 (Solvent A). After injection of the sample, the column was eluted at a flow rate of 1 ml/min with a programmed concave gradient to 55% of the equilibrated Solvent A and 45% of Solvent B (100% methanol) over 2.5 min. After 5 min, Solvent B was increased linearly to 75% over 15 min and was maintained at this level for an additional 15 min. The UV absorbance at 280 and 235 nm and the UV spectra were recorded simultaneously. The retention times for PGB2, LTC4, 6-trans-LTB4, LTB4, LTE4, and 5-HETE were 21.1, 21.6, 23.2, 24.2, 25.0, and 30.7 min, respectively. LTC4, 6-trans-LTB4, LTB4, LTE4, and 5-HETE were quantitated by calculating the ratio of each peak area to the peak area of the internal standard PGB2.

Zymosan A-induced Peritoneal Inflammation-- Each mouse received an intravenous injection of 0.5% Evans blue dye (10 ml of dye solution/kg of body weight) in PBS immediately before the intraperitoneal injection of 1 ml of zymosan A suspension (1 mg/ml in PBS; Sigma). Mice were euthanized by CO2 at time points of 10, 30, 60, and 120 min and underwent peritoneal lavage with 4 ml of cold PBS. Cells were sedimented from the lavage fluid by centrifugation at 500 × g for 5 min, and Evans blue dye extravasation was assessed by light spectrophotometry of the supernatants at 610 nm. In separate experiments to determine the levels of LTB4 and cysLTs in the lavage fluid, mice were injected with zymosan A suspension without intravenous injection of Evans blue dye. After 2 h, the peritoneal lavage fluid was collected and centrifuged at 500 × g for 5 min. After the addition of ~10,000 dpm of [3H]LTB4 and 100 ng of PGB2 in ethanol (4 times the volume of the supernatant), the lavage fluid supernatant was incubated on ice for 30 min and centrifuged at 10,000 × g for 10 min at 4 °C. The ethanolic supernatant was evaporated by vacuum centrifugation, dissolved in 200 µl of 50 mM HEPES buffer, pH 7.1, containing 50% methanol, and analyzed by RP-HPLC as described above. The UV absorbance at 280 nm and the UV spectra were recorded simultaneously. The fraction containing [3H]LTB4 was collected, evaporated by vacuum centrifugation, and dissolved in 200 µl of enzyme immunoassay buffer for detection of LTB4 (Cayman Chemical). LTE4, the only cysLT detected, was quantitated against the internal standard as described above.

Passive Cutaneous Anaphylaxis-- LTC4S -/- and LTC4S +/+ mice received intradermal injections of 25 ng of mouse monoclonal anti-DNP IgE in 25 µl of saline in the right ear and 25 µl of saline only in the left ear. After 20 h, mice were injected intravenously with 100 µg of DNP-human serum albumin in 100 µl of PBS. At 0, 15, 30, 45, 60, 120, and 240 min after the intravenous injection, ear thickness was measured with calipers (Dyer Company). The difference between the thickness of the right and left ears at each time point reflects the extravasation of plasma proteins because of the alteration in vascular permeability induced by the activation of the local mast cells.

