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The Journal of Immunology, 2008, 181, 3558 -3566
Copyright © 2008 by The American Association of Immunologists, Inc.
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Modulation of Lipid and Protein Mediators of Inflammation by Cytosolic Phospholipase A2{alpha} during Experimental Sepsis1

Naonori Uozumi2, Yoshihiro Kita and Takao Shimizu3

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytosolic phospholipase A2{alpha} (cPLA2{alpha}) is one of the key enzymes in lipid mediator generation. It preferentially hydrolyzes arachidonoyl-phospholipid in response to cellular stimuli, liberating arachidonic acid, the shared precursor of PGs and leukotrienes. Mice with disruption of the cPLA2{alpha} gene exhibit a more than 80% decrease in the generation of these lipid mediators, leading to dramatic phenotypes in various models of inflammatory and allergic disease. In this study, we use the cecal ligation and puncture model of sepsis along with multiplex quantitation systems to explore interactions between eicosanoids and protein mediators. cPLA2{alpha}-deficient mice exhibited significantly less weight loss accompanied by decreased generation of PGs, leukotriene B4, IL-6, and CCL2. Despite these differences, genetic ablation of cPLA2{alpha} did not provide any survival advantage. Unexpectedly, abundant production of 12-hydroxy-eicosatetraenoic acid, another arachidonic acid-derived lipid mediator, was found to be unaffected by disruption of the cPLA2{alpha} gene. Eicosanoid production preceded the production of cytokines. Eicosanoid modulation of IL-6 and CCL2 expression was suggested by scattergram analyses. These results provide in vivo evidence for the rapid generation of eicosanoids, regulatory role(s) for cPLA2{alpha}-derived lipid mediators on protein mediator production, and the existence of a robust cPLA2{alpha}-independent pathway(s) of eicosanoid generation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a disease of systemic inflammatory response associated with infection (1). Its occurrence is rising, and the incidence rate for severe cases (i.e., sepsis with organ dysfunction) lies between 50 and 100 cases per 100,000 individuals in industrialized nations (2, 3). Although a gradual decrease in mortality rate has been achieved in recent decades, it still exceeds 30% according to recent reports (3). The pathogenesis of sepsis is thought to include an initial hyperinflammatory reaction evoked by infectious pathogens, followed by a hypo- or dysregulated inflammatory state (4, 5, 6). TLR and their ligands, various cytokines, and coagulation factors have all been identified as key molecules in this process, but our knowledge of sepsis pathogenesis remains incomplete. Cecal ligation and puncture (CLP)4 is an experimental procedure commonly used to model sepsis (7, 8, 9). In CLP, primary polymicrobial infectious foci cause septicemia, septic shock, and mortality.

Arachidonic acid metabolites, or eicosanoids, are a class of lipid mediators involved in inflammatory as well as other physiological processes (10, 11). PGs and leukotrienes (LT) are well characterized both in metabolic pathways and in signaling pathways through their respective cell surface G protein-coupled receptors (12, 13, 14, 15). They are generated from a single molecule of C20 polyunsaturated fatty acid, arachidonic acid, through multistep reactions catalyzed by oxygenases (cyclooxygenases (COX) and lipoxygenases (LO)), and terminal synthases. The rate-limiting step for eicosanoid generation is the availability of arachidonic acid. Phospholipase A2 (PLA2, EC 3.1.1.4) hydrolyzes membrane phospholipid at the sn-2 position to liberate fatty acid (16, 17). Among more than 20 mammalian PLA2s, cytosolic PLA2{alpha} (cPLA2{alpha}; official gene symbol, Pla2g4a) is unique in its substrate preference for arachidonoyl-phospholipid, and gene-targeted mice have demonstrated its critical contribution to eicosanoid production from cells and in disease models (18, 19, 20, 21, 22, 23). We have reported attenuated disease symptoms for cPLA2{alpha}-deficient mice in diverse models, including anaphylaxis (20), lung injury (24, 25), pulmonary embolism (26), arthritis (27), inflammatory bone resorption (28), experimental encephalomyelitis (29), and intestinal polyposis (30). We have also described an enhanced diabetic phenotype for cPLA2{alpha}-deficient mice in NOD (31), indicating that cPLA2{alpha} does not always promote disease.

In this study, we investigated the phenotype of cPLA2{alpha}-deficient mice in the CLP model of sepsis. We observed suppressed accumulation of IL-6 and CCL2 in the peritoneum and serum, and a smaller decrease in body weight after surgery, as symptoms of attenuated disease. Interestingly, these milder responses did not result in a survival advantage for cPLA2{alpha}-deficient mice. This contrasts to a recent report that 5-LO-deficient mice are protected from mortality in the CLP model (32). We conducted multiplex analyses of lipid mediators and cytokines in an attempt to obtain mechanistic insights into the disease phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6J mice were from CLEA Japan. Male cPLA2{alpha}-deficient (cPLA2{alpha}–/–) mice (20) had more than 12 generations of backcrosses to the C57BL/6J strain. cPLA2{alpha}–/– mice were obtained by crossing cPLA2{alpha} heterozygotes (cPLA2{alpha}+/–). Mouse genotype was determined by PCR, as described (20). When comparisons by cPLA2{alpha} genotype were attempted, wild-type (cPLA2{alpha}+/+) mice as well as cPLA2{alpha}–/– mice were prepared from cPLA2{alpha}+/– intercrosses. Commercially available C57BL/6J male mice (CLEA Japan) were used when comparisons among wild-type groups were intended. Animals were maintained in a light-dark cycle, with light from 7:00 a.m. to 8:00 p.m. at 22°C. Mice were fed with a standard laboratory diet and water ad libitum. All of the mice in this study were used under a protocol approved by the University of Tokyo Ethics Committee for Animal Experiments.

