Role of group V phospholipase A2 in zymosan-induced eicosanoid generation and vascular permeability revealed by targeted gene disruption.

Conclusions regarding the contribution of low molecular weight secretory phospholipase A2 (sPLA2) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA2 enzymes. To elucidate the role of group V sPLA2, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C4 and prostaglandin E2 was attenuated approximately 50% in peritoneal macrophages from group V sPLA2-null mice compared with macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA2-null mice compared with wild-type controls. These data provide clear evidence of a role for group V sPLA2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity.

The first step in the biosynthesis of eicosanoids is the release of arachidonic acid from cell membrane phospholipids by phospholipase A 2 . Several classes of phospholipase A 2 have been described in mammals (1,2). Cytosolic phospholipase A 2 (cPLA 2 ) 1 ␣ is an 85-kDa cytosolic enzyme that uses a catalytic serine residue and preferentially cleaves arachidonic acid from cell membrane phospholipids (3). The Ca 2ϩ -dependent translocation of cPLA 2 -␣ from the cytosol to the nuclear envelope (4), a prominent site of eicosanoid biosynthesis, is dependent on a Ca 2ϩ -dependent lipid binding (C-2) domain. Paralogues of cPLA 2 -␣ (cPLA 2 -␤ and cPLA 2 -␥) have been described previously (5,6). cPLA 2 -␤ has a M r of 110,000 and shares 30% identity with cPLA 2 -␣, including a functional C-2 domain. cPLA 2 -␥ has a M r of 61,000, shares 29% sequence identity with cPLA 2 -␣, lacks a C-2 domain, and is Ca 2ϩ -independent. Mammalian low molecular weight secretory phospholipase A 2 (sPLA 2 ) enzymes, which are now 10 in number, are characterized by a conserved motif containing a catalytic histidine residue, by their relatively small size of ϳ14 kDa, and by their highly disulfide-linked tertiary structures (7)(8)(9)(10)(11)(12)(13). They are distinguished from one another by their structures, their biochemical properties, and their tissue distribution. Calcium-independent phospholipase A 2 enzymes have been described in myocardium and in leukocytes (14,15). They have been implicated in membrane remodeling, regulation of store operated calcium channels, apoptosis, and release of arachidonic acid. The fourth group of phospholipase A 2 enzymes comprises the acetyl hydrolases of platelet activating factor (16).
Given the complexity and size of the phospholipase A 2 family, targeted gene disruption is a suitable approach to elucidating the role(s) of individual enzymes and proved fruitful in determining the role of cPLA 2 -␣ in regulating eicosanoid biosynthesis. Disruption of the gene encoding cPLA 2 -␣ led to almost complete abrogation of the rapid generation of prostaglandin (PG) E 2 , leukotriene (LT) C 4 , and LTB 4 from peritoneal macrophages in response to A23187 and a more delayed generation of PGE 2 in response to lipopolysaccharide (LPS) (17,18). Separate studies demonstrated a marked attenuation of the release of labeled arachidonic acid from cPLA 2 -␣-null peritoneal macrophages in response to zymosan, A23187, phorbol myristate acetate, and okadaic acid (19) and the critical role of this enzyme in immediate and delayed phases of eicosanoid generation by mouse bone marrow culture-derived mast cells (20,21).