Statistical Analysis-- Results of the experiments were expressed as means ± S.E. Student's t test was used for the statistical analysis of the results. P values < 0.05 were considered to be significant.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Generation of LTC4S -/- Mice-- The strategy to disrupt the LTC4S gene is shown in Fig. 1A. The neo insertion interrupts the coding sequence in exons II, III, and IV, including Arg-51 in exon II and Tyr-93 in exon IV, respectively, which are the critical residues for LTC4S activity (38). Mouse embryonic stem cells derived from the 129/Sv strain were transfected with the linearized targeting vector and selected with G418 and ganciclovir. Of 228 G418- and ganciclovir-resistant colonies isolated, 12 clones were identified as targeted clones by Southern blot analysis. The verified embryonic stem cell clones were microinjected into blastocysts from C57BL/6 and BALB/c mice for subsequent transfer to pseudopregnant ICR female mice. 8 and 4 chimeric males with more than 50% chimerism were obtained from C57BL/6 and from BALB/c blastocysts, respectively, and bred to C57BL/6 or BALB/c female mice. Of 12 chimeric males, 10 were found to be fertile and provided only 2 male heterozygotes from a total of 478 offspring, one from a C57BL/6 mother and the other from a BALB/c mother, as genotyped by Southern blot analysis. These heterozygotes were backcrossed to the respective wild-type females to produce heterozygotes (N2 generation), and N2 or N3 heterozygotes were interbred to generate LTC4S -/- homozygous mice. Southern blot analysis of EcoRI-digested DNA from the N2F1 progeny demonstrated a 4.5-kilobase (kb) band for the disrupted gene and a 5.8-kb band for the wild-type gene and revealed that the ratio of wild-type, heterozygote, and homozygote offspring was 1:2:1 as illustrated for one litter (Fig. 1B). The LTC4S -/- mice developed normally without any apparent defects, and both genders were fertile. Thus, we concluded that the disruption of LTC4S gene did not cause embryonic lethality, developmental defects, or abnormalities in fertility or parturition in the mouse.


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Fig. 1.   Generation of LTC4S gene-disrupted mice. A, genomic organization of the mouse LTC4S gene (upper), structure of the targeting vector (middle), and organization of the putative recombinant LTC4S allele (lower). The five exons are shown as boxes with the coding regions in black. Restriction enzyme sites include BamHI (B), EcoRI (E), HindIII (H), PstI (P), PmlI (Pm), and XmnI (X). The location of a 400-base pair fragment used for Southern blot analysis is shown as a thick line. B, Southern blot analysis of EcoRI-digested tail DNAs from LTC4S -/-, LTC4S +/-, and LTC4S +/+ mice. C, Northern blot analysis of total RNAs from BMMC of LTC4S -/- and LTC4S +/+ mice. Hybridizations were performed with a 32P-labeled mouse LTC4S cDNA probe (upper) and then with a 32P-labeled mouse glyceraldehyde-3-phosphate (GAPDH) dehydrogenase cDNA probe (lower). Molecular size markers are shown at left.

We used BMMC, which are known to generate LTC4 (39), to confirm that the mRNA for LTC4S is not expressed because of the homologous recombination of the targeting construct. Northern blot analysis with total RNAs from BMMC of LTC4S -/- and LTC4S +/+ mice revealed that the mature LTC4S mRNA with a size of 0.8 kb present in BMMC from the LTC4S +/+ mice was not detected in BMMC from the LTC4S -/- mice (Fig. 1C). A 1.4-kb band detected in BMMC from the LTC4S +/+ mice was considered to be an unspliced LTC4S mRNA. Because we used a LTC4S cDNA probe that included exon V, a 1.8-kb band detected in BMMC from the LTC4S -/- mice was considered to be an unstable transcript that contained exon V and presumably the neo gene.

Functional Disruption of LTC4S in Mouse Tissues and BMMC Assayed in Vitro-- We sought to establish that LTC4S function was absent in BMMC from the LTC4S gene-disrupted mice. No LTC4 biosynthesis was detected in extracts of BMMC from LTC4S -/- mice, whereas BMMC from LTC4S +/- and LTC4S +/+ mice showed LTC4 biosynthesis of 2.6 ± 0.78 pmol/min/106 cells (n = 3) and 15.1 ± 0.77 pmol/min/106 cells (n = 3), respectively. To examine the contribution of LTC4S to the production of LTC4 in various mouse tissues, we measured the LTC4S activity by monitoring the conjugation of LTA4-ME with glutathione in tissues from LTC4S -/-, LTC4S +/-, and LTC4S +/+ mice (Fig. 2). In the LTC4S -/- mice, no GST activity specific for LTA4-ME was detected in the tongue, spleen, and brain, and only slight activity was detected in the lung (~3% of the activity of wild-type), stomach (~10% of the activity of wild-type), and colon (~3% of the activity of wild-type). These results indicate that targeted disruption of the LTC4S gene effectively eliminated the conjugation of LTA4-ME with glutathione in these tissues and establish LTC4S as the dominant enzyme for this reaction in these tissues.


