Macrophage-Expressed Group IIA Secretory Phospholipase A2 Increases Atherosclerotic Lesion Formation in LDL Receptor–Deficient Mice

	Abstract

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Objective— Transgenic mice expressing human group IIA secretory phospholipase A₂ (group IIA sPLA₂) spontaneously develop atherosclerotic lesions. The mechanism for this proatherogenic effect is likely multifactorial, because HDL-cholesterol is significantly lower and LDL/VLDL cholesterol is slightly higher in transgenic mice compared with nontransgenic littermates. In the present study, we show for the first time that elicited peritoneal macrophages from transgenic mice express human group IIA sPLA₂. This study tested whether macrophage-expressed sPLA₂ contributes to atherogenesis.

Methods and Results— Bone marrow cells from either sPLA₂ transgenic mice or control C57BL/6 mice were transplanted into LDL receptor–deficient mice. After hematopoietic engraftment, animals were fed a diet enriched with saturated fat and cholesterol for 12 weeks. Despite a lack of effect on serum lipoprotein concentrations, the presence of bone marrow–derived cells expressing human group IIA sPLA₂ resulted in a significant increase in the extent of atherosclerosis in the aortic arch (12.8±1.4% versus 7.4±0.9%; P<0.005) and aortic sinus (0.3±0.03 mm² versus 0.2±0.04 mm²; P<0.05).

Conclusions— Group IIA sPLA₂ can contribute to atherosclerotic lesion development through a mechanism that is independent of systemic lipoprotein metabolism.

Key Words: atherosclerosis bone marrow transplant  transgenic mice  group IIA secretory phospholipase A₂  macrophages

	Introduction

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Group IIA secretory phospholipase A₂ (group IIA sPLA₂) is a member of a family of secreted phospholipases that hydrolyzes the sn-2 fatty acyl ester bond of glycerophospholipids to generate free fatty acids (FFAs) and lysophospholipids.¹ Group IIA sPLA₂ (traditionally referred to as nonpancreatic or synovial sPLA₂) is thought to be responsible for amplifying the inflammatory component of many disease processes, including atherosclerosis. During acute or chronic inflammation, the concentration of group IIA sPLA₂ can increase more than 100-fold in inflammatory fluids and plasma.² In humans, serum concentration of sPLA₂ is an independent risk factor for coronary artery disease and a predictor of cardiovascular events.³ Group IIA sPLA₂ has been detected in human atherosclerotic lesions by immunocytochemistry. In two studies, group IIA distribution corresponded mainly to that of -actin, suggesting that the primary cellular source of the enzyme in the vessel wall is smooth muscle cells.^4,5 However, some sPLA₂ staining associated with lipid-laden CD68-positive macrophages was also detected. These findings contrast with two other reports, where sPLA₂ was present primarily in areas with macrophage-derived foam cells.^6,7 These discrepant results could be explained by differences in tissue preservation or the extent of atherosclerosis. It is also possible that the antibodies used for immunostaining may exhibit varying amount of cross-reactivity with a related sPLA₂, group V.^8,9

C57BL/6 transgenic mice that express human group IIA sPLA₂ provide a useful model to investigate the role of this enzyme in atherosclerotic lesion development.¹⁰ The C57BL/6 strain lacks endogenous group IIA sPLA₂ protein because of a frame-shift mutation in exon 3.¹¹ Human group IIA sPLA₂ is present in a variety of tissues in the transgenic mice, including liver, kidney, lung, skin, and intima/media of the aorta.¹² Although serum enzymatic activity is elevated 8-fold compared with nontransgenic littermates,¹⁰ there is no evidence of systemic inflammation in the transgenic mice.¹³ Notably, human group IIA sPLA₂ transgenic mice maintained for 12 weeks on a diet containing 1.25% cholesterol, 15.75% fat, and 0.5% sodium cholate have significantly increased vascular lipid deposition compared with nontransgenic littermates.¹⁴ The transgenic mice also develop spontaneous lesions when fed a low-fat diet.¹⁴ Thus, the relationship in humans between sPLA₂ and atherosclerosis may be a causal one.