Cecal ligation and puncture

Male mice (20–30 g), anesthetized with sodium pentobarbital, 60 mg/kg, i.p., were given a 1- to 1.5-cm incision in the lower left abdominal wall. The cecum was exposed and ligated with a silk suture ~5 mm below the ileocecal valve, and punctured twice with a 23-G needle. The cecum was placed in the abdomen, and the incision was closed with silk suture by two layers. Mice were returned to their cages with free access to food and water. Survival and body weight were monitored daily. As controls for the CLP-operated groups, sham and ligation groups were prepared; sham-operated mice did not receive ligation or puncture, and ligation-operated mice were cecum ligated, but not punctured.

Serum and peritoneal lavage fluid collection

Mice were anesthetized with sodium pentobarbital, 60 mg/kg, i.p., and placed in the supine position. Blood, collected from incisions made on femoral vessels, was either allowed to clot at room temperature for 1 h to obtain serum, or introduced into a heparinized tube (Mini Collect; Funakoshi) for plasma preparation. Serum was recovered by centrifugation at 1000 rpm for 15 min at 4°C. Plasma was obtained as the supernatant from spinning at 3000 x g for 10 min at room temperature, following the manufacturer’s instructions. These samples were stored at –80°C until use. Once bleeding had ceased, peritoneal lavage was conducted with 5 ml of ice-cold Dulbecco’s PBS without calcium chloride and magnesium chloride (D(–)-PBS) (Sigma-Aldrich) supplemented with 2 mM EDTA. Aliquoted peritoneal lavage fluid was stored at –80°C until use in mediator quantitations. Cell pellets were hemolyzed with ACK lysing buffer (Lonza) and resuspended to 1 ml with D(–)-PBS and 2 mM EDTA. The total cell number was counted with a hemocytometer, and the differential count was made with a Cytospin slide: Cytospin 3 (Thermo Shandon; 3 x 104 cells, 300 rpm for 5 min), stained with Diff-Quik (International Reagents), and at least 300 cell counts in randomly selected areas.

Quantitation of mediators

The concentrations of inflammatory cytokines in serum and lavage samples were determined by a multiplex cytometric bead array (Mouse Inflammation CBA; BD Biosciences). This kit is designed to measure IL-6, IL-10, CCL2, IFN-{gamma}, TNF-{alpha}, and IL-12 p70. Lipid mediators in peritoneal lavage fluid were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as described (33), allowing for the simultaneous monitoring of 14 mediators, as follows: 5 PGs (PGD2, E2, F2{alpha}, I2 (as 6-keto-PGF1{alpha}), and thromboxane (Tx) A2 (as TxB2)), 3 LTs (LTB4, C4, and D4), 5 hydroxy-eicosatetraenoic acids (HETEs; 5-, 8-, 11-, 12-, and 15-HETE), and platelet-activating factor (PAF).

Pharmacological intervention

After 1.5 h of surgery, mice were given 5 µg of carbaprostacyclin (cPGI2; a stable analog of prostacyclin (PGI2) with an agonist activity; Cayman Chemical) or 1 µg of PGE2 (Cayman Chemical) by i.p. injection of 0.2 ml of saline (0.9% w/v NaCl) containing 5% (v/v) ethanol as vehicle.

Statistical analysis

Statistical calculations were performed with Prism-4 (GraphPad software). A value of p < 0.05 was considered to have statistical significance. The values for measurements were expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Survival and body weight changes after CLP surgery

Survival after CLP surgery was not altered by deletion of cPLA2{alpha} (Fig. 1A). With an impression that cPLA2{alpha}–/– mice had a better coat appearance during the first several days after surgery, body weight was monitored daily in a second experiment (Fig. 1B). A 9% reduction in body weight was observed in both genotypes in the first 24 h after CLP surgery. This decrease was considered to be nonspecific because sham-operated mice showed equivalent transient body weight decreases during the same time period, followed by quick recovery to the original body weight within 1 wk (data not shown). Over the next 24 h, cPLA2{alpha}+/+ mice lost an additional 6% (total 15%) of their original body weight compared with the cPLA2{alpha}–/– loss of additional 3% (total 9%) in that period. These differences became larger for days 3 and 4, and were statistically significant, as follows: p < 0.0001, two-way ANOVA for comparisons of two curves, and p < 0.01 and p < 0.05 for days 3 and 4, respectively, by Bonferroni’s posthoc test. Despite the smaller body weight losses in cPLA2{alpha}–/– mice, this set of mice did not show a survival advantage either (Fig. 1B, inset).