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Fig. 2.   GST activity for LTA4-ME in tissues isolated from LTC4S -/-, LTC4S +/-, and LTC4S +/+ mice. Tissues isolated from LTC4S -/-, LTC4S +/-, and LTC4S +/+ mice were assayed for conjugation of LTA4-ME with glutathione with quantitation of LTC4-ME by RP-HPLC. Values represent means ± S.E. (n = 3).

GST activity for LTA4-ME in the testis of LTC4S -/- mice was essentially the same as that of the LTC4S +/- and LTC4S +/+ mice. The LTC4-ME generated by the testis likely reflects the activity of MGST2, MGST3, and/or Mu-class GSTs (40, 41). However, it appears that cysLTs are not involved in reproduction, because both 5-LO- and FLAP-deficient mice, which lack the cellular enzymatic capacity to generate the required LTA4 substrate, had normal fertility and parturition (42-44).

The activation of BMMC through their high affinity receptor for IgE (Fcepsilon RI) was used to demonstrate the absence of LTC4S in an integrated response of the 5-LO/LTC4S pathway and to delineate the impact of that absence on arachidonic acid metabolism by 5-LO, by PG endoperoxide synthase-1, and by the terminal pathway enzymes, LTA4 hydrolase and hematopoietic PGD synthase. As shown in a representative RP-HPLC assay (Fig. 3), no LTC4 was detected after activation of the BMMC from the LTC4S -/- mice; LTB4 was relatively unaffected, and the 6-trans-LTB4 diastereoisomers were increased in this analysis at 280 nm. The data for three independent experiments, including the quantitation of 5-HETE at 235 nm, are shown in Table I. The BMMC from the LTC4S -/- mice generated no LTC4, or its metabolites, LTD4 and LTE4, produced 5-HETE, a decay product of 5-hydroperoxyeicosatetraenoic acid, and LTB4, at the same level as the BMMC from the LTC4S +/+ mice but did exhibit a 2-fold increment in the elaboration of 6-trans-LTB4 diastereoisomers, the nonenzymatic breakdown products of LTA4. BMMC from LTC4S -/- and LTC4S +/+ mice released comparable amounts of beta -HEX, a marker for exocytosis (p = 0.2535), and generated comparable amounts of PGD2, another mast cell-derived eicosanoid (p = 0.3243). These results indicate that LTA4, a common substrate for LTA4 hydrolase and LTC4S, is not converted to additional LTB4 but rather undergoes non-enzymatic hydrolysis to 6-trans-LTB4 diastereoisomers in BMMC from the LTC4S -/- mice and that the loss of LTC4S does not affect the IgE-mediated exocytosis or major cyclooxygenase pathway in BMMC from the LTC4S -/- mice. We conclude that LTC4S is the major LTC4-producing enzyme in mouse mast cells and tissues and that the disruption of the LTC4S gene does not affect release of mast cell granules or shunt substrate to other eicosanoid pathways of BMMC.


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Fig. 3.   HPLC analyses of the leukotrienes secreted from BMMC of LTC4S -/- and LTC4S +/+ mice. BMMC from LTC4S -/- and LTC4S +/+ mice were stimulated with IgE/anti-Ig, and the supernatants were analyzed by RP-HPLC. The results are representative of the three independent experiments. Retention times corresponding to the various eicosanoids are depicted and labeled.

                              
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Table I
Release of beta -HEX and production of LTC4, 5-HETE, 6-trans LTB4, LTB4, and PGD2 by BMMC from LTC4S-/- and LTC4S+/+ mice
BMMC were sensitized with 2 µg/ml of mouse monoclonal IgE and stimulated with 10 µg/ml of goat anti-mouse Ig. The supernatants were analyzed by RP-HPLC as described under "Experimental Procedures." Values are means ± S.E. (n = 3).

