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• W2149804875 abstract "Secreted phospholipase A2 group X (sPLA2-X) is one of the most potent enzymes of the phospholipase A2 lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA2-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA2-X enzyme activity in vitro and in vivo, by generating sPLA2-X-overexpressing macrophages and transgenic macrophage-specific sPLA2-X mice. Our results show that sPLA2-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2). Moreover, we found that overexpression of active sPLA2-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA2-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA2-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA2-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA2-X in inflammatory lung disease. Secreted phospholipase A2 group X (sPLA2-X) is one of the most potent enzymes of the phospholipase A2 lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA2-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA2-X enzyme activity in vitro and in vivo, by generating sPLA2-X-overexpressing macrophages and transgenic macrophage-specific sPLA2-X mice. Our results show that sPLA2-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2). Moreover, we found that overexpression of active sPLA2-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA2-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA2-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA2-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA2-X in inflammatory lung disease. Secretory phospholipases A2 (sPLA2) 3The abbreviations used are: sPLA2, secretory phospholipase A2; PLA2, phospholipase A2; PC, phosphatidylcholine; LPS, lipopolysaccharide; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; HPTLC, high performance thin layer chromatography; TNF, tumor necrosis factor; IL, interleukin; PGE2, prostaglandin E2; 15d-PGJ2, 15-deoxy-12,14 prostaglandin J2; PAS, periodic acid-Schiff; PAS/D, PAS after diastase digestion; SP, surfactant phospholipid; iNOS, inducible nitric-oxide synthase; AA, arachidonic acid; Cox, cyclooxygenase; Wt, wild type; PGH2, prostaglandin H2; PPARγ, peroxisome proliferator-activated receptor γ; ARD, acute respiratory distress; GAPDH, glyeraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay. represent an important, continuously growing subclass of the phospholipase A2 (PLA2) lipolytic enzyme superfamily. sPLA2 produces free fatty acids and lysophospholipids, important second messengers in cell signaling and signal transduction, by hydrolyzing glycerophospholipids present in cell membranes and plasma lipoproteins. sPLA2 can be distinguished from other PLA2 by their low molecular mass (13-18 kDa), their high disulfide bond content (6-8 bridges), and the requirement for millimolar concentrations of Ca2+ for catalysis. Among the different sPLA2, the group X sPLA2 (sPLA2-X) is the most potent in hydrolyzing phospholipids with different polar head groups and fatty acids (1Balsinde J. Winstead M.V. Dennis E.A. FEBS Lett. 2002; 531: 2-6Crossref PubMed Scopus (407) Google Scholar, 2Ghesquiere S.A. Hofker M.H. de Winther M.P. Cardiovasc. Toxicol. 2005; 5: 161-182Crossref PubMed Scopus (14) Google Scholar). Phospholipases have been implicated in the pathogenesis of lipid-mediated inflammatory diseases such as atherosclerosis. Atherosclerosis, a slow progressing inflammation of the large arteries, is characterized by the build up of lipid-rich material in the inner vessel wall. The macrophage is the most prominent cell type of an atherosclerotic plaque and crucial in lipid accumulation and the concomitant inflammatory process. Because the hydrolysis products of sPLA2 enzymes lead to the generation of atherogenic lipoprotein particles and the production of various inflammatory lipid mediators subtypes, several of these enzymes (e.g. human group IIA, V, and X) have been implicated in atherosclerosis development (2Ghesquiere S.A. Hofker M.H. de Winther M.P. Cardiovasc. Toxicol. 2005; 5: 161-182Crossref PubMed Scopus (14) Google Scholar). More specifically, for both sPLA2-IIA and sPLA2-V, functional studies in mice showed that overexpression results in enhanced atherosclerotic lesion formation(3Bostrom M.A. Boyanovsky B.B. Jordan C.T. Wadsworth M.P. Taatjes D.J. de Beer F.C. Webb N.R. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 600-606Crossref PubMed Scopus (113) Google Scholar, 4Ghesquiere S.A. Gijbels M.J. Anthonsen M. van Gorp P.J. Van der Made I. Johansen B. Hofker M.H. de Winther M.P. J. Lipid Res. 2005; 46: 201-210Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 5Tietge U.J. Pratico D. Ding T. Funk C.D. Hildebrand R.B. Van Berkel T. Eck Van. M. J. Lipid Res. 2005; 46: 1604-1614Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 6Webb N.R. Bostrom M.A. Szilvassy S.J. Van der Westhuyzen D.R. Daugherty A. de Beer F.C. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 263-268Crossref PubMed Scopus (78) Google Scholar). The potent group X sPLA2 was discovered in 1997 by Cupillard et al. (7Cupillard L. Koumanov K. Mattei M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) and is expressed in spleen, thymus, and peripheral blood leukocytes, and in low quantities in lung, pancreas, and colon cells. Under inflammatory conditions, the prepropeptide is cleaved to yield the fully active enzyme. sPLA2-X is structurally related to both sPLA2-IB and -IIA, and once active, it displays potent activity on both anionic and zwitterionic phospholipids, and hydrolyzes PC (8Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Although comparable with group V in terms of cell binding, the hydrophobic nature of sPLA2-X makes the enzyme far more potent. Immunohistochemical staining of atherosclerotic plaques revealed marked sPLA2-X expression of sPLA2-X in foam cell lesions in the arterial intima and in smooth muscle cells of the arterial media in both mice and humans (9Hanasaki K. Yamada K. Yamamoto S. Ishimoto Y. Saiga A. Ono T. Ikeda M. Notoya M. Kamitani S. Arita H. J. Biol. Chem. 2002; 277: 29116-29124Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 10Karabina S.A. Brocheriou I. Le Naour G. Agrapart M. Durand H. Gelb M. Lambeau G. Ninio E. Faseb. J. 2006; 20: 2547-2549Crossref PubMed Scopus (81) Google Scholar). However, to date, no functional studies have been performed investigating the role of sPLA2-X in macrophages during atherogenesis. The present study was performed to investigate in vitro and in vivo the function of the active sPLA2-X enzyme in atherogenic macrophage features like foam cell formation and inflammation. To this end, we generated a macrophage-specific sPLA2-X construct to create macrophages overexpressing active group X, as well as transgenic mice overexpressing the active sPLA2-X enzyme in their macrophages. Our results show that overexpression of the sPLA2-X enzyme in macrophages in vitro reduces macrophage activation and inflammatory responses and mediates enhanced foam cell formation. In vivo experiments show that enhanced sPLA2-X activity in macrophages leads to neonatal death due to severe lung pathology characterized by an interstitial pneumonia with infiltration of surfactant-laden alveolar macrophages and edema. Generation of a Macrophage-specific sPLA2-X Construct—A macrophage-specific sPLA2-X construct was generated by cloning the active human sPLA2-X cDNA (a generous gift from Dr. Lambeau) starting at the second methionine initiator site (Met-32 construct) (7Cupillard L. Koumanov K. Mattei M.G. Lazdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15745-15752Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) behind the macrophage-specific CD68 promoter (2.9-kb CD68 + IVS-1 sequence) (11Gough P.J. Gordon S. Greaves D.R. Immunology. 2001; 103: 351-361Crossref PubMed Scopus (77) Google Scholar) into the pcDNA 3 vector (Invitrogen) using convenient restriction sites (Fig. 1A). Verification was performed by sequencing start and end of the Met-32 construct and boundaries of the CD68 promoter region (data not shown). Generation of RAW264.7 Cells Overexpressing hu-sPLA2-X—The murine macrophage cell line RAW264.7 was cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and 50 μm β-mercaptoethanol. Experiments were performed in medium without β-mercaptoethanol. To establish sPLA2-X-overexpressing RAW 264.7 cells, 10 μg of CD68-sPLA2-X plasmid DNA and 2 μg of neomycin resistance plasmid DNA were added to 10 × 106 RAW264.7 cells resuspended in 500 μl of Optimem (Invitrogen) and electroporated with a Bio-Rad Gene Pulser II at 1070 μF and 280 V in 4-mm cuvettes. Following electroporation, the cells were replated in pre-warmed RAW medium. Two days after transfection, cells were placed on culture medium containing 0.1 mg/ml geneticin for 1 week. Surviving cells were replated in 96-well plates for clonal selection, and individual colonies were selected and screened with PCR for the presence of the human sPLA2-X construct. At least three PCR sPLA2-X-positive and -negative RAW264.7 clones were used for all analyses. Northern Blotting—To