Group IIA sPLA₂ may promote atherosclerosis, at least in part, by altering systemic lipoprotein metabolism. The sPLA₂ transgenic mice have significantly lower plasma concentrations of HDL-cholesterol and phospholipids and slightly higher VLDL/LDL compared with nontransgenic littermates.^13–15 Differences in plasma lipoprotein concentrations do not seem to account entirely for the proatherogenic effect of sPLA₂, however, because transgenic mice maintained on a normal diet spontaneously developed atherosclerotic lesions despite having relatively low LDL and VLDL.¹⁴ Immunocytochemical analysis showed the presence of human group IIA sPLA₂ in aortic lesions of transgenic mice,¹⁴ providing the possibility that sPLA₂ may provide a local proatherogenic effect in the microenvironment of the developing lesion.

We now demonstrate the new finding that group IIA sPLA₂, driven by its natural promoter, is expressed in macrophages in human group IIA transgenic mice. We hypothesized that increased sPLA₂ expression in macrophages within the microenvironment of the developing lesion may contribute to atherogenesis. To test this possibility, we transplanted bone marrow cells derived from human sPLA₂ transgenic mice into LDL receptor–deficient (LDLR^-/-) mice that lack group IIA sPLA₂. We reasoned that this approach would allow for a better mechanistic definition of the proatherogenic effects of sPLA₂, because systemic influences on lipoprotein metabolism should be minimized. Our results show that expression of human group IIA sPLA₂ in bone marrow–derived cells promotes atherosclerotic lesion formation in the absence of any measurable changes in plasma lipoprotein concentrations.

	Methods

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Animals
Animals were maintained in a pathogen-free facility with equal light/dark cycle and free access to food and water. Male LDLR^-/- and C57BL/6 mice were obtained from the Jackson Laboratory. LDLR^-/- mice were backcrossed 10 times on a C57BL/6 background. Human sPLA₂ transgenic mice (backcrossed 12 times into C57BL/6) were obtained from Taconic. Recipient LDLR^-/- mice were maintained on drinking water containing Sulfatrim (275 µg/mL) 1 week before and 2 weeks after bone marrow transplantation. Six weeks after transplantation, animals were maintained on a modified diet containing 20% fat and 0.15% cholesterol (wt/wt) (Harlan Teklad No. 88137) for 12 weeks. All animal procedures were approved by the Veteran’s Administration Institutional Animal Care and Use Committee.

Bone Marrow Transplantation
Ten-week-old LDLR^-/- recipient mice were lethally irradiated with 9 Gy using a cesium  source. Irradiated recipient mice were transplanted by tail vein injection of 1x10⁷ bone marrow cells harvested from tibia and femurs of age-matched C57BL/6 or sPLA₂ transgenic donor mice. To assess engraftment of donor hematopoietic cells, DNA was isolated from bone marrow cells of recipient mice using the DNeasy Tissue Kit (Qiagen). Polymerase chain reaction analysis was performed using DNA isolated from bone marrow of transplanted mice. Donor (LDLR^+/+) DNA and recipient (LDLR^-/-) DNA were amplified in a single reaction using a mixture of 3 oligonucleotide primers that distinguish the 2 alleles, as described by the Jackson Laboratory. In all mice analyzed, the only amplification product detected corresponded to the LDLR^+/+ allele, indicating successful engraftment.

Removal of Tissue and Blood Samples
Recipient animals were bled via the retroorbital sinus 6 weeks after bone marrow transplantation, before initiation of modified diet. Animals were anesthetized using xylazine (4 mg/kg) and chloral hydrate (350 mg/kg). At the end of the study, bone marrow cells were flushed from the femur of each recipient mouse into 1 mL PBS. Terminal blood samples were collected, and samples were spun for 5 minutes at 10 000 rpm to obtain plasma. Animals were perfused with PBS by puncture of the left ventricle. The heart was separated from the aorta and frozen in OCT. The aortas were removed and immersed in 4% paraformaldehyde for 24 hours. After removal of adventitial tissue, the intimal aortic surface was exposed by a longitudinal cut and the tissue was pinned en face.

