Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase.

Leukotriene C(4) synthase (LTC(4)S), 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 LTC(4)S (-/-) mice developed normally and were fertile. LTC(4)S activity, assessed by conjugation of leukotriene (LT) A(4) methyl ester with glutathione, was absent from tongue, spleen, and brain and > or = 90% reduced in lung, stomach, and colon of the LTC(4)S (-/-) mice. Bone marrow-derived mast cells (BMMC) from the LTC(4)S (-/-) mice provided no LTC(4) in response to IgE-dependent activation. Exocytosis and the generation of prostaglandin D(2), LTB(4), and 5-hydroxyeicosatetraenoic acid by BMMC from LTC(4)S (-/-) mice and LTC(4)S (+/+) mice were similar, whereas the degraded product of LTA(4), 6-trans-LTB(4), was doubled in BMMC from LTC(4)S (-/-) 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 LTC(4)S (-/-) mice. These observations indicate that LTC(4)S, but not microsomal or cytosolic glutathione S-transferases, is the major LTC(4)-producing enzyme in tissues and that its integrated function includes mediation of increased vascular permeability in either innate or adaptive immune host inflammatory responses.

The cysteinyl leukotrienes (cysLTs), 1 leukotriene (LT) C 4 , and its metabolites, LTD 4 and LTE 4 , 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 per-meability (3)(4)(5)(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 LTC 4 requires activation with Ca 2ϩ -dependent translocation of cytosolic phospholipase A 2 and 5-lipoxygenase  to the perinuclear and endoplasmic reticular membranes. There, in the presence of 5-LO-activating protein (FLAP), the arachidonic acid released by cytosolic phospholipase A 2 is converted to 5-hydroperoxyeicosatetraenoic acid and then to LTA 4 (9 -11). LTA 4 is processed either to the dihydroxy leukotriene, LTB 4 , by LTA 4 hydrolase (12) or to LTC 4 through conjugation with reduced glutathione by LTC 4 synthase (LTC 4 S) (13)(14)(15). After carrier-mediated export of LTC 4 (16), glutamic acid and glycine are sequentially cleaved by ␥-glutamyl transpeptidase and dipeptidase to form LTD 4 and LTE 4 , respectively (17,18). Two cysLT receptors, termed CysLT 1 and CysLT 2 receptors, are presently known. Whereas the CysLT 1 receptor has a marked preference for signal activation by LTD 4 , the CysLT 2 receptor has a similar recognition of LTC 4 and LTD 4 with a higher K d value relative to that of the CysLT 1 receptor (19,20). Mast cells also metabolize the released arachidonic acid to prostaglandin (PG) D 2 by the successive action of PG endoperoxide synthase-1 or PG endoperoxide synthase-2 (21,22) and hematopoietic PGD synthase (23,24). Whereas a PGD 2 receptor, termed DP, is prominent on smooth muscle such as airways and microvasculature, a recently identified PGD 2 receptor, termed CRTH2, is localized to hematopoietic cells such as T helper type 2 (Th2) cells, basophils, and eosinophils (25,26). LTC 4 S is an 18-kDa integral membrane protein that shows glutathione S-transferase (GST) activity that is strictly specific for LTA 4 as a substrate (13)(14)(15). The human LTC 4 S 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). LTC 4 S 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 LTA 4 to form LTC 4 , and they are ubiquitously expressed even in cells lacking the capacity to provide LTA 4 . LTC 4 can be formed through the transcellular metabolism of LTA 4 by cells that express LTC 4 S, such as platelets (30), or MGST2, such as endothelial cells (31). With the exception of platelets, LTC 4 S has been identified only in hematopoietic cells that also express 5-LO.