verify expression of sPLA2-X in transfected RAW264.7 cells, RNA was isolated from positive and negative sPLA2-X clones using Tri-Reagent (Sigma) according to the manufacturer's instructions. Equal amounts (7.5 μg) of RNA were separated by electrophoresis through denaturing agarose gel (1% w/v) containing 7.5% formaldehyde, transferred to Hybond N, and baked for 2 h at 80 °C. Hybridization was performed on the blots with a 32P-labeled sPLA2-X fragment at 54 °C in hybridization mixture (50% formamide, 1% SDS, 10% dextran sulfate, 5× SSC, 1× Denhardt's, 0.2 m NA2PO4, 50 mg/ml sonicated salmon sperm DNA). Phospholipase Activity—Phospholipase activity was measured in culture medium from sPLA2-X and control RAW264.7 cells using a sPLA2 assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Quantitative PCR—For quantitative PCR analysis, total RNA was isolated from hu-sPLA2-X and control cells using Tri-Reagent (Sigma). Quantity and quality of the RNA were checked on the nanodrop (Witec AG) according to the manufacturer's instructions. RNA (500 ng) was converted into cDNA using the iScript™ cDNA synthesis kit (Bio-Rad) according to the manufacturer's protocol. MyIQ analysis was performed on 10 ng of cDNA using the qPCR iQ™ Custom SYBR® Green Supermix (Bio-Rad) and 300 nm primer. Analysis was performed on an iCycler® thermal cycler (Bio-Rad). Relative expression levels were determined by correction with the housekeeping gene GAPDH for RNA concentration differences. Primer sequences are available upon request. Western Blotting—Cells were lysed in radioimmune precipitation assay buffer containing 1 mm phenylmethylsulfonyl fluoride. After centrifugation for 10 min at 13.2 krpm, protein concentration was determined using a BCA protein assay kit (Pierce). Equal amounts of protein (20 μg) were boiled in Laemmli buffer and loaded on 10-12% polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride membranes. After blocking with 10% nonfat dry milk or 5% bovine serum albumin in phosphate-buffered saline containing 0.1% Tween-20, membranes were incubated overnight at 4 °C with antibodies against hu-sPLA2-X (1 μg/ml, ab47111 Abcam), CD36 (1:1000, MAB1258 Chemicon), and SR-A (1 μg/ml, 2F8). After extensive washing, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using enhanced chemiluminescence (PerkinElmer Life Sciences). β-Actin was used as a loading control. ELISA and Nitric Oxide (NO) Quantification—sPLA2-X and control RAW264.7 cells were activated with 250 ng/ml LPS. ELISA was performed for tumor necrosis factor (TNF), and interleukins (IL) 6 and 10 according to the manufacturer's protocol (BIOSOURCE Inc., Camarillo, CA). Prostaglandin E2 (PGE2) and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) were measured with competitive immunoassay kits according to the manufacturer's instructions (Assay Designs, Ann Arbor, MI). Production of nitric oxide was assessed by measuring the accumulation of nitrite in the cell culture supernatants by the Griess reaction as described (12Schmidt H.H.H.W. Kelm M. Feelisch M. Stamler J. Methods in Nitric Oxide Research. Wiley, Chichester1996: 491-498Google Scholar). Cell Activation—To quantify the cellular response to LPS stimulation, sPLA2-X and control RAW264.7 cells were incubated with 250 ng/ml LPS for 24 h. Activated macrophages, defined as flattened cells with extensions of pseudopodia, were determined based on an average count of three fields along a vertical cross-section (top, middle, bottom) of each well on an inverted microscope (Eclipse E800, Nikon) using a 20× objective lens. LDL Loading and High Performance Thin Layer Chromatography (HPTLC)—Normal low-density lipoproteins (LDL) were isolated from the serum of healthy blood donors (obtained from De Stichting Sanquin Bloedvoorziening, the Netherlands). LDL isolation and CuSO4 oxidation of LDL were performed as described previously (13Hendriks W.L. Van der Boom H. van Vark L.C. Havekes L.M. Biochem. J. 1996; 314: 563-568Crossref PubMed Scopus (49) Google Scholar). LDL and oxidized LDL (oxLDL) were stored under N2 at 4 °C and used within 2 weeks after isolation/modification. sPLA2-X and control RAW264.7 cells were plated at a density of 2 × 106 cells in 6-well plates in serum-free medium (Optimem, Invitrogen) constituted with 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine. After a 4-h adherence period, 25 μg/ml LDL or oxLDL was added to the medium. The cells were incubated for 24 h after which they were washed, lifted, and processed for HPTLC as described previously (14Groeneweg