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• W2029383635 abstract "It has recently been shown that serglycin is essential for maturation of mast cell secretory granules. However, serglycin is expressed also by other cell types, and in this study we addressed the role of serglycin in macrophages. Adherent cells were prepared from murine peritoneal cell populations and from spleens, and analyzed for proteoglycan synthesis by biosynthetic labeling with [35S]sulfate. Conditioned media from serglycin–/– peritoneal macrophages and adherent spleen cells displayed a 65–80% reduction of 35S-labeled proteoglycans, compared with corresponding material from serglycin+/+ cells, indicating that serglycin is the dominant secretory proteoglycan in macrophages of these origins. In contrast, the levels of intracellular proteoglycans were similar in serglycin+/+ and serglycin–/– cells, suggesting that serglycin is not stored intracellularly to a major extent in macrophages. This is in contrast to mast cells, in which serglycin is predominantly stored intracellularly. Transmission electron microscopy revealed that the absence of serglycin did not cause any major morphological effects on peritoneal macrophages, in contrast to dramatic defects in intracellular storage vesicles in peritoneal mast cells. Several secretory products were not found to be affected by the lack of serglycin. However, the secretion of tumor necrosis factor-α in response to lipopolysaccharide stimulation was markedly higher in serglycin–/– cultures than in those of serglycin+/+. The present report thus demonstrates that serglycin is the major proteoglycan secreted by peritoneal macrophages and suggests that the macrophage serglycin may have a role in regulating secretion of tumor necrosis factor-α. It has recently been shown that serglycin is essential for maturation of mast cell secretory granules. However, serglycin is expressed also by other cell types, and in this study we addressed the role of serglycin in macrophages. Adherent cells were prepared from murine peritoneal cell populations and from spleens, and analyzed for proteoglycan synthesis by biosynthetic labeling with [35S]sulfate. Conditioned media from serglycin–/– peritoneal macrophages and adherent spleen cells displayed a 65–80% reduction of 35S-labeled proteoglycans, compared with corresponding material from serglycin+/+ cells, indicating that serglycin is the dominant secretory proteoglycan in macrophages of these origins. In contrast, the levels of intracellular proteoglycans were similar in serglycin+/+ and serglycin–/– cells, suggesting that serglycin is not stored intracellularly to a major extent in macrophages. This is in contrast to mast cells, in which serglycin is predominantly stored intracellularly. Transmission electron microscopy revealed that the absence of serglycin did not cause any major morphological effects on peritoneal macrophages, in contrast to dramatic defects in intracellular storage vesicles in peritoneal mast cells. Several secretory products were not found to be affected by the lack of serglycin. However, the secretion of tumor necrosis factor-α in response to lipopolysaccharide stimulation was markedly higher in serglycin–/– cultures than in those of serglycin+/+. The present report thus demonstrates that serglycin is the major proteoglycan secreted by peritoneal macrophages and suggests that the macrophage serglycin may have a role in regulating secretion of tumor necrosis factor-α. Proteoglycans (PGs) 5The abbreviations used are: PG, proteoglycan; CS, chondroitin sulfate; cABC, chondroitinase ABC; CTL, cytotoxic T lymphocyte; DS, dermatan sulfate; GAG, glycosaminoglycan; HS, heparan sulfate; IL-1α, interleukin-1α; LPS, lipopolysaccharide; MIP-1α, macrophage inflammatory protein-1α; MMP, matrix metalloproteinase; NK, natural killer; NDST-2, N-deacetylase/N-sulfotransferase-2; SG, serglycin; TACE, TNF-α-converting enzyme; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline; ANOVA, analysis of variance. are multifunctional molecules, residing both on cell surfaces, in the extracellular matrix, and in intracellular compartments (1Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1343) Google Scholar, 2Kolset S.O. Prydz K. Pejler G. Biochem. J. 2004; 379: 217-227Crossref PubMed Scopus (139) Google Scholar, 3Lander A.D. Selleck S.B. J. Cell Biol. 2000; 148: 227-232Crossref PubMed Scopus (225) Google Scholar, 4Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (593) Google Scholar). PGs are composed of a protein core to which glycosaminoglycan (GAG) side chains are attached, and many of the functions of PGs have been ascribed to these chains (5Handel T.M. Johnson Z. Crown S.E. Lau E.K. Proudfoot A.E. Annu. Rev. Biochem. 