Functional Assessment of LTC4S Disruption in Vivo-- To elucidate the possible role of cysLTs in acute inflammation, we examined the in vivo responses of LTC4S -/- and LTC4S +/+ mice to the intraperitoneal injection of zymosan A by measurement of plasma protein extravasation and of cysLTs and LTB4 concentrations in the peritoneal lavage fluid. Protein extravasation in the LTC4S -/- mice was significantly reduced at 10, 30, and 60 min after zymosan injection as compared with the LTC4S +/+ mice (Fig. 4A). RP-HPLC analyses of the lavage fluids collected 120 min after zymosan injection revealed a prominent peak of LTE4 (80.7 ± 4.1 ng/ml; n = 4) as the only cysLT detected in the wild-type mice but no LTE4 in the lavage fluid of the LTC4S -/- mice (Fig. 4, B and C). However, the LTB4 level in the peritoneal lavage fluid at this time point was comparable for the LTC4S -/- (1.34 ± 0.53 ng/ml; n = 4) and LTC4S +/+ mice (0.82 ± 0.10 ng/ml; n = 4) (p = 0.3722) (Fig. 4C).


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Fig. 4.   Plasma protein extravasation in zymosan A-induced peritoneal inflammation. A, LTC4S -/- and LTC4S +/+ mice were injected intraperitoneally with 1 ml of zymosan A in PBS (1 mg/ml) immediately after the intravenous injection of 0.5% Evans blue dye. At 10, 30, 60, and 120 min, peritoneal lavage was performed. The cells in the lavage fluid were removed by centrifugation, and the absorbance of the lavage fluid supernatant at 610 nm was measured to quantitate Evans blue dye extravasation. Error bars indicate S.E. (n = 3-4). *, p < 0.05 as compared with LTC4S +/+. B, RP-HPLC analysis at 280 nm of the peritoneal lavage fluid supernatants from LTC4S -/- (left) and LTC4S +/+ (right) 120 min after zymosan A injection. Arrows depict the retention times for PGB2 and LTE4. *, a peak detected at a retention time of about 25.2 min in the LTC4S -/- lavage fluid was not a cysLT as assessed by its UV spectrum. Results are representative of four independent experiments summarized in panel C. C, leukotrienes in the peritoneal lavage fluid supernatants 120 min after zymosan injection of LTC4S -/- (white column) and LTC4S +/+ (black column) were resolved by RP-HPLC and quantitated for LTE4 by UV absorbance at 280 nm relative to the PGB2 standard and for LTB4 by enzyme immunoassay of the fraction eluting at the correct time as defined by the [3H]LTB4. Error bars indicate S.E. (n = 4).

Zymosan is a yeast cell wall polysaccharide that can directly stimulate monocytes to generate leukotrienes (45) or can act indirectly via activation of the alternative complement pathway to provide peptides (46) capable of eliciting leukotrienes (47). Together with the ability of peritoneal macrophages to generate LTC4 in response to zymosan ex vivo (45), the findings that the zymosan-induced elaborations of LTC4 in the peritoneal cavity are similar in time course and concentration in C5a-deficient and sufficient mice and also in mast cell-deficient and control mice (48) suggest that the monocyte/macrophages in the peritoneal cavity are directly activated with zymosan to produce leukotrienes. In the zymosan-elicited peritoneal inflammation model, LTC4 was generated with a peak at 30 min and was converted to LTE4 with a peak at 60 min, whereas vascular permeability, assessed by protein concentration, increased rapidly after injection and reached a peak at 120 min (45). In other studies, the level of LTB4 peaked at 120-180 min (45), followed by the recruitment of neutrophils to a plateau 360-420 min after zymosan injection (48, 49). We focused on the vascular permeability component of the zymosan effect, which was significantly impaired from 10 to 60 min in the LTC4S -/- mice compared with their controls and noted an absence of LTE4, the most stable of the cysLTs (50). That the cysLTs are major mediators of the increase in vascular permeability induced by zymosan is supported by the previous findings of a similar attenuation of this response in 5-LO-deficient and FLAP-deficient mice (44, 49) but not in LTA4 hydrolase-deficient mice (49). Thus, we conclude that the initial phase of zymosan-elicited plasma protein extravasation in the peritoneal cavity is in large part because of the cysLTs and not because of LTB4 or even other 5-LO metabolites.