M. Kanters E. Vergouwe M.N. Duerink H. Kraal G. Hofker M.H. de Winther M.P. J. Lipid Res. 2006; 47: 2259-2267Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Generation of sPLA2-X Transgenic Mice—The same macrophage-specific hu-sPLA2-X construct as described above was also used to generate transgenic mice. Oocyte injection was performed according to standard procedures in C57BL/6J mice (15Hogan B. Beddington R. Constantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). The local Committee for Animal Welfare approved all experiments. The animals were housed under specific pathogen-free conditions, and microbiological status was checked regularly. Histology—Whole mice were formalin-fixed and transversal sections either of a whole mouse or of the different tissues were embedded in paraffin. 3-micron sections were cut and used for general histological examination based on hematoxylin and eosin staining. To determine the presence of human sPLA2-X, lung sections were stained with polyclonal rabbit anti-human sPLA2-X antisera (1:50), a generous gift from Dr. Gelb (University of Washington). Periodic acid-Schiff (PAS) and periodic acid-Schiff after diastase digestion (PAS/D) were performed on lung sections to determine and discriminate the presence of glycogen and mucus. For the detection of surfactant phospholipids (SP), lung sections were stained with a polyclonal rabbit anti-SP-B antibody (1:100), a generous gift from Dr. van Iwaarden (University Maastricht). An experienced animal pathologist (M. G.) performed all pathology and histology of the mice. Statistics—All data are expressed as mean ± S.D. of duplicate or triplicate measurements as indicated for each measurement. Statistical analysis was performed using the Student's t test using GraphPad PRISM software. Statistical significance was set at p < 0.05. Generation of a CD68 hu-sPLA2-X Construct and Establishment of Stable hu-sPLA2-X-overexpressing RAW264.7 Cells—A macrophage-specific human sPLA2-X construct was generated to study the function of sPLA2-X in murine RAW264.7 macrophages. The human CD68 promoter was selected for its ability to deliver high expression in murine macrophages specifically (11Gough P.J. Gordon S. Greaves D.R. Immunology. 2001; 103: 351-361Crossref PubMed Scopus (77) Google Scholar). The Met-32 construct of the human sPLA2-X gene has proven to deliver potent sPLA2-X activity without necessary processing to activate the enzyme (8Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The Met-32 construct was cloned behind the CD68 promoter (Fig. 1A). Additional verification was performed using various restriction enzymes and subsequent gel electrophoresis (data not shown). The expression of human sPLA2-X in stably transfected RAW264.7 cell lines was confirmed at the RNA and protein level (Fig. 1, B and C). At least three independent clones overexpressing sPLA2-X and three negative clones (controls) were selected for subsequent experiments. For transparency purposes, results presented are of a representative negative (ctrl) and positive (grX) clone. First, phospholipase activity was measured in the medium of transfected and control cells. As expected, overexpression of the Met-32 construct of hu-sPLA2-X resulted in enhanced phospholipase activity (∼5-fold) compared with control cells (Fig. 1D). sPLA2-X Overexpression Inhibits Macrophage Activation—The grX macrophages already revealed differences in culturing. Upon incubation with LPS, grX cells showed increased cellular activation characteristics such as cell flattening, increased adherence, and extension of pseudopodia (Fig. 2, A and B). Further elaborating on the morphological differences between activated control and grX cells, we measured NO production (Fig. 2C). In agreement with the morphological features of the cells, grX macrophages produced ∼50% less NO after 24 h of LPS stimulation. The inhibition in NO production resulted from a reduced inducible nitric-oxide synthase (iNOS) expression in sPLA2-X cells (Fig. 2D). The LPS treatment did not alter sPLA2-X activity (data not shown). sPLA2-X Overexpression Enhances the Anti-inflammatory Response of Macrophages—To investigate the effects of sPLA2-X overexpression on macrophage function further, we measured expression and secretion of the inflammatory cytokines TNF, IL-10, and IL-6 upon LPS exposure. Differences in mRNA expression and protein secretion between ctrl and grX cells were observed at all time points. In Fig. 3, both expression and secretion of TNF, IL-10, and IL-6 are shown at their peak