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Volume 272, Number 52, Issue of December 26, 1997 pp. 32792-32797

Resistance to Endotoxic Shock in Phospholipase A2 Receptor-deficient Mice*

(Received for publication, September 16, 1997, and in revised form, October 9, 1997)

Kohji Hanasaki Dagger , Yasunori Yokota , Jun Ishizaki , Takeshi Itoh and Hitoshi Arita

From the Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammals possess various types of secretory phospholipase A2, which differ in the primary structure and tissue distribution. The phosholipase A2 receptor (PLA2R) recognizes group IB phospholipase A2 (PLA2-IB) and mediates the PLA2-IB-induced biological responses in non-digestive organs, including eicosanoid production and contraction of airway smooth muscles. In this study, we generated PLA2R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA2-IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA2R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA2-IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-alpha and interleukin-1beta elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA2R in the progression of endotoxic shock.

INTRODUCTION

Phospholipase A2 (PLA2)1 comprises a diverse family of enzymes that cleave the sn-2 fatty acyl ester bond of glycerophospholipids to yield a free fatty acid and a lysophospholipid (1, 2). The group IB and group IIA PLA2s (PLA2-IB and PLA2-IIA) are two sets of enzymes in a highly conserved family of secretory PLA2 (sPLA2) found in mammals (3-5), which have a number of features different from the other major PLA2 families such as group IV PLA2, including a relatively low molecular mass (13-15 kDa), high disulfide bond content, and a requirement for relatively high concentrations of Ca2+ for catalysis (5, 6). PLA2-IIA is found in many cells and tissues and its expression is modulated by various inflammatory cytokines (4). Since local and systemic levels of PLA2-IIA are elevated in numerous inflammatory conditions such as sepsis, PLA2-IIA has been thought to play pivotal roles in the pathogenesis and/or progression of inflammation (4). PLA2-IB, on the other hand, is abundant in pancreatic juice in many mammals, and thus is frequently referred to as pancreatic type PLA2 (7). PLA2-IB is produced as an inactive pro-enzyme (pro-PLA2-IB) and activated by proteolytic enzymes such as trypsin and plasmin (8, 9). The major physiological function of PLA2-IB has been thought to be digestion of glycerophospholipids in nutrients, given its abundance in digestive organs (7). However, significant quantities of message and protein levels of this enzyme are found in non-digestive organs including the lung, spleen, kidney, and ovary (10), thus prompting us to identify novel biological functions of PLA2-IB exerted through its specific receptor, the PLA2-receptor (PLA2R) (11).

PLA2R is a type I transmembrane glycoprotein of 180-200 kDa (12), and is present in a wide variety of cells and tissues in mouse, rabbit, and human (13-15). It is composed of a large extracellular N-terminal portion, consisting of a N-terminal cystein-rich region, a fibronectin-like type II domain, a tandem repeat of eight carbohydrate-recognition domains essential for ligand binding, and short intracellular C-terminal region. Its overall molecular organization is related to a unique member of the C-type animal lectin family such as the macrophage mannose receptor (16) and DEC-205 in dendritic cells (17), both of which mediate the endocytosis of glycosylated complexes through the carbohydrate-recognition domain structures. Murine PLA2R recognizes an active form of PLA2-IB with a binding affinity of about 1-5 nM, but does not bind pro-PLA2-IB and PLA2-IIA (13). Recent studies have demonstrated a variety of biological responses to PLA2-IB mediated via PLA2R in non-digestive organs, including cell proliferation (18), cell invasion (19), chemokinesis (20), eicosanoid production (21, 22), airway and vascular smooth muscle contraction (23, 24), and fertilization (25). After binding to the PLA2R, PLA2-IB is rapidly internalized and degraded, possibly via the clathrin-coated pit-mediated pathway (26, 27), implicating a possible role of the receptor in the clearance of extracellular PLA2-IB.

In this study, we generated PLA2R mutant mice to define the biological roles of the receptor. Mice deficient for PLA2R exhibited resistance to lethal effects of lipopolysaccharide (LPS), suggesting that PLA2R plays a role in promoting endotoxic shock.

