Targeted disruption of the leukotriene B(4) receptor in mice reveals its role in inflammation and platelet-activating factor-induced anaphylaxis

Abstract

Leukotrienes are derived from arachidonic acid and serve as mediators of inflammation and immediate hypersensitivity. Leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) act through G protein-coupled receptors LTB(4) receptor (BLTR) and Cys-LTR, respectively. To investigate the physiological role of BLTR, we produced mice with a targeted disruption of the BLTR gene. Mice deficient for BLTR (BLTR(-/-)) developed normally and had no apparent hematopoietic abnormalities. Peritoneal neutrophils from BLTR(-/-) mice displayed normal responses to the inflammatory mediators C5a and platelet-activating factor (PAF) but did not respond to LTB(4) for calcium mobilization or chemotaxis. Additionally, LTB(4) elicited peritoneal neutrophil influx in control but not in BLTR(-/-) mice. Thus, BLTR is the sole receptor for LTB(4)-induced inflammation in mice. Neutrophil influx in a peritonitis model and acute ear inflammation in response to arachidonic acid was significantly reduced in BLTR(-/-) mice. In mice, intravenous administration of PAF induces immediate lethal anaphylaxis. Surprisingly, female BLTR(-/-) mice displayed selective survival (6 of 9; P = 0.002) relative to male (1 of 11) mice of PAF-induced anaphylaxis. These results demonstrate the role of BLTR in leukotriene-mediated acute inflammation and an unexpected sex-related involvement in PAF-induced anaphylaxis.

Figure 1

Targeted disruption of mouse BLTR. (A) Wild-type genomic locus of BLTR, targeting vector, and the recombinant mutant genomic locus. Coding region of the BLTR gene is indicated as a solid box. 706 bp of the coding region between the XhoI and SacII sites was replaced with PGK-Neo in the targeting vector. The final construct contained homology arms of 4.0 and 1.5 kb. A BglII–BamHI fragment served as an external probe for Southern blot analysis of genomic DNA from ES cells and mouse tails. (B) Southern blot showing correct targeting and germline transmission of the mutated BLTR gene. Genomic DNA samples prepared from F2 offspring were digested with BamHI and XhoI, separated on 0.6% agarose gels, blotted onto nylon membranes, and hybridized with the ³²P-labeled, 0.9-kb BglII–BamHI fragment. The genotypes of the mice are indicated above the lanes. (C) A three-primer PCR reaction was designed to identify the wild-type, heterozygous, and homozygous mutant alleles. The genotypes of the mice identified by this method confirmed the identifications by the Southern blot method in all cases tested.

Figure 3

Peritoneal leukocyte accumulation in BLTR^+/+ and BLTR^−/− mice. (A) Neutrophil and (B) macrophage accumulation after zymosan injection. The mean number of each leukocyte type (±SEM) in peritoneal lavage samples isolated at the times indicated after zymosan injection are presented. The number of animals for each time point consisted of five to nine animals. Statistically significant differences are indicated (*P < 0.05 and **P < 0.01).

Figure 2

Loss of BLTR function in neutrophils from BLTR^−/− mice. (A) Calcium mobilization was monitored in INDO-1–loaded, zymosan-elicited peritoneal neutrophils stimulated with 100 nM LTB₄, 100 nM C5a, or 100 nM PAF as indicated. Each tracing represents an analysis of 3 × 10⁶ cells from a single mouse with the indicated BLTR genotype, and the data shown is representative of at least six each of BLTR^+/+ and BLTR^−/− animals. (B) Neutrophil chemotaxis. The chemotactic response of zymosan-elicited peritoneal neutrophils from a single BLTR^+/+ or BLTR^−/− mouse to LTB₄ (100 nM) and C5a (100 nM) was determined by counting the number of migrated cells within five high-powered fields (400×) representing three individual wells. Results are representative of at least four mice for each genotype. Differences in LTB₄ responses between BLTR^+/+ and BLTR^−/− mice is statistically significant (*P < 0.05). (C) LTB₄-induced peritonitis. Six mice of each genotype were injected intraperitoneally with 1.0 ml of saline containing 10 μg of LTB₄, and 4 h later, peritoneal exudate cells were harvested. Values represent the mean ± SEM. The difference in neutrophil accumulation between BLTR^+/+ and BLTR^−/− mice is statistically significant (*P < 0.05).

Figure 4

AA-induced ear inflammation. AA (2 mg) was applied to the mouse ears and analyzed for edema and protein extravasation after 90 min as described in Materials and Methods. (A) Edema: wet weight of a 5-mm ear punch of the control (white bars) and AA-treated ears (black bars) was measured. (B) Extravasation: Evans blue dye leakage was measured at a wavelength of 610 nm (OD 610): open bar, control ears; closed bars, AA-treated ears. The decreased inflammatory response in the AA-treated ears of BLTR^−/− mice compared with BLTR^+/+ mice was statistically significant (*P < 0.05) for data in A and B. (C and D) Histology: hematoxylin and eosin–stained tissue sections from BLTR^+/+ (C) and BLTR^−/− (D) mice treated with AA for 90 min.

Figure 4

AA-induced ear inflammation. AA (2 mg) was applied to the mouse ears and analyzed for edema and protein extravasation after 90 min as described in Materials and Methods. (A) Edema: wet weight of a 5-mm ear punch of the control (white bars) and AA-treated ears (black bars) was measured. (B) Extravasation: Evans blue dye leakage was measured at a wavelength of 610 nm (OD 610): open bar, control ears; closed bars, AA-treated ears. The decreased inflammatory response in the AA-treated ears of BLTR^−/− mice compared with BLTR^+/+ mice was statistically significant (*P < 0.05) for data in A and B. (C and D) Histology: hematoxylin and eosin–stained tissue sections from BLTR^+/+ (C) and BLTR^−/− (D) mice treated with AA for 90 min.