Atherosclerotic Lesion Analysis
Lesion size was quantified in both the aortic arch and aortic root, as described previously.¹⁶ The aortic arch was defined as the region from the ascending arch to 3 mm distal to the subclavian artery.¹⁶ Percent lesion area was calculated using Image Pro software (Media Cybernetics). For analysis of the aortic root, tissue was frozen in OCT. Ventricular tissue was sectioned from the apical aspect until the aortic valves were visible. At this point, tissues were cut at 8-µm intervals and placed sequentially on 8 slides. Tissues were cut until 10 sections had been acquired per slide. The aortic sinus was defined as a region in which leaflet values or cusps were present, while the ascending aorta was defined as the distal region of the root. Lesion areas were quantified by Image Pro Software using the luminal edge and internal elastic lamina as boundaries. Oil red O staining was used to assist in the visualization of lesions but not in the quantification of lesion size. Immunocytochemistry was performed on acetone-fixed, frozen sections as described previously.^17,18 A macrophage-specific antisera was obtained from Accurate (AI-AD31240). Anti-human group IIA sPLA₂ was provided by Dr T. Nevalainen¹⁹ and used at a dilution of 1:1000. This rabbit antiserum was raised against recombinant human group IIA sPLA₂ and has been used extensively to stain group IIA sPLA₂ in human¹⁹ and transgenic mouse tissues.¹² Previously, this antiserum was shown to exhibit no immunoreactivity with any tissue from nontransgenic C57BL/6 mice that lack endogenous group IIA sPLA₂,¹² suggesting that this reagent does not cross-react with any other mouse sPLA₂, including group V. Tissues were incubated with a species-specific biotinylated secondary antibody followed by an avidin-biotin peroxidase complex (Elite kits, Vector Laboratories). Immunoreactivity was visualized using the red chromogen amino-ethyl carbazole (Biomeda), and sections were counterstained with hematoxylin.

Lipid, Lipoprotein, and Phospholipase Analysis
Six weeks after transplant and before initiation of high-fat diet, plasma from 4 mice was pooled (3 pools per group) for quantifying lipids and phospholipase activity. For terminal samples, individual mouse plasmas were analyzed separately (C57BL/6LDLR^-/-, n=11; sPLA₂ tgLDLR^-/-, n=12). Total and HDL cholesterol concentrations were determined using an enzymatic assay (Wako Chemicals Inc). Aliquots of plasma pooled from 2 to 4 mice (200 µL) were clarified by centrifugation and resolved by size exclusion chromatography using a Superose 6 column (Pharmacia LKB Biotechnology Inc). The column was eluted at a flow rate of 0.5 mL/min in buffer containing 150 mmol/L NaCl and 10 mmol/L Tris/HCl, pH 7.4, 0.01% sodium azide. The cholesterol content of fractions (0.5 mL) was determined enzymatically (Wako Chemicals). Phospholipase activity in plasma was determined using a colorimetric assay (Wako Chemicals) with mixed micelles comprising 1-palmitoyl, 2-oleoyl phosphatidylglycerol, deoxycholate, and Nonidet-40 as substrate.²⁰ Values were expressed as the amount of FFA released in the assay per microliter of plasma.

Immunoblot Analysis
Peritoneal macrophages were collected from untreated human group IIA sPLA₂ transgenic mice or mice 5 days after intraperitoneal injection of 1% biogel (1 mL). Adherent cells were cultured for 16 hours in DMEM containing 10% FBS. Postnuclear supernatants of cell lysates were separated by nonreducing SDS-PAGE, electroblotted onto 0.2 µmol/L pore-size PVDF membrane (Schleicher and Schuell), and immunoblotted using rabbit anti-human synovial sPLA₂.⁸ Antibody binding was visualized by chemiluminescence detection (ECL, Amersham Corp).

Statistical Analysis
All data are presented as mean±SEM. Student’s t test was performed using Sigma Stat 2.03 (SPSS, Inc). All data met the constraints of normality and equivalence of variance to permit parametric analysis.