EXPERIMENTAL PROCEDURES

Materials

Sodium [125I]iodine (carrier-free, 3.7 GBq/ml) was purchased from Amersham Corp. Porcine pancreatic PLA2-IB was obtained from Boehringer Mannheim, and exhibited a single 14-kDa band on SDS-polyacrylamide gel electrophoresis. The PLA2-IB solution was dialyzed against phosphate-buffered saline (PBS), sterilized by filtration through a 0.22-µm filter, and stored at -20 °C in small aliquots until use. This PLA2-IB preparation was free of endotoxin. LPS (Salmonella typhosa 0901) was purchased from Difco Laboratories. Protein concentrations were determined using a BCA protein assay reagent kit (Pierce Chemical Co.).

Targeting Vector Construction

The mouse PLA2R gene was cloned from a 129 SVJ genomic library (Stratagene) using a 0.3-kb cDNA fragment encompassing the initial ATG codon of mouse PLA2R (13) as a probe. The 3.25-kb XbaI-NsiI and 3.15-kb MluI-EcoRI genomic fragments derived from the isolated clone were utilized for the construction of the targeting vector together with a neomycin-resistance gene driven by the phosphoglycerate kinase-1 (pgk-1) promoter (pgk-neor), as well as a diphtheria toxin A fragment gene driven by the MC1 promoter, as positive and negative selection markers, respectively. Using this construct, homologous recombination results in the replacement of the NsiI-MluI genomic fragment including the translation starting codon in the pgk-neor cassette, resulting in abolition of PLA2R expression.

Generation of PLA2R Mutant Mice

The ES cell line used in this study was E14 derived from 129/Ola (28), in which we confirmed a natural disruption of the PLA2-IIA gene.2 The targeting experiment and generation of mutant mice were performed essentially as described previously (29). In brief, the E14 cells (1.7 × 107 cells) were electroporated with a Bio-Rad Gene Pulser (0.8 kV, 3 microfarads) using 30 µg of NotI-linearized targeting vector. The electroporated cells were selected in medium containing G418 (125 µg/ml). The cells of surviving colonies were screened for homologous recombination by Southern blot analysis. The mutant cells were microinjected into 3.5-day-old C57BL/6J blastocysts, and the embryos were transferred into the uteri of pseudopregnant ICR mice. Chimeric mice were bred with C57BL/6J mice, in which the PLA2-IIA gene is naturally disrupted (30). The heterozygous F1 offspring were then interbred to generate homozygotes. The genotypes of mice were determined by Southern blot analysis of DNA prepared from tails.

Southern Blot Analysis

Genomic DNAs were digested with BamHI overnight, and electrophoresed through 0.8% agarose gels. The DNAs were transferred to GeneScreen Plus membranes (NEN Life Science Products) and probed with a 1.12-kb BamHI-XbaI fragment. Membranes were then washed and analyzed using a Fujix BAS2000 Bio-Image Analyzer.

Northern Blot Analysis

Total RNA was prepared from tissues with RNeasy (QIAGEN). Poly(A)+ RNA, purified with a QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, Inc.), was electrophoresed under denaturing conditions, and transferred onto GeneScreen Plus membrane. The blot was hybridized with the following cDNA probes in the order; murine PLA2R, rat PLA2-IB, rat PLA2-IIA, and beta -actin, which were labeled with [alpha -32P]dCTP using Megaprime DNA labeling systems (Amersham Corp.). The blot was analyzed and quantified with the BAS2000 Analyzer.

PLA2-IB Binding Assay

Iodination of porcine PLA2-IB was performed by the chloramine-T method (26), and the specific radioactivity of [125I]PLA2-IB obtained was about 500 cpm/fmol. Preparation of crude membranes from various tissues and binding of [125I]PLA2-IB (2 nM) to the crude membranes (300 µg) were performed as described previously (26). The specific binding was calculated as the difference between binding in the presence and absence of unlabeled porcine PLA2-IB (500 nM).