We sought to establish that LTC 4 S was the dominant constitutive enzymatic source of LTC 4 in situ in the mouse by targeted gene disruption of the LTC 4 S gene. The loss of function in the gene-disrupted mice relative to their controls was * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Generation of LTC 4 S Ϫ/Ϫ Mice-A 5.7-kb 129/Ola mouse genomic fragment containing the LTC 4 S gene (32) was subcloned into a pcDNA3 vector (Invitrogen). After the endogenous neomycin resistance gene (neo) of the pcDNA3 and ϳ400 base pairs of the 5Ј 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 LTC 4 S gene. The herpes simplex virus thymidine kinase (TK) gene was inserted at the 3Ј-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 g/ml; Life Technologies, Inc.) and ganciclovir (2 M), and homologous recombination was confirmed by Southern blot analysis of EcoRI-digested genomic DNA from each embryonic stem cell clone with the ϳ400-base pair 5Ј 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 N 2 or N 3 generation were intercrossed to obtain homozygotes. All the experiments were performed with LTC 4 S Ϫ/Ϫ 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 LTC 4 S 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 g/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 ϫ 10 5 /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-g sample of the total RNA was resolved by electrophoresis on a formaldehyde-denatured gel and transferred to a nylon membrane (Gelman Sciences) with 20ϫ SSC for 24 h. The membrane was baked at 80°C for 2 h, prehybridized at 42°C for 2 h in 5ϫ SSC, 5 ϫ Denhardt's solution, 50% formamide, 0.2% SDS, 100 g/ml denatured salmon sperm DNA, and then hybridized at 42°C for 16 h with a 32 P-labeled mouse LTC 4 S cDNA fragment prepared with a Megaprime DNA labeling kit (Amersham Pharmacia Biotech). The blot was washed once in 0.5ϫ SSC, 0.1% SDS at 60°C for 30 min and twice in 0.2ϫ 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 Ϫ80°C with an intensifying screen. The probe was stripped, and the blot was hybridized with a 32 P-labeled mouse glyceraldehyde-3-phosphate dehydrogenase cDNA probe.
Enzyme Assay-Mice were euthanized with CO 2 , 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 ϫ g for 5 min at 4°C, and the supernatants were collected for measurements of LTC 4 S activity and protein concentration. To assay LTC 4 S activity, the samples were incubated in 200 l of HEPES buffer, pH 7.6, containing 10 mM MgCl 2 , 10 mM glutathione at room temperature. The reaction was started by the addition of 2 l of LTA 4 -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 M. After incubation for 10 min at room temperature, the reactions were terminated by the addition of three volumes of methanol containing 200 ng of PGB 2 . Samples were analyzed for LTC 4 -ME by reverse phase-high performance liquid chromatography (RP-HPLC) (14). Protein concentration was determined by the Bradford method (35) with bovine ␥-globulin as a standard. Enzyme activity was expressed as pmol of LTC 4 -ME/mg/10 min.
IgE-dependent Activation of BMMC-BMMC were washed, suspended at a concentration of 1 ϫ 10 7 cells/ml in Hanks' balanced salt solution containing 1 mM CaCl 2 , 1 mM MgCl 2 , and 0.1% bovine serum albumin (HBSA 2ϩ ), and sensitized with 2 g/ml monoclonal anti-dinitrophenyl (DNP) IgE (Sigma) for 1 h at 4°C. After being washed with HBSA 2ϩ , the cells were resuspended at a concentration of 1 ϫ 10 7 cells/ml in HBSA 2ϩ and stimulated with 10 g/ml goat anti-mouse immunoglobulin (Jackson ImmunoResearch). After 15 min, the reaction was stopped by centrifugation at 120 ϫ g for 5 min at 4°C, and the supernatants were retained for assays of ␤-hexosaminidase (␤-HEX) and eicosanoids. The cell pellets were suspended in HBSA 2ϩ and disrupted by repeated freeze-thawing. ␤-HEX, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis of the hydrolysis of p-nitrophenyl-␤-D-2-acetamido-2-deoxyglucopyranoside (36). The percent release of ␤-HEX was calculated by the formula [S/(SϩP)] ϫ 100, where S and P are the ␤-HEX contents of equal portions of supernatant and cell pellet, respectively. PGD 2 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 PGB 2  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 CO 2 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 ϫ 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 LTB 4 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 ϫ g for 5 min. After the addition of ϳ10,000 dpm of [ 3 H]LTB 4 and 100 ng of PGB 2 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 ϫ g for 10 min at 4°C. The ethanolic supernatant was evaporated by vacuum centrifugation, dissolved in 200 l 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 [ 3 H]LTB 4 was collected, evaporated by vacuum centrifugation, and dissolved in 200 l of enzyme immunoassay buffer for detection of LTB 4 (Cayman Chemical). LTE 4 , the only cysLT detected, was quantitated against the internal standard as described above.