	Results

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Immunoblot Analysis of Human sPLA₂ Transgenic Mouse Peritoneal Macrophages
In humans, group IIA sPLA₂ mRNA is not detected in monocytes or terminally differentiated, unstimulated macrophages.⁷ However, group IIA sPLA₂ mRNA transcription is induced in human monocyte-derived macrophages incubated with minimally modified or mildly oxidized LDL.⁷ To assess whether macrophages from transgenic mice harboring the entire human group IIA sPLA₂ gene express human sPLA₂, immunoblot analysis of peritoneal macrophages was performed . Human sPLA₂ protein was not detected in resident macrophages from the transgenic mice. In contrast, an 14-kDa immunoreactive band, which comigrated with recombinant human group IIA sPLA₂, was detected in elicited macrophages that were collected 5 days after peritoneal injection of a 1% solution of Biogel. As expected, group IIA sPLA₂ was not detected in either resident or biogel-elicited macrophages from nontransgenic littermates (data not shown). These results suggest that macrophage-derived sPLA₂ may contribute to the intense sPLA₂ staining in aortic lesions of human sPLA₂ transgenic mice.¹⁴

fig.ommitted Western blot analysis of peritoneal macrophages from human group IIA sPLA2 transgenic mice. Macrophages were collected from duplicate untreated mice (resident) or from triplicate mice 5 days after intraperitoneal injection of 1% biogel (elicited) and cultured as described in the Experimental Procedures section. Aliquots corresponding to 10 µg protein were separated by nonreducing SDS-PAGE, immunoblotted with rabbit anti-human group IIA sPLA2, and visualized by chemiluminescence detection. Purified recombinant human group IIA (rGIIA) sPLA2 (100 ng) was analyzed for comparison. Similar results were obtained in 2 additional experiments.

Plasma Cholesterol, Phospholipase Activity, and Lipoprotein Distributions in Mice After Bone Marrow Transplantation
To test the hypothesis that macrophage-expressed sPLA₂ contributes to atherosclerotic lesion development, 10-week-old LDLR^-/- mice were transplanted with bone marrow cells derived from either human group IIA sPLA₂ transgenic mice or wild-type mice. Six weeks after bone marrow transplantation, mice were maintained for 12 weeks on a modified diet containing 20% fat and 0.15% cholesterol (wt/wt). Plasma concentrations of total cholesterol, HDL-cholesterol, and phospholipase activity were quantified before and after feeding a high-fat diet. In both sPLA₂ tgLDLR^-/- and C57BL/6LDLR^-/- mice, total cholesterol concentrations were highly elevated after modified diet feeding  None of the parameters measured were significantly different between the two groups of mice, either before or after the atherogenic diet. Separation of plasma lipoproteins by size exclusion chromatography showed that most plasma cholesterol in mice fed the modified diet was in the VLDL/LDL fractions . There was no significant difference in lipoprotein cholesterol distribution in C57BL/6LDLR^-/- and sPLA₂ tgLDLR^-/- mice.

fig.ommitted  Total Cholesterol (TC), HDL Cholesterol (HDL-C), and Phospholipase (PLase) Concentrations in C57BL/6LDLR-/- and sPLA2 tgLDLR-/- Mice

fig.ommitted  Lipoprotein cholesterol distributions of C57BL/6 LDLR-/- and sPLA2 tgLDLR-/- mice fed a modified diet for 12 weeks. Values represent the mean cholesterol content of each fraction (±SEM) for 4 pools per group, with 2 to 3 mice in each pool.

Quantification of Atherosclerotic Lesions
The extent of atherosclerosis was measured on the intimal surface of the aortic arch and in the aortic root. No discernible lesions were detected in either the abdominal aorta or thoracic aorta in any of the recipient mice. En face analysis of the aortic arch showed a highly significant 73% increase in lesion area in mice transplanted with bone marrow cells derived from human group IIA sPLA₂ transgenic mice compared with control bone marrow (percent lesion area, 12.8±1.4% versus 7.4±0.9% for transgenic and nontransgenic, respectively; P<0.005;). In addition, there was a significant increase in mean atherosclerotic lesion area within the aortic sinus of the aortic root (mean lesion area per section, 0.2±0.04 versus 0.3±0.03 mm² for nontransgenic and transgenic, respectively; P<0.05; ).

fig.ommitted    A, En face lesion area in aortic arch of C57BL/6LDLR-/- and sPLA2 tgLDLR-/- mice as percent of total intimal area. Horizontal bars indicate the mean for each group (P<0.005). B, Lesion size in the aortic root of C57BL/6LDLR-/- and sPLA2 tgLDLR-/- mice (n=8). Lesion area was determined in 8-µm-thick sections at 80-µm intervals after Oil red O staining. Values represent mean lesion areas (±SEM) for each section. The transition zone between the aortic sinus and the ascending aorta, defined by disappearance of the valve cusps, is 0 on the x-axis.