Assay for PLA2-IB Clearance

Mice matched for gender and age (10-15 weeks) were used in this experiment. Sterile porcine PLA2-IB (20 mg/kg) was intravenously injected into mouse tail, and plasma was prepared after the indicated times. The amount of porcine PLA2-IB in plasma was then quantified as follows. Plasma samples were diluted in PBS containing 0.5% bovine serum albumin and 4 mM EDTA, and mixed with [125I]PLA2-IB (20 ng/ml) and anti-porcine PLA2-IB polyclonal antibody we had previously prepared. After the incubation for 4 h at room temperature, goat anti-rabbit IgG antibody-coupled agarose (Sigma) was added and incubated for 30 min. After the addition of PBS, the reaction mixture was centrifuged and the radioactivity of the resulting pellet was counted. This radioimmunoassay detected porcine PLA2-IB at concentrations ranging from 1 ng/ml to 1 µg/ml.

Endotoxic Shock

Mice matched for gender and age (10-15 weeks) were used in the following LPS shock experiments. Mice were intraperitoneally injected with LPS at a dose of 20 or 30 mg/kg in saline, and their survival was monitored. In separate experiments, mice were injected intraperitoneally with LPS at a sublethal dose of 10 mg/kg. After 17 h, sterile porcine PLA2-IB (50 mg/kg) was intravenously injected into mice, and their survival was monitored.

Assay of Plasma Levels of TNF-alpha , IL-1beta , and Nitric Oxide (NO) Metabolites

Mice were injected intraperitoneally with LPS at a dose of 30 mg/kg. Blood was collected 1 h later for the assay of TNF-alpha , and 5 h later for the assay of IL-1beta or nitrate plus nitrite. The plasma levels of cytokines were measured with a murine enzyme-linked immunosorbent assay kit (Endogen Inc.) and the plasma levels of nitrate plus nitrite were measured with a Nitrate/Nitrite colorimetric assay kit (Cayman Chemical Co.).

RESULTS

Generation and Characterization of PLA2R-/- Mice

The targeting strategy for disruption of the PLA2R gene is shown in Fig. 1A. A neomycin resistance gene was inserted between a 3.25-kb XbaI-NsiI fragment and a 3.15-kb MluI-EcoRI fragment, both derived from the genomic clone of PLA2R isolated from the 129 SVJ genomic library. This insertion interrupts the coding sequence in exon 1, including the translation starting codon. This DNA construct was introduced into E14 embryonic stem cells, and transfectants were selected with G418. Of 464 G418-resistant colonies, 2 were determined to have undergone homologous recombination. Cells from the two targeted clones were injected into C57BL/6J blastocysts, and the embryos were reimplanted into foster animals. Chimeric mice derived from 1 clone transmitted the mutation to offspring. Heterozygotes were interbred to generate PLA2R- homozygotes. Southern blot analysis of BamHI-digested tail DNA from F2 progeny revealed the 30- and/or 5.7-kb DNA fragments expected for the wild-type, heterozygous, and homozygous mutant genotypes (Fig. 1B).

Fig. 1. Generation and characterization of PLA2R-deficient mice. A, partial restriction map of the wild-type PLA2R allele (top), the targeting construct (middle), and the predicted homologous recombinant allele (bottom). Exon 1 including the ATG translation starting codon is indicated as a thick box in the upper panel. Thick bar indicates the position of the probe used for Southern blot hybridization. B, Southern blot analysis of PLA2R locus. Genomic DNA was extracted from tail biopsies of F2 offspring and digested with BamHI. The blot was hybridized with the probe. The wild-type allele gives a 30-kb fragment and the mutated allele gives a 5.7-kb fragment. +/+, wild-type mice; +/-, heterozygotes; and -/-, homozygotes. C, Northern blot analysis. Poly(A)+ RNA from the indicated tissues were probed with PLA2R cDNA.

[View Larger Version of this Image (35K GIF file)]

Northern blot analysis revealed the 5.4-kb PLA2R mRNA present in the lung, kidney, and ovary in wild-type mice, but absent in the homozygous mutant mice (Fig. 1C). Disruption of PLA2R allele did not alter the expression pattern and the level of PLA2-IB mRNA in PLA2R-deficient mice. The expression of PLA2-IIA mRNA was not detected in both wild-type and mutant mice (data not shown). Western blot analysis using a polyclonal antibody against the recombinant soluble form of mouse PLA2R detected the 180-kDa PLA2R protein in lung, kidney, and ovary in wild-type mice, but not in the homozygous mutant mice. Immunohistochemical analysis using the antibody revealed that positive staining of renal glomerula in wild-type mice was not detected in the mutant mice (data not shown).