Figure 1
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  		FIGURE 1. Survival curves and body weight changes after CLP. A, Adult (14- to 17-wk-old) male cPLA2{alpha}+/+ (+/+, n = 24) and cPLA2{alpha}–/– (–/–, n = 18) mice were subjected to CLP operation, and survival was monitored daily for 15 days. No significant difference was observed by genotype (p = 0.69, log-rank test). B, Adult (14- to 15-wk-old) male cPLA2{alpha}+/+ (+/+, n = 7) and cPLA2{alpha}–/– (–/–, n = 11) mice were CLP operated and weighed daily. Significant differences were observed for days 4 and 5 (*, p < 0.05; {dagger}, p < 0.01; two-way ANOVA with Bonferroni’s post-test). Inset, Shows the survival curves for the same experimental groups (no difference by genotype, p = 0.27, log-rank test).

 
Serum cytokine concentration

Previous studies of CLP-operated mice noted inflammatory cytokine accumulations within hours after surgery with impacts on mortality of CLP-operated mice (7, 9, 34). We chose a 5-h time point at which to measure inflammatory cytokine concentrations in sera using a multiplex cytometric bead array. IL-6 and CCL2 were the two most abundant mediators observed, followed by TNF-{alpha} (Fig. 2). The remaining three cytokines (IFN-{gamma}, IL-10, and IL-12p70) were under the detection limits for the majority of serum samples, and were excluded from further analyses. IL-6 concentrations increased in a stepwise manner in cPLA2{alpha}+/+ mice, indicating that cecal ligation alone evoked inflammatory responses and that perforation of the cecum wall and subsequent leakage of its content further enhanced the response (Fig. 2A). cPLA2{alpha}–/– mice showed similar IL-6 responses to ligation. Additional responses to cecal puncture appeared suppressed in the cPLA2{alpha}–/– group, resulting in a statistically significant difference by cPLA2{alpha} genotype, as follows: 18.7 ± 3.6 ng/ml in the cPLA2{alpha}+/+ group (n = 20, mean ± SEM), and 11.5 ± 1.5 ng/ml in the cPLA2{alpha}–/– group (n = 15, mean ± SEM), p < 0.05, ANOVA, with Newman-Keuls’ multiple comparison test for pairwise comparison (Fig. 2A). Upon cecal ligation, CCL2 concentrations increased to a similar extent in both genotypes of mice. Although cecal perforation in cPLA2{alpha}+/+ mice did not cause marked changes in serum CCL2 concentrations, cPLA2{alpha}–/– mice showed a decreasing trend (Fig. 2B). TNF-{alpha} concentrations were mostly of the same patterns in both cPLA2{alpha}+/+ and cPLA2{alpha}–/– mice (Fig. 2C). Cecal puncture did not induce any further increase in TNF-{alpha} concentrations compared with ligation groups for both genotypes.


Figure 2
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  		FIGURE 2. Serum cytokine concentrations after 5 h of surgery. Adult (14- to 37-wk-old) male cPLA2{alpha}+/+ (+/+, n = 18, 19, and 20) and cPLA2{alpha}–/– (–/–, n = 9, 14, and 15) mice were subjected to sham, ligation, or CLP operation, and blood was collected 5 h after surgery. A, IL-6; B, CCL2; and C, TNF-{alpha}. *, p < 0.05; {dagger}, p < 0.01; and {ddagger}, p < 0.001; ANOVA with Neuman-Keuls’ multiple comparison test for pairwise comparisons (A and B), and Kruskal-Wallis test with Dunn’s multiple comparison test for pairwise comparisons (C).

 
Peritoneal lavage fluid cytokine concentration

To test whether the observed changes in inflammatory cytokine concentrations were restricted to serum samples, we examined peritoneal lavage fluid at the same time point as above, i.e., 5 h after surgery. Concentrations of peritoneal lavage fluid IL-6 showed almost the same pattern as that of serum IL-6 (Fig. 3A). cPLA2{alpha}–/– mice responded in a similar manner to cecal ligation compared with cPLA2{alpha}+/+ mice. When the cecum was punctured, cPLA2{alpha}–/– mice stayed at the equivalent average concentrations of IL-6 (11.7 ± 1.8 and 12.0 ± 2.8 ng/ml for ligation and CLP groups, respectively, mean ± SEM, n = 8 both groups), in contrast to cPLA2{alpha}+/+ mice, whose IL-6 increased (10.8 ± 1.8 and 20.6 ± 3.4 ng/ml for ligation and CLP groups, mean ± SEM, n = 12 and 13, respectively,). CLP-operated cPLA2{alpha}+/+ mice contained a statistically significantly higher concentration of IL-6 compared with the ligation group of the same genotype and with CLP-operated cPLA2{alpha}–/– mice (p < 0.05, ANOVA with Newman-Keuls’ multiple comparison test for pairwise comparison). We did not detect differences between ligation groups. The pattern of lavage CCL2 concentrations mirrored that of lavage IL-6; statistical significance was limited to comparison of sham-operated and CLP-operated cPLA2{alpha}+/+ mice (Fig. 3B). Concentrations of CCL2 in cPLA2{alpha}–/– peritoneal lavage fluid for ligation-operated and CLP-operated groups were close to those of the cPLA2{alpha}+/+ ligation group (2.9 ± 0.3, 2.7 ± 0.5, and 2.9 ± 0.4 ng/ml for ligation and CLP groups of cPLA2{alpha}–/– and ligation group of cPLA2{alpha}+/+, mean ± SEM, n = 8, 8, and 12, respectively). Only the CLP-operated cPLA2{alpha}+/+ group showed an elevated CCL2 concentration (4.4 ± 0.6 ng/ml, mean ± SEM, n = 13). TNF-{alpha}, IFN-{gamma}, IL-10, and IL-12p70 were excluded from analyses because of rare occurrence of these molecules above the lower detection limit.