Whereas these data demonstrate the essential function of cPLA 2 -␣ in supplying arachidonic acid for leukotriene and prostaglandin biosynthesis, several lines of evidence have suggested that low molecular weight sPLA 2 enzymes amplify the actions of cPLA 2 -␣ in eicosanoid generation. Transfection of HEK293 cells with the heparin-binding group IIA sPLA 2 , group V sPLA 2 , or group IID sPLA 2 amplified the cPLA 2 -␣-dependent release of arachidonic acid and PGE 2 generation in response to A23187 or to LPS and interleukin-1␤ (22,23). The action of these enzymes was attributed to their ability to bind to glypican in caveoli, leading to internalization and co-localization with prostaglandin endoperoxide synthase enzymes (22). Similarly, adenoviral transfection of group V sPLA 2 or group IIA sPLA 2 into mouse mesangial cells amplified H 2 O 2 -induced release of arachidonic acid. This was observed only in cPLA 2 -␣sufficient and not in cPLA 2 -␣-null cells (24). When P388D 1 macrophages were primed with LPS for 1 h and then activated with platelet activating factor, there was biphasic release of labeled arachidonic acid (25). Studies with pharmacological inhibitors and with antisense oligonucleotides suggested that the initial intracellular release of arachidonic acid was dependent upon cPLA 2 -␣, whereas the subsequent extracellular release was dependent upon group V sPLA 2 , which was dependent upon the initial action of cPLA 2 -␣ (26,27). In certain subclones of the P388D 1 macrophage, LPS stimulation alone elicited a delayed phase of arachidonic acid release and prostaglandin endoperoxide synthase-2-dependent PGE 2 generation. Both cPLA 2 -␣ and group V sPLA 2 were required; however, in contrast to the primed-immediate response to LPS and platelet activating factor, activation of cPLA 2 -␣ led to induction of group V sPLA 2 expression that in turn induced prostaglandin endoperoxide synthase-2 (28).
The data implicating low molecular weight sPLA 2 enzymes in eicosanoid generation have thus relied on either transfection experiments or pharmacological and antisense inhibition experiments. The former, although informative with regard to the potential functions of a sPLA 2 , fail to address the role of the endogenous enzymes. The latter have lacked specificity (25,27), failed to discriminate between individual low molecular weight phospholipase A 2 enzymes (29), or yielded conflicting data (30,31). To definitively test the role of group V sPLA 2 in vivo and in vitro, we have generated mice with targeted disruption of the gene encoding this enzyme.

EXPERIMENTAL PROCEDURES
Materials-Restriction enzymes were from Roche Molecular Biochemicals. Human serum albumin, zymosan A, Evans blue dye, and paraformaldehyde were from Sigma.
Targeted Disruption of the Gene for Group V sPLA 2 -We used a group V sPLA 2 cDNA (32) to screen a 129 mouse genomic DNA library in Lambda FIX II (Stratagene). An ϳ11-kb portion of the group V sPLA 2 gene, composed of two contiguous SacI restriction fragments, was isolated and used to construct a targeting vector in pBluescript (Fig. 1A) in which the fourth exon was excised with BclI and replaced with a neomycin resistance gene that lacked a polyadenylation signal. This leads to loss of the histidine residue essential for the catalytic function of the sPLA 2 enzymes (33) and will lead to a premature stop codon if exons III and V are spliced. A thymidine kinase gene was inserted at a SalI site within pBluescript upstream of the BamHI site.
Embryonic stem cells were transfected with the linearized targeting construct. Clones with homologous recombination of the targeting vector were selected in gancyclovir and neomycin according to established protocols and identified by genomic Southern blotting. Two 129 embryonic stem cell clones heterozygous for disruption of the group V sPLA 2 gene were injected separately into blastocysts from C57BL/6 mice and transferred to the uteri of C57BL/6 pseudopregnant females. Pups that were chimeric for 129 and C57BL/6 cells were detected by coat color. Five mice (three males and two females) with Ͼ50% chimerism were obtained. Two of the males, each derived from different embryonic stem cell clones, were bred back to C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME). Offspring were screened for transmission of the disrupted allele of group V sPLA 2 by Southern blotting of DNA derived from tail snips. Breeding pairs from this F 1 generation of group V sPLA 2 ϩ/Ϫ mice were established, and their F 2 group V sPLA 2 -null and group V sPLA 2 -wild-type progeny were used in subsequent experiments.
Southern Blotting-DNA extracted from mouse tail snips was digested with NcoI or EcoRI, resolved in separate lanes of 1% agarose gels, transferred to Immobilon NY membranes (Millipore), and probed with a [ 32 P]dCTP random prime-labeled (Stratagene) 306-bp portion of DNA from the 3Ј-untranslated region of the group V sPLA 2 gene that lies outside the targeting construct (Fig. 1A).