Binding of PLA2-IB to the tissue membrane fractions was examined using porcine [125I]PLA2-IB as a radioligand, which recognizes mouse, rat, and bovine PLA2R with the same binding specificity (11, 31). As shown in Fig. 2, specific binding of PLA2-IB was detected in each membrane fraction of wild-type mice, but not in homozygous mutant mice. Taken together, these results demonstrate that the PLA2R gene was completely inactivated by our gene disruption strategy.

Fig. 2. PLA2-IB binding activity in tissue membranes of PLA2R-deficient mice. Specific binding of [125I]PLA2-IB to the crude membrane preparations of various tissues was examined as described under "Experimental Procedures." +/+, wild-type mice; and -/-, PLA2R-deficient mice. Data represent the mean value performed in two mice.

[View Larger Version of this Image (21K GIF file)]

Phenotype of PLA2R-deficient Mice

Under specific pathogen-free conditions, PLA2R-deficient mice survived at the expected Mendelian ratio: genotyping of 169 F2 mice revealed 26% knockout, 49% heterozygous, and 25% wild-type mice. The PLA2R-deficient mice were fertile and appeared healthy. There was no difference in blood cell composition or plasma lipid composition between wild-type and mutant mice. Necropsy and microscopic examination of major tissues revealed no significant pathology in PLA2R-deficient mice.

PLA2-IB Clearance in Blood

Since PLA2-IB is rapidly internalized and degraded after the receptor binding in several types of cultured cells (26, 27), PLA2R might play a role in clearance of PLA2-IB, selectively withdrawing it from the extracellular fluid. The cells composing blood vessels, including endothelial cells and smooth muscle cells, express high levels of PLA2R (26). The metabolism of intravenously injected porcine PLA2-IB was then examined by radioimmunoassay using polyclonal antibody specific for this type of PLA2. As shown in Fig. 3, PLA2-IB rapidly disappeared from blood within 5 min, and almost disappeared after 30 min in wild-type mice. PLA2R-deficient mice exhibited almost the same degradation kinetics, indicating that vascular PLA2R does not play a role in the clearance of circulating PLA2-IB.

Fig. 3. PLA2-IB clearance in PLA2R-deficient mice. Sterile porcine PLA2-IB (20 mg/kg) was intravenously injected into mouse tail, and plasma was prepared after the indicated times. The amount of porcine PLA2-IB in plasma was then quantified as described under "Experimental Procedures." Each point represents the mean value performed in two mice. The data are representative of three experiments. open circle , PLA2R+/+; bullet , PLA2R-/-.

[View Larger Version of this Image (19K GIF file)]

Endotoxic Shock

PLA2-IB was found to elicit the production of proinflammatory eicosanoids in lung parenchyma (24) as well as glomerular mesangial cells (22) through binding to PLA2R, suggesting the involvement of PLA2R in the progression of pulmonary and renal inflammatory diseases. We therefore investigated the role of PLA2R in a model of endotoxic shock, a systemic inflammatory response syndrome. In wild-type mice, LPS (30 mg/kg) induced 46% lethality by 17 h after challenge, whereas PLA2R-deficient mice all survived (Fig. 4). At 24 h after LPS injection, survival rate was only 8% for wild-type mice compared with 50% for PLA2R-deficient mice. At 20 mg/kg, LPS lethality was 36% for wild-type mice, in contrast to 0% for PLA2R-deficient mice after 24 h treatment (Table I), demonstrating the participation of PLA2R in LPS-induced lethality.

Fig. 4. Endotoxic shock in PLA2R-deficient mice. Each point represents the survival rate (%) of mice at the indicated times after LPS injection (30 mg/kg). Statistical significance was determined by the Log-rank test (p = 0.0123). open circle , PLA2R+/+ (n = 13); bullet , PLA2R-/- (n = 14).