Figure 3
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  		FIGURE 3. Peritoneal lavage fluid cytokine and eicosanoid concentrations after 5 h of surgery. Adult (14- to 37-wk-old) male cPLA2{alpha}+/+ (+/+, n = 11, 12, and 13) and cPLA2{alpha}–/– (–/–, n = 4, 8, and 8) mice were subjected to sham, ligation, or CLP operation, and the peritoneum was lavaged with 5 ml of ice-cold D(–)-PBS supplemented with 2 mM EDTA at 5 h after surgery. A, IL-6; B, CCL2; C, 6-keto-PGF1{alpha}; D, 12-HETE; E, PGE2; F, LTB4. *, p < 0.05; {dagger}, p < 0.01; and {ddagger}, p < 0.001; ANOVA with Neuman-Keuls’ multiple comparison test for pairwise comparisons (A–E), and Kruskal-Wallis test with Dunn’s multiple comparison test for pairwise comparisons (F).

 
Peritoneal lavage fluid lipid mediator concentration

The dominant contribution of cPLA2{alpha} to eicosanoid generation is well documented (18, 19). Using a LC-MS/MS device, we determined lipid mediator concentrations in the same peritoneal lavage fluid, as described above (33). Among the 14 mediators analyzed, PGI2 (detected as 6-keto-PGF1{alpha}, the stable natural derivative of PGI2) and 12-HETE were the most abundant molecular species in CLP-operated cPLA2{alpha}+/+ mice, followed by PGE2 (Fig. 3, C–E). LTB4 was the major LT detected in peritoneal lavage (Fig. 3F). Concentrations of 6-keto-PGF1{alpha} for cPLA2{alpha}+/+ mice showed a stepwise pattern similar to those for serum and peritoneal lavage IL-6 concentrations (Figs. 2A and 3, A and C). cPLA2{alpha}–/– mice generated 14–19% of the amount of 6-keto-PGF1{alpha} of cPLA2{alpha}+/+ mice (Fig. 3C). Significant differences were detected even for comparison of ligation groups (3.4 ± 0.3 and 0.6 ± 0.1 ng/ml for cPLA2{alpha}+/+ and cPLA2{alpha}–/– groups, mean ± SEM, n = 12 and 8, respectively, p < 0.01, ANOVA with Newman-Keuls’ multiple comparison test for pairwise comparison), as well as between CLP groups (5.2 ± 1.0 and 0.8 ± 0.1 ng/ml for cPLA2{alpha}+/+ and cPLA2{alpha}–/– groups, mean ± SEM, n = 12 and 8, respectively, p < 0.001, ANOVA with Newman-Keuls’ multiple comparison test for pairwise comparison). This differs from the observation for IL-6 and CCL2, in which no differences by genotypes were detected between ligation groups. Interestingly, 12-HETE showed a distinct pattern (Fig. 3D). No significant differences were observed for any comparisons by genotype or surgical operation. PGE2 behaved in a similar pattern as 6-keto-PGF1{alpha}, i.e., a stepwise increase upon ligation and cecal puncture on cPLA2{alpha}+/+ mice, and a more than 80% decrease upon cPLA2{alpha} gene disruption (Fig. 3E). The absolute values of PGE2 were smaller than those of 6-keto-PGF1{alpha} by a factor of ~1/6. LTB4 was detected in abundance in cPLA2{alpha}+/+ mice when the cecum was ligated and punctured, but not when the cecum was only ligated. The concentrations of LTB4 from cPLA2{alpha}–/– mice stayed near the lower limit of detection, 40 pg/ml, for all three groups (Fig. 3F). Results for the remaining lipid mediators were shown as Table I.


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  		Table I. Peritoneal lavage fluid eicosanoid concentrations after 5 h of surgerya

 
In summary, cPLA2{alpha}–/– mice showed attenuated generation of lipid and protein inflammatory mediators, but no survival advantage compared with cPLA2{alpha}+/+ mice.

Mediator quantitation from an earlier time point

Having observed robust accumulations of both cytokines and lipid mediators in the peritoneum at 5 h after ligation and CLP surgery, we sought to assay the differential timelines of lipid and protein mediators. We examined 1.5-h samples from C57BL/6J male mice, seven mice for each surgical group. LC-MS/MS analysis of peritoneal lavage samples revealed accumulation of 6-keto-PGF1{alpha}, 12-HETE, and PGE2 to levels comparable to those seen 5 h after surgery (Fig. 4 and Table II). However, inflammatory cytokines were undetectable or under reliable ranges for quantitation for all six cytokines in either peritoneal lavage or serum samples. These findings indicate that lipid mediators are generated earlier than inflammatory cytokines in the peritoneum. LTB4 rarely (one and two of seven mice for ligation-operated and CLP-operated groups gave reliable measurement, respectively) exceeded the quantitation limit (40 pg/ml) at the 1.5-h time point (Table II), suggesting that production of LTB4 starts later than other eicosanoids.