2005; 74: 385-410Crossref PubMed Scopus (432) Google Scholar). However, several functions have been reported to reside in the protein cores of PGs, as shown e.g. for aggrecan (6Kiani C. Chen L. Wu Y.J. Yee A.J. Yang B.B. Cell Res. 2002; 12: 19-32Crossref PubMed Scopus (478) Google Scholar) and perlecan (7Makatsori E. Lamari F.N. Theocharis A.D. Anagnostides S. Hjerpe A. Tsegenidis T. Karamanos N.K. Anticancer Res. 2003; 23: 3303-3309PubMed Google Scholar). The GAG chains, either of chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), or heparin type, have been shown to interact with a series of molecules, and to regulate their activities (8Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1243) Google Scholar, 9Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1678) Google Scholar). For example, PGs are known to interact with such diverse molecules as mast cell chymases (10Pejler G. Maccarana M. J. Biol. Chem. 1994; 269: 14451-14456Abstract Full Text PDF PubMed Google Scholar), antithrombin (11Princivalle M. Hasan S. Hosseini G. de Agostini A.I. Glycobiology. 2001; 11: 183-194Crossref PubMed Scopus (37) Google Scholar), cytokines (12Schonherr E. Hausser H.J. Dev. Immunol. 2000; 7: 89-101Crossref PubMed Scopus (234) Google Scholar), lipoprotein lipase (13Kolset S.O. Salmivirta M. Cell Mol. Life Sci. 1999; 56: 857-870Crossref PubMed Scopus (69) Google Scholar), and fibroblast growth factor (14Vlodavsky I. Miao H.Q. Medalion B. Danagher P. Ron D. Cancer Metastasis Rev. 1996; 15: 177-186Crossref PubMed Scopus (270) Google Scholar). PGs in intracellular granules and vesicles are receiving increasing attention (2Kolset S.O. Prydz K. Pejler G. Biochem. J. 2004; 379: 217-227Crossref PubMed Scopus (139) Google Scholar). Studies in which the gene coding for N-deacetylase/N-sulfotransferase-2 (NDST-2), an important enzyme in HS/heparin biosynthesis, was targeted, showed that the lack of heparin in mast cells led to generation of storage granules without heparin and that the lack of heparin caused major defects in the storage of other granule constituents, such as proteases and histamine (15Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellen L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (406) Google Scholar, 16Humphries D.E. Wong G.W. Friend D.S. Gurish M.F. Qiu W.T. Huang C. Sharpe A.H. Stevens R.L. Nature. 1999; 400: 769-772Crossref PubMed Scopus (361) Google Scholar). The heparin chains in mast cell secretory granules have commonly been thought to be attached to the serglycin (SG) core protein and, indeed, the recent targeting of the SG gene resulted in similar defects in mast cell granule storage as those observed after the knock-out of NDST-2 (17Abrink M. Grujic M. Pejler G. J. Biol. Chem. 2004; 279: 40897-40905Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Together, these data provide strong support for an essential role of SG in mediating storage of mast cell secretory granule compounds. However, SG expression has been detected in a multitude of cells other than mast cells (reviewed in Ref. 2Kolset S.O. Prydz K. Pejler G. Biochem. J. 2004; 379: 217-227Crossref PubMed Scopus (139) Google Scholar), e.g. macrophages, cytotoxic T lymphocytes (CTLs) and neutrophils. In a recent report it was shown that CTLs from serglycin knock-out (SG–/–) mice displayed defects in the storage of granzyme B along with morphological defects of the lytic granule, although storage of several other granule constituents were not affected (18Grujic M. Braga T. Lukinius A. Eloranta M.L. Knight S.D. Pejler G. Abrink M. J. Biol. Chem. 2005; 280: 33411-33418Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In this study we specifically addressed the role of SG in macrophages. Our results show that macrophage SG, in contrast to SG in mast cells and CTLs, is predominantly secreted, and that it may have a role in regulating the secretion of tumor necrosis factor-α (TNF-α). Animals—Wild type, SG–/–, and NDST-2 knock-out (NDST-2–/–) mice on C57BL/6J genetic background were as described previously (15Forsberg E. Pejler G. Ringvall M. Lunderius C. Tomasini-Johansson B. Kusche-Gullberg M. Eriksson I. Ledin J. Hellman L. Kjellen L. Nature. 