[View Larger Version of this Image (19K GIF file)]

Table I. Lethality of PLA2R-deficient mice in endotoxic shock

Mice were intraperitoneally administered at the indicated doses of LPS. The lethality in each experiment was scored at 24 h after LPS injection.
LPS Lethality: dead/total (% mortality)
PLA2R+/+ PLA2R-/-
mg/kg
20  5/14  (36%) 0/15  (0%)a
30 12/13  (92%) 7/14  (50%)a
a p < 0.05 versus wild-type mice, as determined by Fisher's exact test.

To test for involvement of PLA2-IB in the LPS shock model, mice were sensitized with a sublethal dose of LPS (10 mg/kg) for 17 h, and then injected with PLA2-IB (50 mg/kg). Administration of PLA2-IB did not by itself produce any visible signs of endotoxemia in both types of mice (data not shown). As shown in Fig. 5A, LPS alone induced 11% lethality by 24 h after challenge in wild-type mice. Administration of PLA2-IB caused a significant enhancement of the LPS-induced lethality (p < 0.05). In contrast, LPS did not alone induce lethal effect, and exogenously added PLA2-IB did not significantly affect the survival rate in PLA2R-deficient mice (Fig. 5B). These results suggest that the enhanced lethal effects by PLA2-IB in LPS-sensitized wild-type mice are mediated via PLA2R.

Fig. 5. Effect of exogenous PLA2-IB on endotoxic shock. After administration of a sublethal dose of LPS (10 mg/kg), sterile PBS (open circle ) or porcine PLA2-IB (bullet ; 50 mg/kg) was injected at 17 h in wild-type mice (A, PBS: n = 9; PLA2-IB: n = 9) or PLA2R-deficient mice (B, PBS: n = 5; PLA2-IB: n = 11). Each point represents the survival rate (%) of mice after PLA2-IB injection. Statistical significance was determined by the Log-rank test (A, p = 0.0175; B, p = 0.3286).

[View Larger Version of this Image (15K GIF file)]

Elevation of TNF-alpha , IL-1beta , and NO Levels in Plasma After Challenge with LPS

Treatment of mice with LPS results in up-regulation of the production of various proinflammatory cytokines and inflammatory factors, including TNF-alpha , IL-1beta , and NO, which play crucial roles in the pathogenesis of endotoxic shock (32). The maximum levels of TNF-alpha and IL-1beta elevated in plasma following the LPS administration (1 and 5 h later, respectively) were then examined. As shown in Fig. 6A, the plasma level of TNF-alpha in LPS-treated PLA2R-deficient mice was significantly lower than that in wild-type mice (male PLA2R+/+, 3.64 ± 1.17 ng/ml; male PLA2R-/-, 1.54 ± 0.49 ng/ml, p = 0.0143, female PLA2R+/+, 9.61 ± 5.19 ng/ml; female PLA2R-/-, 1.96 ± 0.95 ng/ml, p = 0.0092). As shown in Fig. 6B, the plasma level of IL-1beta was also reduced in endotoxin-treated mutant mice compared with that in wild-type mice (male PLA2R+/+, 1.48 ± 0.67 ng/ml; male PLA2R-/-, 0.65 ± 0.45 ng/ml, p = 0.0321, female PLA2R+/+, 2.51 ± 1.02 ng/ml; female PLA2R-/-, 0.82 ± 0.19 ng/ml, p = 0.0005). In contrast, there were no differences in the levels of NO metabolites between both types of mice after the challenge with endotoxin (data not shown).

Fig. 6. Plasma levels of TNF-alpha and IL-1beta elevated after LPS treatment in PLA2R-deficient mice. Mice were injected intraperitoneally with LPS (30 mg/kg). Blood was collected 1 h later for the assay of TNF-alpha (A, male PLA2R+/+: n = 5; male PLA2R-/-: n = 4, female PLA2R+/+: n = 13; female PLA2R-/-: n = 4) or 5 h later for the assay of IL-1beta (B, male PLA2R+/+: n = 7; male PLA2R-/-: n = 6, female PLA2R+/+: n = 11; female PLA2R-/-: n = 9). Levels of these cytokines in plasma of wild-type mice (open symbols) and PLA2R-deficient mice (closed symbols) were determined by enzyme-linked immunosorbent assay (squares for males; circles for females). Statistical significance was determined by the Mann-Whitney test (A, male: p = 0.0143; female: p = 0.0092; B, male: p = 0.0321; female: p = 0.0005).