Figure 4
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  		FIGURE 4. Peritoneal lavage fluid eicosanoid concentrations 1.5 h after surgery. Male C57BL/6J (n = 7 for each group, 8- to 9-wk-old) mice were subjected to sham, ligation, or CLP operation, and the peritoneum was lavaged with 5 ml of ice-cold D(–)-PBS supplemented with 2 mM EDTA at 1.5 h after surgery. A, 6-keto-PGF1{alpha}; B, 12-HETE; and C, PGE2. *, p < 0.05; {dagger}, p < 0.01; ANOVA with Neuman-Keuls’ multiple comparison test for pairwise comparisons.

 

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  		Table II. Peritoneal lavage fluid eicosanoid concentrations 1.5 h after surgerya

 
Plasma 12-HETE concentration

The distinct pattern of peritoneal lavage 12-HETE compared with other mediators at 5 h after surgery prompted us to ask whether this pattern is preserved in plasma. The average 12-HETE concentrations were found to be 100–300 ng/ml, with no statistically significant differences (p = 0.29, ANOVA, n = 7 each group; Table III). Basal 12-HETE concentrations in plasma and in serum of wild-type mice were determined in parallel to be lower than 10 ng/ml and higher than 3.5 µg/ml, respectively (n = 3).


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  		Table III. Plasma 12-HETE concentration after 5 h of surgerya

 
Inflammatory cell infiltration into peritoneal cavity

Some eicosanoids, such as LTs, are known for strong chemoattractivity. Although one possible mechanism underlying decreased inflammatory cytokine accumulations in cPLA2{alpha}–/– mouse is changes in infiltrating cell populations into the site of inflammation, neither cellularity, nor differential counts of peritoneal exudates collected at 5 h after surgery was found to be significantly different by either genotype or treatment (Table IV).


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  		Table IV. Inflammatory cell counts of peritoneal lavage fluid after 5 h of surgerya

 
Pharmacological intervention

The results described above suggested a model in which lipid mediators modulate responses of the whole body directly and/or indirectly through cytokine production. The 5-LO-deficient mice were protected from CLP-induced death (32), and cPLA2{alpha}–/– mice could be regarded as mice with both 5-LO and COX pathway deficiency given their attenuated productions of both LTs and PGs. Complementation experiments were conducted where we treated CLP-operated mice i.p. with 5 µg of cPGI2, a stable PGI2 analog, or 1 µg of PGE2, at 1.5 h after surgery. Smaller body weight losses for cPLA2{alpha}–/– mice were reproduced, as described earlier, after 2 days of surgery (Fig. 5, A and B). Among the same genotype groups, neither cPGI2 treatment nor PGE2 treatment had any effect on body weight changes for 2 wk (Fig. 5, A and B). We could not find significant effects on survival for any combination of genotypes and treatments (Fig. 5, C and D).


Figure 5
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  		FIGURE 5. Body weight changes and survival curves of mediator-treated mice. Adult (8- to 16-wk-old) male mice were subjected to CLP operation and i.p. injected with 5 µg of cPGI2, 1 µg of PGE2, or vehicle (0.5% v/v ethanol in saline) in a volume of 0.2 ml after 1.5 h of surgery. Body weight changes (A and B) and survival (C and D) were monitored daily until 14 days after surgery. A and C, cPLA2{alpha}+/+ mice (n = 12, 19, and 13 for vehicle, cPGI2, and PGE2 treatment, respectively). B and D, cPLA2{alpha}–/– mice (n = 15, 16, and 15 for vehicle, cPGI2, and PGE2 treatment, respectively). No significant differences were observed for any combination of treatment for both genotypes on body weight changes (two-way ANOVA) and survival curves (log-rank test).