1999; 400: 773-776Crossref PubMed Scopus (406) Google Scholar, 17Abrink M. Grujic M. Pejler G. J. Biol. Chem. 2004; 279: 40897-40905Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In some experiments SG heterozygote (SG+/–) mice were also used. Age-matched littermates (8–12 weeks) of both genders were used in all experiments, but the same gender was used consistently within each experiment. Animals were maintained according to the guidelines established by the Swedish National Board for Laboratory Animals, and all experiments were approved by the local ethical committee. Isolation and Culture of Peritoneal Macrophages—Mice were sacrificed by CO2 asphyxiation, and resident peritoneal cells were isolated by washing the peritoneal cavities with cold PBS (one mouse yielded 2–3 × 106 viable peritoneal cells). Isolated cells were washed three times in PBS by centrifugation at 300 × g for 10 min at 4 °C and resuspended in serum-free RPMI 1640 culture medium containing 2 mm l-glutamine and 50 μg/ml gentamicin. Cell culture reagents were purchased from Sigma, unless otherwise stated. Cell viability was determined by trypan blue exclusion. The cells were seeded at either 1 × 106 cells/500 μl of culture medium in 24-well plates, 1 × 106 cells/ml culture medium in 6-well plates, or 8 × 106 cells/10 ml of culture medium in 9-cm Petri dishes, and cultured at 37 °C in 5% CO2. After incubation for 2 h, non-adherent cells were removed by washing with Ca2+/Mg2+-free PBS three times. Peritoneal macrophages were obtained at a purity of greater than 95% by this procedure (19Uhlin-Hansen L. Eskeland T. Kolset S.O. J. Biol. Chem. 1989; 264: 14916-14922Abstract Full Text PDF PubMed Google Scholar, 20Uhlin-Hansen L. Wik T. Kjellen L. Berg E. Forsdahl F. Kolset S.O. Blood. 1993; 82: 2880-2889Crossref PubMed Google Scholar). Adherent cells were cultured further in the serum-free medium specified above, unless otherwise stated. In some experiments, peritoneal cells were depleted from mast cells by density gradient centrifugation on metrizamide (21Sterk A.R. Ishizaka T. J. Immunol. 1982; 128: 838-843PubMed Google Scholar), followed by culturing of mast cell-depleted peritoneal cells as well as mast cell-depleted peritoneal cells reconstituted with purified mast cells. Isolation and Culture of Spleen Cells—Spleens were aseptically removed and placed in Petri dishes containing 5 ml of cold PBS. The spleens were then cut in pieces and the spleen cells were recovered using the plunger of a 10-ml syringe. Red blood cells were lysed for 5 min at room temperature in a lysis buffer containing 150 mm NH4Cl, 10 mm KHCO3, and 0.1 mm Na2EDTA (pH 7.4). Cells were washed three times in PBS by centrifugation at 300 × g for 10 min at 4 °C and resuspended in the same culture medium as was used for peritoneal macrophages. Cell viability was determined by trypan blue exclusion. One spleen yielded 6–9 × 107 viable cells. The cells were seeded at a concentration 4 × 106 cells/500 μl culture medium in 24-well plates or 1 × 107 cells/10 ml of culture medium in 9-cm Petri dishes and cultured at 37 °C in 5% CO2. After incubation for 2 h, non-adherent cells were removed by washing with Ca2+/Mg2+-free PBS three times followed by the addition of fresh culture medium. 35S Labeling of Macromolecules—For radiolabeling of macromolecules, adherent cells were cultured in sulfate-free RPMI 1640 culture medium (Invitrogen), with 2 mm l-glutamine added, and exposed to [35S]sulfate (50 μCi/ml) (PerkinElmer Life Sciences, Boston, MA) for 24 h. PG Analyses—Conditioned media and cell fractions from peritoneal macrophages and adherent spleen cells were collected, and purified using DEAE Sephacel anion exchange chromatography (Amersham Biosciences), as previously described (17Abrink M. Grujic M. Pejler G. J. Biol. Chem. 2004; 279: 40897-40905Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The fractions were analyzed for 35S radioactivity using liquid