Immunocytochemical Analysis of Lesions
Immunocytochemical analysis of aortic root sections confirmed the presence of human group IIA sPLA₂ in lesions of sPLA₂ tgLDLR^-/- mice that was associated with lipid-laden macrophages. As expected, human sPLA₂ was not detected in C57BL/6LDLR^-/- mice (data not shown). Inspection of all sections from aortic roots of each animal did not reveal overt differences in the cellular characteristics of lesions from C57BL/6LDLR^-/- and sPLA₂ tgDLR^-/- mice.

fig.ommitted  Immunocytochemical analysis of sections from the same aortic sinus region of a sPLA2 tgLDLR-/- mouse stained with rabbit anti-human group IIA sPLA2 (A) or anti-mouse macrophage antisera (B). Immunoreactivity was visualized using the red chromogen, amino-ethyl carbazole. Sections were counterstained with hematoxylin. Magnification, x200.

	Discussion

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C57BL/6 transgenic mice expressing human group IIA sPLA₂ have increased vascular lipid deposition compared with nontransgenic littermates.¹⁴ The present study demonstrates for the first time that macrophage cells in these transgenic mice express human group IIA sPLA₂. This finding suggested the interesting possibility that macrophage-expressed sPLA₂ contributes to atherosclerotic lesion formation in the transgenic mice. To test this possibility, lethally irradiated LDLR^-/- mice were reconstituted with bone marrow cells derived from either human sPLA₂ transgenic mice or wild-type mice. Both donor and recipient mice were C57BL/6 background and thus lack the expression of mouse group IIA sPLA₂ because of a natural mutation in the endogenous gene.¹¹ We selected male mice for our analysis because studies in sPLA₂ transgenic mice have shown that the magnitude of the sPLA₂ effect is more pronounced in this sex compared with females.¹⁴ Our results demonstrate that mice reconstituted with bone marrow cells from human group IIA sPLA₂ transgenic mice develop significantly larger lesions in both the aortic sinus and aortic arch compared with mice repopulated with wild-type bone marrow cells.

Immunocytochemical analysis of lesions from sPLA₂ tgLDLR^-/- mice verified the presence of sPLA₂ associated with macrophage foam cells. Group IIA sPLA₂ has similarly been detected in human lesions.^4–7 It is possible that such lesional sPLA₂ immunostaining represents enzyme that has infiltrated into the intima from another source rather than macrophage expression. However, it seems likely that group IIA detected in our transplantation experiment is primarily macrophage derived, because (1) group IIA was not detected in any other region in the vessel wall that appeared to be devoid of macrophage cells; (2) we would expect that most donor-derived cells present in lesions would be macrophages; and (3) we have demonstrated that macrophage cells in the transgenic mice do indeed express human group IIA sPLA₂. It is notable that in situ hybridization analyses of sPLA₂ transgenic mouse tissues showed no hybridization signal in spleen or lymph nodes, suggesting that the human group IIA promoter is not constitutively active in transgenic mouse macrophages.¹² In this study, we show that human sPLA₂ protein is also not present in resident peritoneal macrophages from the transgenic mice but can be detected in biogel-elicited peritoneal macrophages. There is evidence that the endogenous group IIA sPLA₂ promoter is under transcriptional regulation in human monocyte macrophages.⁷ Taken together, the evidence suggests that macrophage cells can be a source of group IIA sPLA₂. This conclusion is consistent with the finding that macrophage expression of group IIA sPLA₂ has been reported in association with other human inflammatory diseases in addition to atherosclerosis, including Crohn’s disease²¹ and acute pancreatitis.²²

Our data showed regional-specific differences in the proatherogenic response to group IIA sPLA₂ expression. Whereas lesion formation was significantly enhanced in the aortic sinus and aortic arch, there was no difference in the extent of lesions in the ascending aorta. A similar region-specific difference in response to macrophage expression of lipoprotein lipase has also been reported.²³ In fact, in the relatively few studies where atherosclerosis has been quantified in the aortic root as well as throughout the aorta, many have reported nonuniform alterations in atherogenesis in response to an intervention.^24–27 Whether such discrepancies are attributable to regional differences in hemodynamics or other factors requires additional investigation.