 
Scattergram

As described in Materials and Methods, peritoneal lavage was preceded by blood collection. Thus, each mouse used to generate Fig. 3 has corresponding data points in Fig. 2. Scattergrams were plotted to find correlations between the concentrations of major mediators (Fig. 6). Serum IL-6 and CCL2 showed a moderate linear correlation (R2 = 0.440, all mice combined, n = 75) that appeared more profound for cPLA2{alpha}+/+ mice (R2 = 0.529, cPLA2{alpha}+/+, n = 46, and R2 = 0.234, cPLA2{alpha}–/–, n = 29; Fig. 6A). The correlation was more evident for lavage IL-6 and CCL2 concentrations (R2 = 0.866, all mice combined, n = 54), and this linear correlation was not affected by cPLA2{alpha} genotype (R2 = 0.862, cPLA2{alpha}+/+, n = 34, and R2 = 0.875, cPLA2{alpha}–/–, n = 20; Fig. 6B). Concentrations of 6-keto-PGF1{alpha} and PGE2 in peritoneal lavage had the strongest linear correlations, less so in cPLA2{alpha}–/– mice (R2 = 0.881, cPLA2{alpha}+/+, n = 36, and R2 = 0.171, cPLA2{alpha}–/–, n = 18; Fig. 6, C and D). When serum mediator concentrations were plotted against peritoneal lavage IL-6 concentrations (Fig. 6, E and F), they showed a different trend from those between serum (Fig. 6A) and lavage (Fig. 6B) IL-6 and CCL2. Correlations between IL-6 concentrations appeared to have a higher-order relationship rather than a linear one. Although bar graphs of lavage IL-6 and 6-keto-PGF1{alpha} from cPLA2{alpha}+/+ mice shown in Fig. 3, A and C, had certain similarities, the plot for these two mediators turned out to have only a modest linear correlation (R2 = 0.469, cPLA2{alpha}+/+, n = 36; Fig. 6G). We could not find any apparent correlations for 12-HETE concentrations with any of the mediators quantitated in this study (data not shown).


Figure 6
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  		FIGURE 6. Scattergram. The data set described in Figs. 2 and 3 was replotted to make scattergrams. Lipid mediator concentrations are of peritoneal lavage fluid. A data point was excluded from the panel, when the concentration of either mediator for that data point was below the lower detection limit: 0.32 ng/ml for IL-6 and CCL2, 0.08 ng/ml for lipid mediators; dilution factor of 1/4 for the measurement taken into account. A and B, Correlations between IL-16 and CCL2 concentrations (A, serum; B, peritoneal lavage fluid). C and D, Correlations between peritoneal lavage fluid 6-keto-PGF1{alpha} and PGE2 concentrations; cPLA2{alpha}–/– data points are replotted in D from C. E–G, Correlations between peritoneal lavage IL-6 concentration and serum cytokine concentrations (E and F) and peritoneal lavage fluid 6-keto PGF1{alpha} concentration (G).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Lipid mediators generated through cPLA2{alpha} are undoubtedly involved in inflammatory and allergic responses. Previous studies demonstrated a marked reduction in disease in cPLA2{alpha}–/– mice for various inflammatory and allergic disease models, as follows: anaphylaxis (20), brain infarction (21), acute lung injury (24), lung fibrosis (25), arthritis (27), inflammatory bone resorption (28), and experimental encephalomyelitis (29). This strongly suggests that cPLA2{alpha} is one of the key molecules in immunological pathophysiology. In this study of experimental sepsis, we demonstrated attenuated inflammatory responses in cPLA2{alpha}–/– mice. Accumulations of IL-6 and CCL2 in the peritoneum and serum were significantly suppressed, and smaller body weight losses were observed compared with cPLA2{alpha}+/+ mice. The amounts of PGs and LTB4 in the peritoneum also showed marked decreases relevant to the lack of cPLA2{alpha} activity. One might expect better prognosis of cPLA2{alpha}–/– mice for all of these circumstances; however, we could not find any differences in the survival curves by cPLA2{alpha} genotype (Figs. 1–3).

The diverse eicosanoids include products with counteracting activity in many systems, including the following: 1) pro- and anti-aggregatory effects on platelets by TxA2 and PGI2 (26, 35); 2) sleep/awake regulation by PGD2/PGE2 (36); 3) LTs and PGs in asthma (11, 14, 37, 38, 39, 40, 41); and 4) PGI2 and cysteinyl LTs in bleomycin-induced lung injury (25, 42, 43, 44, 45, 46, 47). Products of the 5-LO pathway are thought to have proinflammatory functions in murine CLP (32, 48). The 5-LO-deficient mice are protected against CLP-induced death, and cysteinyl LTs (the collective name for LTC4, D4, and E4) were pharmacologically suggested to be key mediators of CLP lethality acting through effects on vascular permeability (32). Mice deficient in BLT1, the high-affinity receptor for LTB4, were slightly protected against CLP-induced death (48). We noticed dramatic decreases in LTB4 accumulation in the peritoneal lavage fluid of CLP-operated cPLA2{alpha}–/– mice (Fig. 3F). Although the peritoneum did not exhibit marked accumulation of cysteinyl LT in CLP, it seems reasonable to presume decreased production of cysteinyl LTs in cPLA2{alpha}–/– mice as well, because we have documented marked decreases in cysteinyl LT accumulations in bronchoalveolar lavage fluid upon acute lung injury (24) and in bleomycin-induced lung fibrosis models (25). At a cellular level, multiple reports, including ours, have described diminished production of cysteinyl LTs from stimulated macrophages and mast cells (20, 21, 49, 50, 51). Hypothesizing a counteraction model in CLP, we suggest that favorable effects on survival, given decreased 5-LO product(s) in cPLA2{alpha}–/– mice, are cancelled out by lack of another eicosanoid(s) at the same time. LC-MS/MS analyses of peritoneal lavage fluid on 14 different lipid mediators indicated that two COX pathway products, 6-keto-PGF1{alpha}, the stable metabolite of PGI2, and PGE2, were abundantly accumulated in the lavage fluid, and that more than 80% of their productions was mediated by cPLA2{alpha} (Fig. 3, C and E). We attempted to demonstrate roles for these PGs by treating mice with cPGI2, a stable PGI2 analog, or PGE2 1.5 h after the surgery, but detected no differences in body weight changes or survival by the treatment (Fig. 5). We could not find any effects on cytokine production using C57BL/6J male mice treated with cPGI2 or PGE2 following ligation or CLP operation (data not shown). A previous pharmacological study with a selective inhibitor of COX-2 found improvement for early survival in murine endotoxemia, but not in the CLP model (52). Candidate fatty acid-derived lipid mediators on the protective side include arachidonic acid-derived lipoxins, and eicosapentaenoic acid- and docosahexaenoic acid-derived resolvins and protectins, which have anti-inflammatory functions (53, 54, 55, 56). Considering strong antimicrobial effector functions of LTB4 (57), lack of LTB4 production in cPLA2{alpha}–/– mice might have resulted in insufficient bacterial clearance masking the favorable aspects of decreased LT production. Further studies are needed to determine whether this counterregulatory model of lipid mediators holds for CLP and sepsis.