based on protein secretion. The pro-inflammatory cytokine TNF was strongly induced after 4 h of LPS, but grX cells showed a marked reduction in TNF expression and subsequent release compared with ctrl cells (Fig. 3, A and B). In addition to TNF, the anti-inflammatory cytokine IL-10 was also measured. IL-10 mRNA levels in grX cells were already higher at basal conditions, and this difference increased upon LPS exposure (Fig. 3C). IL-10 secretion was highest after 24 h of LPS, and a 2-fold increase was observed in the grX cells compared with ctrl cells (Fig. 3D). These data suggest that overexpression of sPLA2-X leads to a more antiinflammatory macrophage phenotype. Finally, IL-6 expression and secretion was measured. IL-6 is a key cytokine in activation and regulation of the immune system during infections and inflammation. grX cells were more responsive in LPS-induced IL-6 expression and secretion (Fig. 3, E and F). Interestingly, overexpression of another secretory phospholipase, sPLA2-IIa (4Ghesquiere S.A. Gijbels M.J. Anthonsen M. van Gorp P.J. Van der Made I. Johansen B. Hofker M.H. de Winther M.P. J. Lipid Res. 2005; 46: 201-210Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), in macrophages did not affect cytokine production (supplemental data).FIGURE 3Cytokine profiles after activation of RAW264.7 cells expressing hu-sPLA2-X. Graphs depicted on the left side (A, C, E) show the respective gene expression levels (corrected for GAPDH) of TNF, IL-10, and IL-6 in unstimulated and LPS (250 ng/ml)-stimulated RAW264.7 cells expressing hu-sPLA2-X. On the right side (B, D, F) are the corresponding protein levels of the cytokines as measured by ELISA. The results shown are of control cells (ctrl), which are negative for hu-sPLA2-X expression, and hu-sPLA2-X-expressing macrophages (grX). These results are representative of two independent experiments with n = 3; *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Enhanced Release of PGE2 and 15d-PGJ2 in sPLA2-X Macrophages during Activation—One of the mechanisms by which the inflammatory process is controlled is via the regulatory interaction of prostaglandin E2 and IL-6. sPLA2-X activity leads to the generation of arachidonic acid (AA), an essential substrate in the production of anti-inflammatory prostaglandins like PGE2 and 15d-PGJ2 through the action of cyclooxygenase (Cox)-2. Therefore, we investigated the effects of sPLA2-X overexpression on Cox-2 expression and secretion of PGE2 and 15d-PGJ2 upon activation with LPS. Cox-2 expression was induced upon LPS stimulation, and this induction was significantly higher in grX cells (Fig. 4A). PGE2 and 15d-PGJ2 levels in non LPS-treated macrophages were mildly but significantly higher in the overexpressing cells compared with the control cells (Fig. 4, B and C). After 4 h of LPS activation, PGE2 levels increased, with more PGE2 being produced by the grX cells. At 24 h of activation, PGE2 levels in sPLA2-X-overexpressing macrophages increased more than 8-fold compared with the control macrophages. A similar pattern was seen for 15d-PGJ2, but this prostaglandin was induced later. These results show that sPLA2-X activity in macrophages leads to elevated PGE2 and 15d-PGJ2 levels under basal conditions and that the difference with control cells increases even more dramatically upon LPS stimulation. sPLA2-X Overexpression Enhances Foam Cell Formation—sPLA2-X is potentially proatherogenic because of its capacity to modify lipoproteins. To study the effects of sPLA2-X overexpression on cholesterol accumulation and foam cell formation, sPLA2-X-overexpressing macrophages were incubated with normal or oxidative-modified LDL for 24 h. Results show enhanced cholesterol ester accumulation of group X-overexpressing macrophages (Fig. 5A). Even without addition of (ox)LDL, the grX macrophages accumulated low levels of cholesterol esters, probably derived from the serum present in the culture medium. Incubation with LDL, which can be modified by sPLA2, led to a ∼3-fold increase in cholesterol ester accumulation in the sPLA2-X cells. The difference was even more pronounced upon incubation with oxLDL. Gene expression and protein analyses showed that this difference is most likely due to up-regulation of scavenger receptor CD36 (Fig. 5, B and D), but not SR-A (Fig. 5, C and D) in sPLA2-X macrophages. These data suggest that sPLA2-X enhances lipid accumulation not only via lipid modification, but also through up-regulation of CD36 in macrophages. Generation of Macrophage-specific sPLA2-X Transgenic