Northern Blotting-Mouse hearts were homogenized in Tri Reagent (Molecular Research Center, Cincinnati, OH), and RNA was extracted according to the manufacturer's protocol. Ten g of RNA from each heart were resolved in separate lanes of 1.2% agarose formaldehyde gels, blotted to Immobilon-N (Millipore), and probed with a [ 32 P]dCTPlabeled cDNA spanning the open reading frame of group V sPLA 2 as described previously (34).
Reverse Transcription-PCR (RT-PCR)-Expression of transcripts for group V sPLA 2 was also analyzed by RT-PCR. One g of RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and the Advantage RT for PCR kit (Clontech) for 1 h at 42°C. Five l of the resulting reaction mixture were used in a PCR reaction with Taq Gold polymerase (PerkinElmer Life Sciences), using the following primers that span the open reading frame: forward primer, 5Ј-ACACTGGCTTGGTTCCTGGC-3Ј in exon II; and reverse primer, 5Ј-GACATTAGCAGAGAAGTTGGGG-3Ј in exon V. PCR conditions were as follows: a denaturing step at 95°C for 5 min; followed by 35 cycles of 95°C for 45 s, 65°C for 45 s, and 72°C for 90 s; followed by a final extension step at 72°C for 10 min. Primers for S15 ribosomal protein (Ambion, Austin, TX) were used as a positive control. Products were resolved on 2% agarose gels and visualized with ethidium bromide.
Immunofluorescence Analysis of Group V sPLA 2 Expression-The peritoneal cavities of mice were flushed with 5 ml of ice-cold PCG buffer (25 mM Pipes, 110 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 g/liter glucose, pH 7.4). After washing, 3.3 ϫ 10 5 cells were plated on glass coverslips in 24-well tissue culture plates in 500 l of PCG buffer, 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin and incubated overnight at 37°C with 5% CO 2 . Nonadherent cells were removed by washing three times with PCG buffer. Cells were fixed with 2% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS) for 15 min at room temperature, washed once in Hanks' balanced salt solution without Mg 2ϩ or Ca 2ϩ (HBSSϪ) containing 0.1% bovine serum albumin (HBA), permeabilized with 0.025% saponin (Sigma) in PBS for 10 min at room temperature, and washed twice with HBA. Macrophages were then blocked in HBA containing 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Cells were incubated with 5 g/ml rabbit anti-mouse group V sPLA 2 (32) in blocking buffer for 2 h at room temperature, washed extensively with HBA, and incubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (heavy and light chains; Jackson ImmunoResearch), diluted 1:400. Cells were washed five times with HBA, mounted in Vectashield™ mounting medium (Vector Laboratories, Burlingame, CA), and imaged with a Nikon Eclipse TE2000U microscope coupled with a Spot-RT digital camera.
Isolation and Activation of Mouse Peritoneal Macrophages-The peritoneal cavities of mice sacrificed by CO 2 inhalation were flushed with 5 ml of ice-cold Dulbecco's modified Eagle's medium (Life Sciences, Rockville, MD) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10 units/ml heparin. After two washes in HBSSϪ, 3.75 ϫ 10 5 cells in 500 l of Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin were placed in individual wells of a 48-well tissue culture plate and incubated for 3 h at 37°C with 5% CO 2 . Nonadherent cells were removed by washing with HBSSϪ. Nonadherent cells from each mouse were pooled and counted to determine the fraction of adherent cells, which did not differ between group V sPLA 2 -null cells and wild-type cells. Two hundred l of Dulbecco's modified Eagle's medium containing 0.1% human serum albumin were added to each well, and adherent cells were stimulated in a dose-and time-dependent manner with zymosan A (0 -100 particles/ cell; 0 -6 h) (35). Supernatants were collected for measurements of cysteinyl leukotrienes and PGE 2 by enzyme-linked immunoassays (Amersham Biosciences). In some experiments, supernatants were analyzed for cysteinyl leukotriene content by reverse phase high performance liquid chromatography (RP-HPLC).