[View Larger Version of this Image (11K GIF file)]

DISCUSSION

For the past three decades, PLA2-IB has been thought to act as a digestive enzyme, given its abundance in digestive organs including the pancreas (7). We have shown that PLA2-IB binds to PLA2R to induce a variety of biological responses (11). In the present study, we generated PLA2R-deficient mice, which exhibited significantly longer survival against two lethal doses of bacterial LPS, indicating a potential involvement of the PLA2-IB/PLA2R pathway in the progression of endotoxic shock. In endotoxemia, PLA2-IIA has long been postulated to play important roles, since elevated levels of this type of sPLA2 in serum and extracellular fluids are associated with propagation of inflammatory conditions (4). However, murine PLA2R possesses a strict binding specificity for the active form of PLA2-IB, but not for PLA2-IIA (13). In addition, the PLA2-IIA gene is naturally disrupted in the mouse strains used in this study (30). Our recent studies have shown that a potent sPLA2 inhibitor, one of the 1-oxamoylindolizine derivatives synthesized in our laboratories (33), inhibits the PLA2-IB binding to murine PLA2R and prolongs the survival of PLA2-IIA-deficient mice with a model of endotoxic shock.3 Thus, these findings suggest that, in addition to PLA2-IIA, PLA2-IB also plays a role in promoting murine endotoxic shock through binding to PLA2R. Recently, novel types of mammalian low molecular weight sPLA2 have been identified, and classified into different groups according to their molecular structures and the localization of disulfide bridges (5, 6). Among them, group V sPLA2, highly expressed in heart (34), was reported to involve in mast cell and macrophage activation process (6, 35). The expression of human group X sPLA2 is restricted to spleen, thymus, and peripheral blood leukocytes, indicating a potential role in the immune system and/or inflammation (36). Although there is no information about the binding affinity of these types of sPLA2 to the PLA2R at present, the possibility that they also involve in the progression of endotoxic shock through binding to PLA2R and/or via their enzymatic activities deserves attention in the future.

Endotoxic shock is a systemic inflammatory process that is characterized histologically by cell damage, tissue necrosis, and vascular disruption (37). LPS activates inflammatory cells, causing them to synthesize and release signals and molecules that contribute to the pathophysiologic process of septic shock (32). Especially, TNF-alpha , IL-1beta , and NO are essential molecules, since mice deficient in TNF-alpha , TNF-alpha receptors, IL-1beta -converting enzyme or inducible NO synthase are markedly resistant to LPS-induced mortality (38-42). In PLA2R-knockout mice, the plasma levels of TNF-alpha and IL-1beta after LPS treatment were significantly lowered compared with those in wild-type mice (Fig. 6), which might account for the reduced sensitivity to endotoxin-induced lethality. TNF-alpha is produced by many cell types, such as monocytes and macrophages, lymphocytes, neutrophils, mast cells, and fibroblasts, and is a key regulator for the synthesis of other proinflammatory cytokines, including IL-1beta (43). In endotoxin-treated mice, the production of TNF-alpha was detected in various tissues including lung, spleen, kidney, and uterus/fallopian tubes (44), where both PLA2-IB and PLA2R messages are considerably expressed (13). PLA2-IB induces a rapid eicosanoid formation via the PLA2R in lung parenchyma and vascular endothelial cells (23, 24), and these eicosanoid metabolites are known to play potential roles in the production of proinflammatory cytokines by macrophages in concert with endotoxin stimulation (32). Although the eicosanoid levels in LPS-stimulated cells and tissues of both types of mice remain to be examined, it is tempting to speculate that eicosanoid metabolites produced via PLA2-IB/PLA2R pathway may contribute to the production of TNF-alpha and IL-1beta . Further studies are required to clarify the molecular mechanisms relevant to the PLA2R in endotoxic shock.