Certain mouse strains, in particular the C57BL/6 mouse, lack the expression of functional group IIA protein because of a frame-shift mutation in exon 3.¹¹ The fact that C57BL/6 mice are relatively susceptible to the development of atherosclerosis suggests that group IIA sPLA₂ is not required for atherosclerotic lesion formation in these mice. In a recent study, it was reported that apoE^-/-/sPLA₂^+/+ and apoE^-/-/sPLA₂^-/- mice fed a high-fat diet for 22 weeks had no significant difference in aortic cholesterol content.²⁸ The authors concluded that endogenous mouse sPLA₂ does not significantly affect atherogenesis in apoE^-/- mice. This result contrasts with our finding that human sPLA₂ expression significantly enhances atherogenesis in LDLR^-/- mice. Several factors could account for the discrepancy between our study and the published report, including possible species differences between mouse and human sPLA₂. It is also possible that the lack of effect of the sPLA₂ genotype in the previous study was attributable to deficiencies in immune function in apoE^-/- mice²⁹ that have not been documented in LDLR^-/- mice.

Accumulating data from studies in vitro show that phospholipids on lipoprotein particles are substrates for group IIA sPLA₂ hydrolysis.^30–32 In the human group IIA sPLA₂ transgenic mice, increased sPLA₂ activity is accompanied by a 30% to 40% decrease in HDL-cholesterol concentrations.^13,14 In the present study, we assessed whether mice repopulated with macrophages expressing human group IIA sPLA₂ have altered plasma phospholipase activity or lipoprotein cholesterol concentrations, because such systemic effects could influence the extent of vascular lipid deposition. Our results show that mice repopulated with bone marrow cells expressing human group IIA sPLA₂ had no detectable alterations in plasma cholesterol concentrations, lipoprotein cholesterol distribution, or sPLA₂ activity compared with mice repopulated with wild-type bone marrow. We conclude from these findings that sPLA₂ expressed by macrophages within the microenvironment of developing lesions can promote the atherogenic process.

Local expression of group IIA sPLA₂ in the vessel wall could have multiple proatherogenic effects. One of the potential products of sPLA₂ hydrolysis is lysophosphatidyl choline, which has chemoattractant, chemostatic, and mitogenic effects on monocytes,³³ macrophages,³⁴ smooth muscle cells,^35,36 and T-lymphocytes.³⁷ Lysophospholipids can also serve as the substrate for the generation of potent proinflammatory lipid mediators, including platelet activating factor and lysophosphatidic acid. FFAs released by sPLA₂ hydrolysis may be metabolized into proinflammatory agents such as eicosanoids or undergo oxidative modification. In addition to generating lipid mediators of inflammation, several lines of evidence suggest that sPLA₂ modification of LDL can result in structural alterations of the particle that promotes lipid accumulation in the vessel wall. Studies in vitro indicate that lipolysis of LDL with sPLA₂ results in partial lipoprotein aggregation and increased affinity for proteoglycans.^38,39 In addition, sPLA₂ modification increases the susceptibility of LDL to hydrolysis by secretory sphingomyelinase.⁴⁰ This leads to the accumulation of ceramide within the particles, which can also promote particle aggregation and fusion. Aggregated/fused LDL, which is prominent in atherosclerotic lesions,^41–43 is one of the most potent inducers of macrophage foam-cell formation in vitro.^44–46 Thus, although not directly shown, sPLA₂ hydrolysis of LDL could promote atherogenesis by increasing the retention of LDL particles in the subendothelium and by generating potent inducers of macrophage foam cells. Alternatively, sPLA₂ may promote atherosclerosis by modifying HDL in the vessel well to reduce its protective activity.⁴⁷ Given the multitude of potential mechanisms whereby local expression of sPLA₂ could promote vascular lipid deposition, additional studies are necessary to delineate its proatherogenic effect.

	Acknowledgments

 
This work was supported by National Institutes of Health Grants HL-65730 (to D.R. van der Westhuyzen) and HL-69463 (to F.C. de Beer). The authors thank Dr T. Nevalainen for generously providing rabbit anti-human group IIA sPLA₂. We also thank Jin Yu, Hong Xhu, and Wei Shi for assistance with mouse dissections and Deborah Howatt and John Burckle for tissue sectioning, image analysis, and immunocytochemistry.

Received November 14, 2002; accepted November 22, 2002.

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