Zymosan-induced Peritonitis-Each mouse received an injection of 0.5% Evans blue dye in PBS into the tail vein (100 g dye/g body weight) immediately before injection of 1 ml of zymosan A (1 mg/ml in PBS) into the peritoneal cavity (36). At selected times (up to 2 h later), the mice were euthanized by CO 2 inhalation, and the peritoneal cavity was lavaged with 4 ml of ice-cold PBS. The peritoneal lavage fluid was centrifuged at 500 ϫ g for 5 min, and the optical density of the supernatant was measured at 610 nm to assess extravasation of Evans blue dye. In some experiments, 4 volumes of ethanol and 60 ng of PGB 2 (as internal standard for RP-HPLC) were added to the lavage fluid, mixed well, incubated on ice for 30 min, and centrifuged at 10,000 ϫ g for 10 min at 4°C. The supernatants were then dried by vacuum centrifugation, resuspended in 50 mM HEPES (pH 7.6) and methanol (1:2, w/v), and analyzed for cysteinyl leukotriene content by RP-HPLC.
RP-HPLC Analysis of Cysteinyl Leukotrienes-Cysteinyl leukotrienes were measured by RP-HPLC as described previously (36,37). Briefly, samples were applied to a 5-m, 4.6 ϫ 250-mm C18 Ultrasphere RP column (Beckman) 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 solvent A and 45% methanol over 2.5 min. After 5 min, methanol was increased linearly to 75% over 15 min and maintained at this level for an additional 15 min. The UV absorbance at 280 nm was recorded. The retention times for PGB 2 , LTC 4 , LTD 4 , and LTE 4 were 21.4, 22.2, 23.7, and 25.1 min, respectively. Quantities of cysteinyl leukotrienes were calculated from the ratio of the peak area of each leukotriene to the peak area of the PGB 2 internal standard after correction for the differences in molar extinction coefficients.
Statistical Analyses-Data are presented as arithmetic means and S.E. Statistical differences between groups were calculated using Student's t test.

RESULTS AND DISCUSSION
Generation of Mice Lacking Group V sPLA 2 -To generate mice lacking group V sPLA 2 , we used targeted gene disruption. Southern blotting of wild-type genomic DNA for group V sPLA 2 yielded bands of ϳ5 and ϳ3.7 kb with NcoI (Fig. 1B) and EcoRI (data not shown) digestion, respectively. Southern blotting of DNA from animals with disruption of the group V sPLA 2 gene yielded bands 2.6 and 0.8 kb smaller than wild-type DNA after digestion with with NcoI (Fig. 1B) and EcoRI (data not shown), respectively, as predicted from restriction enzyme mapping of wild-type and mutated alleles (Fig. 1A). The mouse heart is rich in transcripts for group V sPLA 2 (8). To confirm that incorporation of the disrupted allele for group V sPLA 2 led to loss of normal transcripts, RNA was extracted from the hearts of mice born to the F 1 intercrosses and analyzed for group V sPLA 2 transcripts by RNA blotting (Fig. 1C) and RT-PCR (Fig.  1D). RNA from the hearts of wild-type mice and BALB/c mice yielded a band of ϳ2 kb on Northern analysis (Fig. 1C), whereas RNA from mice genotyped as having no wild-type allele for the group V sPLA 2 gene yielded only aberrant transcripts of ϳ4 kb and lacked a transcript of the appropriate size on Northern analysis (Fig. 1C). The finding that the aberrant transcripts observed in group V sPLA 2 -null hearts were nonproductive is revealed by RT-PCR. RT-PCR analysis of wildtype hearts yielded a product of the expected size (ϳ400 bp) and also yielded a smaller product that on sequencing was shown to lack exon IV, indicative of alternate splicing, as described previously for group V sPLA 2 (38). RT-PCR analysis of RNA from group V sPLA 2 -null hearts failed to yield a product in RT-PCR (Fig. 1D).
Immunofluorescence analysis (Fig. 2) confirmed the lack of expression of group V sPLA 2 in peritoneal macrophages from group V sPLA 2 -null mice. By comparison, peritoneal macrophages from wild-type littermates revealed cytoplasmic staining for group V sPLA 2 with perinuclear accentuation, similar to the pattern we reported previously for mouse bone marrow culture-derived mast cells (32).