We can consider another counterregulatory model at the level of cytokines. Both IL-6 and CCL2 showed decreases for CLP-operated cPLA2{alpha}–/– mice compared with their genotype control group (Figs. 2 and 3). IL-6 concentration is generally believed to reflect the strength of inflammatory responses in CLP-operated mice. Higher concentrations of plasma IL-6 after 6 h of CLP surgery were shown to be indicative of early mortality in female BALB/c mice (34). In contrast, CCL2 has been demonstrated to have anti-inflammatory roles in CLP by the use of neutralizing Ab (58, 59, 60) and CCL2-deficient mice (61); mice with deficient or blocked CCL2 functions were more susceptible to CLP-induced mortality. Decreases in both IL-6 and CCL2 responses in CLP-operated cPLA2{alpha}–/– mice might have functionally cancelled each other out. The concentrations of IL-6 and CCL2 in ligation-operated mice did not appear to have any differences by cPLA2{alpha} genotype. When cPLA2{alpha}–/– mice were CLP operated, they showed small changes in the average concentrations of these mediators compared with the ligation group in contrast to cPLA2{alpha}+/+ mice (Figs. 2 and 3). This suggests that IL-6 and CCL2 responses can be divided into two mechanisms, as follows: a cPLA2{alpha}-independent ligation-induced mechanism, and a cPLA2{alpha}-dependent puncture-induced mechanism. The latter might be explained by the ability of LTB4 to transcriptionally activate both IL-6 and CCL2 expression, which was shown with cultured human peripheral blood monocytes (62, 63). Of interest is that LTB4, IL-6, and CCL2 are detected from 5-h but not in the 1.5-h harvests, unlike certain PGs and 12-HETE, which were abundantly detected starting at the earlier time point. Specific detection of LTB4 in the CLP-operated cPLA2{alpha}+/+ mice (Fig. 3F) suggests the existence of a puncture-specific mechanism for lipid mediator generation. This might be related to specific cell population(s), although the current study failed to detect any differences in infiltrating inflammatory cell numbers (Table IV).

TNF-{alpha} is one of the critical mediators for CLP and sepsis (64). At cellular levels, both resident (31) and proteose peptone-elicited (data not shown) peritoneal macrophages produce more TNF-{alpha} by LPS stimulation compared with genotype controls. Lipid mediators suggested to be responsible for this modulation of TNF-{alpha} are PGE2, and PGI2 acting through respective EP2 or EP4, and IP G protein-coupled receptor on the cell surface (65, 66). This comprises an example of a negative regulatory role for lipid mediators on cytokine generation, which might have affected the CLP phenotype in cPLA2{alpha}–/– mice, although we could not detect any changes in the TNF-{alpha} production profile by cPLA2{alpha} genotype in this study (Fig. 2C).

The 12-HETE was unexpectedly revealed to be one of the most abundant eicosanoids in peritoneal lavage fluid (Fig. 3D). Its concentration was in the same range as 6-keto-PGF1{alpha}, and exceeded those of PGE2 and LTB4 by 5- to 10-fold. Notably, 12-HETE concentrations did not show a stepwise pattern upon ligation and puncture of cecum as observed for other mediators in peritoneal lavage fluid and plasma (Table III). Furthermore, cPLA2{alpha} genotype did not affect 12-HETE accumulation. Murine serum contains µg/ml levels of 12-HETE, and we have previously reported that serum from cPLA2{alpha}–/– mice contained significantly less 12-HETE than from wild-type mice in two genetic backgrounds (C57BL/6J and C3H/HeN) (26). This suggests that 12-HETE production in CLP is regulated by a distinct cPLA2{alpha}-independent mechanism(s) apart from platelet-derived 12-HETE accumulation in serum, which is mostly cPLA2{alpha} dependent. The physiological role of 12-HETE, 12-LO, and 12/15-LO products is documented for platelet aggregation (67), cancer metastasis (68), atherosclerosis (69, 70), and neuronal plasticity (71), but is not known for sepsis and CLP. The functions of this abundant lipid mediator, as well as the PLA2 molecule(s) responsible for its production, remain to be elucidated.