The endogenous level of PLA2-IB is largely dependent on the rate of conversion from its inactive form, pro-PLA2-IB, by serine proteases such as trypsin and plasmin (8, 9). Reactive oxygen species, generated from leukocytes after the exposure of endotoxin (45), were reported to stimulate a membrane-associated serine esterase activity in vascular endothelial cells (46), which might participate in the production of active form of PLA2-IB. LPS also induces the production of urokinase-type plasminogen activator that activates the conversion of zymogen plasminogen to plasmin in vascular endothelial cells and fibroblasts (47, 48). In neutrophils, TNF-alpha and IL-1beta were reported to induce the protease cleavage of pro-PLA2-IB (49). Neutrophils are principally involved in the pathogenesis of pulmonary tissue injury and, in patients with acute lung injury, the amount of propeptide released during the PLA2-IB activation process was found to be elevated in plasma and urine (50). PLA2-IB elicits potent contraction in lung parenchyma by producing eicosanoid inflammatory mediators via PLA2R (24). Taken together, these findings suggest that active PLA2-IB produced in pulmonary loci after endotoxin challenge involves in lung injury via PLA2R. PLA2-IB also stimulates prostanoid production through binding to PLA2R in glomerular mesangial cells (22, 51), which might participate in inflammatory responses to renal injury.

After binding to the PLA2R, PLA2-IB is rapidly internalized and degraded in various types of cultured cells, possibly via clathrin-coated pit-mediated pathway (26, 27). In the present study, there was no difference in the degradation kinetics of exogenously added PLA2-IB in plasma between wild-type and PLA2R-deficient mice (Fig. 3), suggesting that vascular PLA2R does not play a potential role in the clearance of circulating PLA2-IB. Exogenously added PLA2-IB alone did not elicit any endotoxic symptoms. However, after sensitization with a sublethal dose of LPS, PLA2-IB induced an enhancement of the lethality in wild-type mice in contrast to no significant effects in PLA2R-deficient mice (Fig. 5), indicating that circulating PLA2-IB promotes endotoxic responses, possibly via vascular PLA2R. Alternatively, PLA2-IB might pass through the blood vessels to promote tissue injury, as a result of the abnormal vascular permeability present during endotoxic shock (52). Since increased systemic levels of PLA2-IB have been observed in patients with acute pancreatitis and renal failure (53), circulating PLA2-IB might play a role in progression of these inflammatory conditions via PLA2R.

In conclusion, our findings presented here suggest that PLA2R plays a role in promoting endotoxic shock. Study of the underlying mechanisms will aid our understanding of the function and signal transduction of PLA2R in the cytokine production as well as in the tissue injury during endotoxemia. To define the pathophysiological significance of the PLA2R further, we are now generating PLA2R-deficient mice that possess the PLA2-IIA gene. These PLA2R-mutant mice will be valuable tools for further elucidation of the in vivo role of the PLA2R in various disease conditions in which PLA2-IB might be involved. Finally, the present study should be of great value for research on the development of PLA2R-blocking agents as therapeutic drugs for septic shock.

FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4 Sagisu, 5-Chome, Fukushima-ku, Osaka 553, Japan. Tel.: 81-6-458-5861; Fax: 81-6-458-1193.
1   The abbreviations used are: PLA2, phospholipase A2; PLA2-IB, group IB phospholipase A2; PLA2-IIA, group IIA phospholipase A2; sPLA2, secretory phospholipase A2; pro-PLA2-IB, group IB prophospholipase A2; PLA2R, phospholipase A2 receptor; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; NO, nitric oxide; TNF-alpha , tumor necrosis factor-alpha ; IL-1, interleukin-1; kb, kilobase(s).
2   Y. Yokota, K. Hanasaki, and H. Arita, unpublished data.
3   Y. Yokota, K. Hanasaki, and H. Arita, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. Itohara (Kyoto University, Japan) for providing E14 cells as well as technical support; Dr. Suzuki, Dr. Arimura, Dr. Hori, and Dr. Tanaka for help in the analysis of PLA2R-deficient mice; H. Watanabe for statistical analysis of data; and K. Nakano and S. Andoh-Sinonome for excellent technical assistance.

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Volume 272, Number 52, Issue of December 26, 1997 pp. 32792-32797
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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