Breeding of mice heterozygous for the disrupted allele of group V sPLA 2 produced approximately equal numbers of male and female mice with a Mendelian pattern of inheritance of the disrupted allele. Group V sPLA 2 -null mice were healthy to at least 6 months of age, indicating that group V sPLA 2 is not required for normal growth and development of mice.
Peritoneal macrophages have been used extensively to study eicosanoid generation (17-19, 39), and they express group V sPLA 2 (Fig. 2). As phagocytes, they are an integral part of the innate immune response. They respond to ingestion of zymosan particles, extracted from yeast, with robust eicosanoid generation in vitro (40 -42), and in vivo administration of zymosan to the peritoneal cavity elicits an inflammatory response with plasma exudation that is dependent in part on the release of cysteinyl leukotrienes acting through the cysteinyl leukotriene type 1 receptor (CysLT 1 R) (36,43). We therefore studied eicosanoid generation in peritoneal macrophages from group V sPLA 2 -null mice in vitro and cysteinyl leukotriene generation and plasma extravasation in response to intraperitoneal injection of zymosan in vivo.
Zymosan-induced Eicosanoid Generation by Mouse Peritoneal Macrophages-Stimulation of group V sPLA 2 -null and wild-type macrophages with unopsonized zymosan particles elicited maximum cysteinyl leukotriene and PGE 2 generation at 3 h (data not shown). Cysteinyl leukotriene generation plateaued at 30 -100 zymosan particles/cell, whereas PGE 2 generation plateaued with a maximal response at 3-10 particles/cell (Fig. 3, A and B). Both cysteinyl leukotriene generation and PGE 2 generation were attenuated at all doses of zymosan in group V sPLA 2 -null peritoneal macrophages compared with wild-type control cells, with no change in the position of the dose-response curves (Fig. 3, A and B). Cysteinyl leukotriene generation in response to 100 zymosan particles/cell was attenuated 58% in group V sPLA 2 -null macrophages (24.6 Ϯ 5.7 ng/10 6 cells compared with 57.4 Ϯ 10.7 ng/10 6 wild-type cells; n ϭ 9; p ϭ 0.01). PGE 2 generation in response to 3 zymosan particles/cell was attenuated 49% in group V sPLA 2 -null macrophages (7.1 Ϯ 1.6 ng/10 6 cells compared with 14.1 Ϯ 1.8 ng/10 6 wild-type cells; n ϭ 9; p ϭ 0.01). RP-HPLC analysis revealed the identity of the cysteinyl leukotrienes in the cell supernatants as LTC 4 , with no detectable extracellular conversion to LTD 4 or LTE 4 (data not shown), and confirmed the attenuation of LTC 4 generation in group V sPLA 2 -null macrophages compared with wild-type macrophages (Fig. 3C). Attenuation of eicosanoid generation in zymosan-stimulated group V sPLA 2 -null peritoneal macrophages was demonstrated for macrophages obtained from mice derived from two separate embryonic stem cell clones.
These data demonstrate a role for group V sPLA 2 in eicosanoid generation by zymosan-stimulated peritoneal macrophages. Nevertheless, there was still significant release of both LTC 4 and PGE 2 from group V sPLA 2 -null macrophages in response to zymosan. Previous work demonstrated complete abrogation of the release of arachidonic acid in response to zymosan in peritoneal macrophages derived from mice lacking cPLA 2 -␣ (19). Combined with our data, this suggests an absolute requirement for cPLA 2 -␣ in eicosanoid generation by zymosan-stimulated peritoneal macrophages and an amplifying role for group V sPLA 2 . This is consistent with the conclusions obtained in other cells. In HEK293 cells, the function of transfected group V sPLA 2 in supplying arachidonic acid for eicosanoid generation was abolished by pharmacological inhibition of cPLA 2 -␣ (22). In primary cultures of mouse mesangial cells, adenoviral transfection with group V sPLA 2 augmented arachidonic acid release in response to H 2 O 2 by cells obtained from cPLA 2 -␣-sufficient mice but not from cPLA 2 -␣-null mice (24). The precise mechanism by which group V sPLA 2 amplifies the essential function of cPLA 2 -␣ is unknown and is the subject of ongoing experiments.