scintillation counting. Fractions containing radioactivity were pooled, and low molecular [35S]sulfate substances and free [35S]sulfate were removed using PD-10 desalting columns (Amersham Biosciences). After desalting, aliquots of the samples were analyzed for 35S radioactivity. Portions of the samples were further treated with papain, as previously described (17Abrink M. Grujic M. Pejler G. J. Biol. Chem. 2004; 279: 40897-40905Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). After desalting, the samples were concentrated using a SpeedVac system. Samples of isolated [35S]sulfate-labeled GAGs from conditioned medium (∼10,000 cpm) and from cell fractions (∼7000 cpm) were mixed with 0.5 mg each of unlabeled internal standards of CS-A and pig mucosal heparin, and applied to a 1-ml DEAE Sephacel anion exchange column connected to a HPLC system, as previously described (17Abrink M. Grujic M. Pejler G. J. Biol. Chem. 2004; 279: 40897-40905Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The samples were eluted using a linear LiCl gradient (50 mm to 2 m). Collected fractions were analyzed for 35S radioactivity and for uronic acid content using the carbazole reaction (22Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5205) Google Scholar) to detect the internal standards. GAGs released from the PG protein cores (∼10,000 cpm), using either NaOH (23Cheung W.F. Eriksson I. Kusche-Gullberg M. Lindhal U. Kjellen L. Biochemistry (Mosc). 1996; 35: 5250-5256Crossref Scopus (49) Google Scholar) or papain, were subjected to HNO2 (pH 1.5) or chondroitinase ABC (cABC) treatment, as previously described (24Svennevig K. Prydz K. Kolset S.O. Biochem. J. 1995; 311: 881-888Crossref PubMed Scopus (41) Google Scholar). cABC (from Proteus vulgaris) was purchased from Seikagaku Corporation (Tokyo, Japan). The amounts of HS and CS were calculated from the proportions of degradation products by gel chromatography on Superose 6 after HNO2 or cABC treatment, respectively. To analyze macromolecular properties of the macrophage PGs, aliquots of the [35S]PGs were analyzed by size exclusion chromatography, before and after treatment with NaOH or papain. Samples (∼7000 cpm) were analyzed by Superose 6 gel chromatography (Amersham Biosciences) using 1 m NaCl as mobile phase. The elution profiles were monitored by liquid scintillation counting of eluted fractions. Pulse-Chase Experiments—Peritoneal macrophages were cultured at a density of 5 × 105 cells/500 μl of sulfate-free RPMI 1640 culture medium, with 2 mm l-glutamine added, and exposed to 500 μCi/ml of [35S]sulfate for 30 min. The cells were then washed five times with culture medium to remove free [35S]sulfate. Thereafter the cells were chased for 30 min to 24 h in RPMI 1640 culture medium containing 2 mm l-glutamine and 50 μg/ml gentamicin. Conditioned media were collected, and loose cells were separated from the media by centrifugation at 350 × g for 3 min. Cells were washed three times with PBS and lysed in 1 m NaCl and 1% (v/v) Triton X-100. All cell fractions and conditioned media were analyzed by Superose 6 gel chromatography using 1 m NaCl and 1% (v/v) Triton X-100 as mobile phase. Some samples were subjected to NaOH treatment to release the GAGs from the PG protein cores. The elution profiles were monitored by liquid scintillation counting of eluted fractions. Lysozyme Analyses—Lysozyme activity was measured using a turbidimetric assay based on the ability of lysozyme to disrupt the cell wall of the bacterium Micrococcus lysodeikticus. Peritoneal macrophages were cultured in vitro in the absence or presence of 1 μg/ml lipopolysaccharide (LPS) from Eschericha coli serotype 055:B5 (Sigma). Conditioned medium was harvested after 24 h incubation, and loose cells were separated from the medium by centrifugation at 350 × g for 3 min. Next, 50 μl of the medium was added to a mixture of 125 μl of PBS (pH 6.2) and 25 μl of a M. lysodeikticus suspension (20 mg/ml in PBS, pH 6.2) in microtiter plate wells and incubated at 37 °C with gentle agitation. Lysis of the bacteria leads to a decrease in absorbance at 595 nm, which was measured at different time points. Matrix Metalloproteinase Analyses—The possible presence of matrix metalloproteinase (MMP) activities was investigated using gelatin zymography. Briefly, conditioned media were separated on 7.5% polyacrylamide gels containing 0.1% gelatin (type A, from porcine skin, Sigma). After electrophoresis, gels were washed with 2.5% (v/v) Triton X-100 and further incubated overnight at 37 °C in 50 mm Tris (pH 7.5), 200 mm NaCl, 5mm CaCl2, and 0.02% (w/v) Brij 35 (Sigma), followed by staining with Coomassie Brilliant Blue. Positive controls of MMP-2 (25Loennechen T. Mathisen B. Hansen J. Lindstad R.I. El Gewely S.A. Andersen K. Maelandsmo G.M. Winberg J.O. Biochem. Pharmacol. 