The lack of differences in infiltrating inflammatory cell populations by cPLA2{alpha} genotype (Table IV) stands apart from previous observations of inflammatory disease model studies, in which marked decreases in infiltrating inflammatory cell numbers were associated with attenuated disease phenotypes (24, 25, 27, 29). We previously reported no significant differences in either cellularity or differential counts for casein-induced inflammatory exudate cells recovered from the peritoneal cavity of cPLA2{alpha}–/– mice compared with cPLA2{alpha}+/+ controls at 4–5 h after i.p. casein injection (72). The degree of eicosanoid contribution to inflammatory cell infiltration appears to differ depending on triggers and/or locations of inflammation.

Scattergram analysis (Fig. 6) is useful to unveil shared regulatory mechanisms for pairs of parameters (73). Linear correlations were best detected for two pairs of mediators: lavage IL-6 and CCL2 (Fig. 6B), and 6-keto-PGF1{alpha} and PGE2 (Fig. 6C). It is tempting to think that correlated mediators are produced by a shared set of cells using common transcription factors and/or metabolic enzymes. Identification of such putative cell types and determination of stimuli and signaling pathways that produce correlated amounts of mediators should greatly advance our knowledge. Serum IL-6 and CCL2 concentrations (Fig. 6A) were less linearly correlated than those of peritoneal lavage fluid (Fig. 6B), observed as smaller R2 values for the former. Much less linear correlations were observed for lavage IL-6 concentrations and serum cytokine and peritoneal lavage 6-keto-PGF1{alpha} concentrations (Fig. 6, E–G). These weaker correlations suggest function(s) of undefined modulating factors for production of these mediators.

The production of multiple lipid mediators in various cell and tissue types is regulated by cPLA2{alpha} activity. One should not assume that cPLA2{alpha} deficiency leads in only one direction, either attenuated or enhanced disease. The attenuated phenotypes of cPLA2{alpha}-deficient mice do not simply indicate one-sided effects of lipid mediators, but rather the dominance of the prosymptomatic side of lipid mediators in the wild-type animals. One can find reports of adverse effects of cPLA2{alpha} deficiency, as follows: intestinal ulcers (30), the diabetic model in NOD background (31), hypoxic pulmonary vasoconstriction (74), renal functions (75), and female reproduction (20, 21, 76). Not only mouse models, but an inherited human cPLA2{alpha} deficiency, reported recently, was associated with impaired eicosanoid biosyntheis, small intestinal ulceration, and platelet dysfunction (77). We do not yet have a global view of eicosanoid functions, because of their multiplicity of active molecular species. Another complexity for lipid mediator functions arises from molecular diversity of mammalian PLA2. Six gene loci were identified for the mammalian cPLA2 (18, 19, 78, 79), and together with secretory PLA2 (sPLA2), calcium-independent PLA2, and PAF acetyl hydrolase categories, they form a large PLA2 family of more than 20 members (16, 17). As we noted for 12-HETE, cPLA2{alpha} does not always contribute to the major part of eicosanoid generation. Is 12-HETE in CLP-operated mice an exception? Many sPLA2s have been claimed to be involved in lipid mediator generation, but little compelling in vivo evidence has been provided to date (17). A recent study using group X sPLA2 (official gene symbol, Pla2g10)-deficient mice demonstrated profound prodisease roles for this sPLA2 in experimental asthma models (80). It seems unlikely that simple inhibition of one specific PLA2 would be of significant benefit to septic patients. Indeed, clinical trials in sepsis targeting two PLA2 molecules (group IIA sPLA2 (official gene symbol, PLA2G2A) and secretory PAF acetylhydrolase (official gene symbol, PLA2G7)) failed (81, 82, 83). Still, we believe that lipid mediators are good therapeutic targets in sepsis for two reasons. First, they are a class of mediators distinct from cytokines and coagulation factors, their production having a distinct regulatory mechanism. Second, they function interactively and specifically with other classes of mediators. Involvement of lipid mediators in various aspects of sepsis pathophysiology is expected, and multiplex approaches such as those described in this study will be powerful tools for uncovering the underlying mechanisms.


   Acknowledgments
 
We thank T. Yokomizo, S. Ishii, and M. Taniguchi for discussion, and C. L. Karp for discussion and critical reading of the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.S. and N.U.), and a grant from Ono Medical Research Foundation (to N.U.). Back

2 Current address: Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. Back

3 Address correspondence and reprint requests to Dr. Takao Shimizu, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail address: tshimizu{at}m.u-tokyo.ac.jp Back

4 Abbreviations used in this paper: CLP, cecal ligation and puncture; COX, cyclooxygenase; cPGI2, carbaprostacyclin; cPLA2, cytosolic phospholipase A2; HETE, hydroxy-eicosatetraenoic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LO, lipoxygenase; LT, leukotriene; PGI2, prostacyclin; PLA2, phospholipase A2; sPLA2, secretory PLA2; D(–)-PBS, Dulbecco’s PBS without calcium chloride and magnesium chloride; PAF, platelet-activating factor; Tx, thromboxane. Back

Received for publication August 13, 2007. Accepted for publication June 27, 2008.


   References
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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