The participation of group V sPLA 2 and its possible crosstalk with cPLA 2 -␣ in the release of arachidonic acid has been demonstrated in the mouse P388D 1 macrophage cell line. When these cells were labeled with [ 3 H]arachidonic acid for 5 h, primed with LPS for 1 h, and then activated with platelet-activating factor, there was a biphasic release of labeled arachidonic acid (25). In the first phase, which was complete within 2 min, arachidonic acid was released and retained within the cell, whereas in the second phase, extracellular release of labeled arachidonic acid occurred over the succeeding 10 min. Studies with pharmacological inhibitors and with antisense oligonucleotides demonstrated that the initial intracellular release of arachidonic acid was dependent upon cPLA 2 -␣, whereas the subsequent extracellular release was dependent upon group V sPLA 2 (26). The initial action of cPLA 2 -␣ appeared necessary for the cellular function of group V sPLA 2 (27). In certain subclones of the P388D 1 macrophage, LPS stimulation alone elicited a delayed phase of arachidonic acid release and prostaglandin endoperoxide synthase-2-dependent PGE 2 generation (28). Both cPLA 2 -␣ and group V sPLA 2 were required, but, in contrast to the primed-immediate response to LPS and PAF, activation of cPLA 2 -␣ led to induction of group V sPLA 2 expression, which in turn induced prostaglandin endoperoxide synthase-2. A third pathway of arachidonic acid release in P388D 1 macrophages was described in response to zymosan (44). Zymosan alone elicited the Ca 2ϩ -dependent activation of cPLA 2 -␣, which elicited an immediate and sustained release of arachidonic acid that was inhibited by methyl arachidonyl fluorophosphonate, an inhibitor of cPLA 2 -␣, and not by LY311717, an inhibitor of group V sPLA 2 (44). Priming with LPS augmented the response to zymosan, providing an increment in release of arachidonic acid that was inhibited by LY311717. Exogenous group V sPLA 2 also augmented the response to zymosan. By comparison, our findings indicate a role for group V sPLA 2 in peritoneal macrophages stimulated with zymosan alone in the absence of LPS priming. The differences between our data and those obtained with the P388D 1 cell line may be due to the transformed nature of P388D 1 cells or may relate to the observation that release of arachidonic acid in response to zymosan was only observed in the MAB clone of this cell line (44) and/or to the observation that P388D 1 cells have low levels of esterified arachidonic acid compared with primary cultures of mouse macrophages (45).
Zymosan-induced Peritonitis-Disruption of the genes encoding LTC 4 synthase (43) and of CysLT 1 R (36), revealed the contribution of the cysteinyl leukotrienes, acting through CysLT 1 R, to plasma exudation in zymosan-induced peritonitis. We therefore assessed cysteinyl leukotriene generation (by RP-HPLC) and plasma exudation (by leak of Evans blue dye) in group V sPLA 2 -null and wild-type mice in response to intraperitoneal injection of a suspension of zymosan A. There was a significant ϳ50% attenuation of the leak of Evans blue dye 15 and 30 min after injection of zymosan in mice lacking group V sPLA 2 compared with that observed in wild-type littermates (Fig. 4). No difference in plasma exudation was apparent at 60 and 120 min. We measured cysteinyl leukotrienes in the peritoneal lavage fluid 30 min after intraperitoneal injection of zymosan, when there was significant attenuation of plasma FIG. 2. Immunofluorescence staining for group V sPLA 2 . Peritoneal macrophages from group V sPLA 2 wild-type (A) and group V sPLA 2 -null (B) mice were fixed in 2% paraformaldehyde, permeabilized with 0.025% saponin, and stained with affinity-purified rabbit IgG anti-mouse group V sPLA 2 , followed by fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG. Nuclei were stained blue with Hoechst stain.

FIG. 3. Eicosanoid generation by peritoneal macrophages in response to zymosan particles in vitro.