Mice—After studying the role of sPLA2-X overexpression in macrophages in vitro, we proceeded to an in vivo model to study the role of macrophage-specific overexpression of sPLA2-X. To establish sPLA2-X transgenic mice, three oocyte injection rounds were performed using the same CD68 sPLA2-X construct as used to generate the cell lines described above. Only three founder mice, of 55 born, were tested positive for hu-sPLA2-X by PCR (data not shown). The success rate of this construct (5.5%) was compared with two other macrophage-specific constructs used to generate transgenic mice (x1 and x2, Table 1) at the same time, and in the same facility under the same conditions. These other two macrophage-specific constructs yielded 18.6 and 8.9% transgenic founder mice, respectively. Moreover, the founder mice from these latter constructs mostly transmitted their transgene over two generations (7/8 and 4/4, respectively). For the sPLA2-X transgenic mice, the first founder (female, no. 1) died within the first 3 weeks after birth. The second founder mouse (male, no. 2) showed very poor growth and died at 3 weeks of age. The third founder mouse (male, no. 3) exhibited no external signs of pathology and reached adulthood. Breeding of this mouse resulted in a total offspring of 62 mice, of which only one animal tested positive for the transgene. Breeding of this F1 mouse resulted in 16 pups of which none tested positive for the sPLA2-X construct.TABLE 1Comparison of three macrophage-specific constructs used to produce transgenic miceConstructMice born[Nest size]PCR-positive miceTransgenesis success rateTransmission efficiency over 2 generations#%CD68 hu-sPLA2 X554.135.50/3x1435.1818.67/8x2455.048.94/4 Open table in a new tab Macrophage-specific sPLA2-X Transgenic Mice Die Because of Severe Lung Pathology—s PLA2-X founder no. 1 and no. 2 died within the first 3 weeks after birth. Pathological examination revealed severe lung pathology (Fig. 6). The cause of death was an interstitial pneumonia with severe infiltration of alveolar macrophages and edema (Fig. 6, A and B). The presence of macrophage-secreted human PLA2-X in lung sections of the transgenic sPLA2-X mice was confirmed by immunohistochemistry (Fig. 6, C and D). Other tissues like liver and bone marrow also showed positive staining for hu-sPLA2-X in monocytes/macrophages (data not shown). Further histological examination of the lungs was performed, and lung sections were stained with PAS and PAS/D. PAS staining detects glycogen stores, which are abundantly present in immature epithelial cells before conversion of glycogen into surfactant, and as such PAS can be used as a marker for immature lung development. However, PAS staining will also detect mucus, of which the presence in lungs often coincides with inflammation. Therefore, we also performed a PAS/D staining, which only stains cytoplasmic neutral mucin droplets, by digesting the glycogen. Whereas lungs of wild-type (Wt) mice were negative for both PAS and PAS/D staining, lungs of the sPLA2-X mice showed strong colocalized equally distributed staining for both PAS (Fig. 6, E and F) and PAS/D (Fig. 6, G and H) showing that the lungs of the sPLA2-X-overexpressing mice were filled with mucus. Several reports have shown that sPLA2s are involved in the hydrolysis of surfactant phospholipids, thereby leading to malfunction of the lung surfactant and subsequent lung failure. The pulmonary surfactant system is a complex mixture of lipids and proteins required for maintenance of the lung alveoli and minimization of the work of breathing during regular volume changes (16Daniels C.B. Orgeig S. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 9-36Crossref PubMed Scopus (46) Google Scholar). Hydrophobic SP-B is an important component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure (17Nogee L.M. Garnier G. Dietz H.C. Singer L. Murphy A.M. deMello D.E. Colten H.R. J. Clin. Investig. 1994; 93: 1860-1863Crossref PubMed Scopus (471) Google Scholar). Therefore, we stained lung sections of the sPLA2-X mice for the presence of SP-B. Results showed clear staining of the type II alveolar epithelial cells, the producers of SP-B, in both Wt and sPLA2-X m" @default.
• W2149804875 created "2016-06-24" @default.
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• W2149804875 title "Macrophage Secretory Phospholipase A2 Group X Enhances Anti-inflammatory Responses, Promotes Lipid Accumulation, and Contributes to Aberrant Lung Pathology" @default.
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