Passive Cutaneous Anaphylaxis-LTC 4  Statistical Analysis-Results of the experiments were expressed as means Ϯ S.E. Student's t test was used for the statistical analysis of the results. P values Ͻ 0.05 were considered to be significant. 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 LTC 4 S 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 (N 2 generation), and N 2 or N 3 heterozygotes were interbred to generate LTC 4 S Ϫ/Ϫ homozygous mice. Southern blot analysis of EcoRIdigested DNA from the N 2 F 1 progeny demonstrated a 4.5kilobase (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 LTC 4 S Ϫ/Ϫ mice developed normally without any apparent defects, and both genders were fertile. Thus, we concluded that the disruption of LTC 4 S gene did not cause embryonic lethality, developmental defects, or abnormalities in fertility or parturition in the mouse.

Generation of LTC 4 S Ϫ/Ϫ Mice-The strategy to disrupt the LTC 4 S gene is shown in
We used BMMC, which are known to generate LTC 4 (39), to confirm that the mRNA for LTC 4 S is not expressed because of the homologous recombination of the targeting construct. Northern blot analysis with total RNAs from BMMC of LTC 4 S Ϫ/Ϫ and LTC 4 S ϩ/ϩ mice revealed that the mature LTC 4 S mRNA with a size of 0.8 kb present in BMMC from the LTC 4 S ϩ/ϩ mice was not detected in BMMC from the LTC 4 S Ϫ/Ϫ mice (Fig. 1C). A 1.4-kb band detected in BMMC from the LTC 4 S ϩ/ϩ mice was considered to be an unspliced LTC 4 S mRNA. Because we used a LTC 4 S cDNA probe that included exon V, a 1.8-kb band detected in BMMC from the LTC 4 S Ϫ/Ϫ mice was considered to be an unstable transcript that contained exon V and presumably the neo gene. respectively. To examine the contribution of LTC 4 S to the production of LTC 4 in various mouse tissues, we measured the LTC 4 S activity by monitoring the conjugation of LTA 4 -ME with glutathione in tissues from LTC 4 S Ϫ/Ϫ , LTC 4 S ϩ/Ϫ , and LTC 4 S ϩ/ϩ mice (Fig. 2). In the LTC 4 S Ϫ/Ϫ mice, no GST activity specific for LTA 4 -ME was detected in the tongue, spleen, and brain, and only slight activity was detected in the lung (ϳ3% of the activity of wild-type), stomach (ϳ10% of the activity of wild-type), and colon (ϳ3% of the activity of wild-type). These results indicate that targeted disruption of the LTC 4 S gene effectively eliminated the conjugation of LTA 4 -ME with glutathione in these tissues and establish LTC 4 S as the dominant enzyme for this reaction in these tissues.

Functional Disruption of LTC 4 S in Mouse Tissues and BMMC Assayed in Vitro-
GST activity for LTA 4 -ME in the testis of LTC 4 S Ϫ/Ϫ mice was essentially the same as that of the LTC 4 S ϩ/Ϫ and LTC 4 S ϩ/ϩ mice. The LTC 4 -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 LTA 4 substrate, had normal fertility and parturition (42)(43)(44).