To examine the contribution of cysLTs to a mast cell-mediated acute allergic reaction in the skin, we performed passive cutaneous anaphylaxis in the ears of the LTC4S -/- and LTC4S +/+ mice (Fig. 5). The intravenous injection of DNP-human serum albumin elicited rapid ear swelling in both the LTC4S -/- and wild-type mice that peaked at 15 min, persisted to 45 min, and then declined with resolution at 240 min. However, ear swelling was significantly reduced from 15 to 60 min by ~50% in the LTC4S -/- mice. As BMMC from the LTC4S -/- mice had no capacity to generate LTC4 in response to IgE-dependent activation while being fully competent in secretory granule exocytosis and generation of LTB4 and PGD2, we conclude that cysLTs are major mediators of the ear swelling initiated in situ by antigen activation of IgE-sensitized mast cells.


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Fig. 5.   Passive cutaneous anaphylaxis in LTC4S -/- mice. LTC4S -/- and LTC4S +/+ mice received intradermal injections of 25 ng of mouse anti-DNP monoclonal IgE in 25 µl of saline in the right ear and 25 µl of saline only in the left ear. After 20 h, mice were injected intravenously with 100 µg of DNP-human serum albumin in 100 µl of PBS, and ear thickness was measured 0, 15, 30, 45, 60, 120, and 240 min later. The difference between the thickness of the right and left ears at each time point is expressed as the mean ± S.E. *, p < 0.05 as compared with LTC4S +/+.

Topical administration of mast cell-derived mediators such as LTC4, LTD4, histamine, and serotonin has been demonstrated to induce ear edema in the mouse as assessed after 30 min by Evans blue dye extravasation. Serotonin and LTC4 were about 100 and 10 times, respectively, more potent than histamine on a weight basis in eliciting increased vascular permeability in the mouse ear (51). The contribution of the cysLTs to the ear edema in a model of passive IgE-induced cutaneous anaphylaxis had been considered to be minimal, because CysLT1 receptor antagonists did not block increased vascular permeability (52, 53). The 5-LO -/-, FLAP -/-, or LTA4 hydrolase -/- mice also failed to show attenuated ear edema after passive systemic sensitization with monoclonal anti-DNP IgE followed in 24 h by antigen challenge (44, 49). In as much as there was a fall in body temperature similar to that of the normal mice, these gene-disrupted mice did experience systemic anaphylaxis. However, our results that IgE-dependent passive cutaneous anaphylaxis was significantly reduced in the LTC4S -/- mice as compared with the wild-type littermates indicate that the cysLTs play an important role in the increased vascular permeability in the IgE-dependent allergic reactions in the skin. Thus, the LTC4S -/- mice, generated by targeted disruption of the gene, do have a discrete phenotype that becomes apparent during the initial phase of altered vascular permeability that accompanies an inflammatory reaction elicited by either an innate or a specific immune stimulus.

    ACKNOWLEDGEMENTS

We thank Dr. Arlene Sharpe and Lina Du (Brigham and Women's Hospital) for blastocyst injection of embryonic stem cells. We thank Dr. Kongyi Xu for technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AI31599 and HL36110 and the Human Frontier Science Program (to Y. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Brigham and Women's Hospital, Smith Bldg., Rm. 628, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1270; Fax: 617-525-1310; E-mail: blam@rics.bwh.harvard.edu.

Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M103562200

    ABBREVIATIONS

The abbreviations used are: cysLT(s), cysteinyl leukotriene(s); LT, leukotriene; 5-LO, 5-lipoxygenase; FLAP, 5-LO-activating protein; LTC4S, LTC4 synthase; PG, prostaglandin; GST, glutathione S-transferase; MGST(s), microsomal GST(s); BMMC, bone marrow-derived mast cells; 5-HETE, 5-hydroxyeicosatetraenoic acid; PBS, phosphate-buffered saline; ME, methyl ester; RP-HPLC, reverse phase high performance liquid chromatography; beta -HEX, beta -hexosaminidase; kb, kilobase; DNP, dinitrophenyl; HBSAa+, Hanks' balanced salt solution containing bovine serum albumin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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