Peritoneal macrophages from group V sPLA 2 -null (Ⅺ) and wild-type control mice (f) were stimulated for 3 h in a dose-dependent manner. Supernatants were analyzed by enzyme-linked immunoassays for cysteinyl leukotrienes (CysLT) (A) and for PGE 2 (B) (n ϭ 9) and analyzed for cysteinyl leukotriene generation by RP-HPLC (C) (n ϭ 6). ND, not detected; ϩ, p Ͻ 0.05; ‫,ء‬ p Ͻ 0.02; ‫,ءء‬ p Ͻ 0.01. extravasation. LTE 4 was the predominant cysteinyl leukotriene present in the lavage fluid, as reported previously (43), and its generation was attenuated in group V sPLA 2 -null animals compared with wild-type controls (Fig. 5).
The attenuation of enhanced vascular permeability in response to zymosan A in group V sPLA 2 -null mice at early time points but not at later time points is somewhat distinct from the findings in mice lacking LTC 4 synthase or CysLT 1 R (36,43). In the former, attenuated vascular permeability was observed up to 1 h after intraperitoneal injection of zymosan, and in the latter, it extended to 4 h. Nevertheless, the greatest attenuation of plasma exudation in both strains of mice was observed at early time points and was less marked at 2 and 4 h, consistent with our findings in group V sPLA 2 -null mice and indicating a significant role for other vasoactive mediators, as would be expected. Furthermore, the attenuation of cysteinyl leukotriene generation in group V sPLA 2 -null mice in vivo, as in vitro, was partial, allowing significant occupancy of CysLT 1 R as cysteinyl leukotriene generation proceeded, even in the face of attenuated leukotriene production.
Whereas our findings using targeted gene disruption clearly identify a role for group V sPLA 2 in eicosanoid generation by peritoneal macrophages and in leukotriene-dependent plasma exudation, the mechanisms of action of group V sPLA 2 are unclear. Our data, in the context of the existing literature, are consistent with a role for group V sPLA 2 in amplifying the response to cPLA 2 -␣ (22,24,27). Nevertheless, the nature of this interaction is poorly defined. It may involve secretion of group V sPLA 2 from the cell with subsequent internalization through binding to caveoli (22). It might be due to the capacity of group V sPLA 2 for interfacial binding to phosphatidylcholine, providing direct release of arachidonic acid from cell membrane phospholipids (46), or, as recently described for human eosinophils (47), group V sPLA 2 may be internalized for direct action at the perinuclear membrane. However, each of these postulated mechanisms of action requires the prior secretion of the enzyme and its subsequent internalization for action in an autocrine or a paracrine manner. Our immunofluorescence data indicate prominent perinuclear staining for group V sPLA 2 (Fig. 2). The perinuclear region is a prominent location for enzymes of eicosanoid biosynthesis, either constitutively (48,49) or after translocation in response to calcium flux (4,50,51). Thus, group V sPLA 2 may act intracellularly, without a requirement for prior secretion, to amplify the effects of translocated cPLA 2 -␣.
In summary, this is the first report of mice with targeted disruption of group V sPLA 2 . Our studies reveal the participation of the enzyme in zymosan-induced eicosanoid generation both in vitro and in vivo and suggest a role for the enzyme in acute innate immune responses. FIG. 5. Leukotriene generation in the peritoneal exudates of mice injected with zymosan A. Group V sPLA 2 -null (Ⅺ; n ϭ 3) and wild-type (f; n ϭ 4) mice were injected with 1 mg/ml zymosan A. The cysteinyl leukotriene content of the exudate was measured by RP-HPLC as described under "Experimental Procedures." Data are provided separately for LTC 4 , LTE 4 , and total cysteinyl leukotriene generation. No LTD 4 was detected. ‫,ء‬ p Ͻ 0.05.

FIG. 4. Plasma exudation in response to intraperitoneal injection of zymosan A.
Group V sPLA 2 -null (E) and wild-type (q) mice were injected with 1 mg/ml zymosan A, and plasma exudation was assessed by the leak of Evans blue dye as described under "Experimental Procedures." Data are reported as absorbance of the peritoneal exudates at 610 nm for individual mice (A) and as the means Ϯ S.E. for each group of mice (B). ‫,ء‬ p Ͻ 0.005.