The activation of BMMC through their high affinity receptor for IgE (Fc⑀RI) was used to demonstrate the absence of LTC 4 S in an integrated response of the 5-LO/LTC 4 S 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, LTA 4 hydrolase and hematopoietic PGD synthase. As shown in a representative RP-HPLC assay (Fig. 3), no LTC 4 was detected after activation of the BMMC from the LTC 4 S Ϫ/Ϫ mice; LTB 4 was relatively unaffected, and the 6-trans-LTB 4 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 LTC 4 S Ϫ/Ϫ mice generated no LTC 4 , or its metabolites, LTD 4 and LTE 4 , produced 5-HETE, a decay product of 5-hydroperoxyeicosatetraenoic acid, and LTB 4 , at the same level as the BMMC from the LTC 4 S ϩ/ϩ mice but did exhibit a 2-fold increment in the elaboration of 6-trans-LTB 4 diastereoisomers, the nonenzymatic breakdown products of LTA 4 . BMMC from LTC 4 S Ϫ/Ϫ and LTC 4 S ϩ/ϩ mice released comparable amounts of ␤-HEX, a marker for exocytosis (p ϭ 0.2535), and generated comparable amounts of PGD 2 , another mast cell-derived eicosanoid (p ϭ 0.3243). These results indicate that LTA 4 , a common substrate for LTA 4 hydrolase and LTC 4 S, is not converted to additional LTB 4 but rather undergoes non-enzymatic hydrolysis to 6-trans-LTB 4 diastereoisomers in BMMC from the LTC 4 S Ϫ/Ϫ mice and that the loss of LTC 4 S does not affect the IgE-mediated exocytosis or major cyclooxygenase pathway in BMMC from the LTC 4 S Ϫ/Ϫ mice. We conclude that LTC 4 S is the major LTC 4 -producing enzyme in mouse mast cells and tissues and that the disruption of the LTC 4 S gene does not affect release of mast cell granules or shunt substrate to other eicosanoid pathways of BMMC.
Functional Assessment of LTC 4 S Disruption in Vivo-To elucidate the possible role of cysLTs in acute inflammation, we examined the in vivo responses of LTC 4 S Ϫ/Ϫ and LTC 4 S ϩ/ϩ mice to the intraperitoneal injection of zymosan A by measurement of plasma protein extravasation and of cysLTs and LTB 4 concentrations in the peritoneal lavage fluid. Protein extravasation in the LTC 4 S Ϫ/Ϫ mice was significantly reduced at 10, 30, and 60 min after zymosan injection as compared with the LTC 4 S ϩ/ϩ mice (Fig. 4A). RP-HPLC analyses of the lavage fluids collected 120 min after zymosan injection revealed a prominent peak of LTE 4 (80.7 Ϯ 4.1 ng/ml; n ϭ 4) as the only cysLT detected in the wild-type mice but no LTE 4 in the lavage fluid of the LTC 4 S Ϫ/Ϫ mice (Fig. 4, B and C). However, the LTB 4 level in the peritoneal lavage fluid at this time point was comparable for the LTC 4 S Ϫ/Ϫ (1.34 Ϯ 0.53 ng/ml; n ϭ 4) and LTC 4 S ϩ/ϩ mice (0.82 Ϯ 0.10 ng/ml; n ϭ 4) (p ϭ 0.3722) (Fig.  4C).
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 LTC 4 in response to zymosan ex vivo (45), the findings that the zymosan-induced elaborations of LTC 4 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, LTC 4 was generated with a peak at 30 min and was converted to LTE 4 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 LTB 4 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 LTC 4 S Ϫ/Ϫ mice compared with their controls and noted an absence of LTE 4 , 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 LTA 4 hydrolasedeficient 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 LTB 4 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 LTC 4 S Ϫ/Ϫ and LTC 4 S ϩ/ϩ mice (Fig. 5). The intravenous injection of DNP-human serum albumin elicited rapid ear swelling in both the LTC 4 S Ϫ/Ϫ 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 ϳ50% in the LTC 4 S Ϫ/Ϫ mice. As BMMC from the LTC 4 S Ϫ/Ϫ mice had no capacity to generate LTC 4 in response to IgE-dependent activation while being fully competent in secretory granule exocytosis and generation of LTB 4 and PGD 2 , we conclude that cysLTs are major mediators of the ear swelling initiated in situ by antigen activation of IgE-sensitized mast cells.
Topical administration of mast cell-derived mediators such as LTC 4 , LTD 4 , 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 LTC 4 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 CysLT 1 receptor antagonists did not block increased vascular permeability (52,53). The 5-LO Ϫ/Ϫ , FLAP Ϫ/Ϫ , or LTA 4 hydrolase Ϫ/Ϫ 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 LTC 4 S Ϫ/Ϫ 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 LTC 4 S Ϫ/Ϫ 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.