2003; 66: 2341-2353Crossref PubMed Scopus (18) Google Scholar, 26Mathisen B. Lindstad R.I. Hansen J. El Gewely S.A. Maelandsmo G.M. Hovig E. Fodstad O. Loennechen T. Winberg J.O. Clin. Exp. Metastasis. 2003; 20: 701-711Crossref PubMed Scopus (58) Google Scholar) and MMP-9 (27Winberg J.O. Kolset S.O. Berg E. Uhlin-Hansen L. J. Mol. Biol. 2000; 304: 669-680Crossref PubMed Scopus (78) Google Scholar, 28Winberg J.O. Berg E. Kolset S.O. Uhlin-Hansen L. Eur. J. Biochem. 2003; 270: 3996-4007Crossref PubMed Scopus (47) Google Scholar) were run with each gel. Cytokine Analyses—Peritoneal macrophages and adherent spleen cells were cultured in vitro in the absence or presence of 1 μg/ml LPS. Conditioned media were collected after 1, 4, and 24 h incubation, and loose cells were separated from the media by centrifugation at 350 × g for 3 min. Macrophage inflammatory protein-1α (MIP-1α), interleukin-1α (IL-1α), and TNF-α levels in conditioned media were assessed by ELISA (R&D Systems, Abingdon, UK), as described by the manufacturer. TNF-α levels were also determined in cell fractions from peritoneal macrophages. In some experiments cells were incubated in the presence of 20 nm of the TNF-α converting enzyme (TACE) inhibitor TIMP-3 (Sigma). Cells were washed three times with PBS and further lysed in PBS containing 1% (v/v) Nonidet P-40 (Sigma), 0.5% (w/v) sodium deoxycholate (Sigma), 0.1% (w/v) SDS and Complete™ protease inhibitor mixture (Roche Applied Science, Mannheim, Germany). Cell debris was pelleted by centrifugation, and the supernatants were collected and subjected to TNF-α analysis. Transmission Electron Microscopy of Peritoneal Cells—Peritoneal cells from SG+/– and SG–/– mice were fixed for 6 h in 2% glutaraldehyde in a 0.1 m sodium cacodylate buffer supplemented with 0.1 m sucrose, followed by 1.5 h of postfixation in 1% osmium tetroxide dissolved in the same cacodylate buffer. After dehydration in ethanol, the cells were embedded in the epoxy resin Agar 100 (Agar Scientific, Stansted, UK). Ultrathin sections were placed on copper grids covered with a film of polyvinyl formal plastic (Formvar, Agar Scientific, Stansted, UK) and contrasted with uranyl acetate and lead citrate. Electron micrographs were taken with a Hitachi electron microscope (Hitachi Ltd., Tokyo, Japan). Statistical Analyses—Statistical significance was tested using a one-way analysis of variance (ANOVA). When more than two groups were compared, a Tamhane's T2 post hoc test was performed for all groups. The significance level was set to 5%, and all analyses were performed using SPSS 12. PGs—Peritoneal macrophages and adherent spleen cells were obtained from SG+/+ and SG–/– mice. Both cell types were cultured in vitro and incubated with [35S]sulfate for 24 h to label PGs. It has previously been established that [35S]sulfate is incorporated almost exclusively into PGs in peritoneal macrophages (29Kolset S.O. Biochem. Biophys. Res. Commun. 1986; 139: 377-382Crossref PubMed Scopus (22) Google Scholar). Labeled [35S]PGs were purified both from the conditioned media and the cell layers. Quantification of the labeled PGs recovered from the respective pools showed that most of the incorporated radioactivity of SG+/+ peritoneal macrophages and adherent spleen cells was in the medium fractions, indicating that a majority of the macrophage PGs are destined for secretion rather than to cell-associated compartments (Table 1, Experiment 1). The absence of SG did not cause any significant effect on the amount of PGs recovered from the cell layers, indicating that intracellular and cell surface-associated PGs in macrophages are mainly of non-SG nature, for example syndecan-1, syndecan-4, and versican (7Makatsori E. Lamari F.N. Theocharis A.D. Anagnostides S. Hjerpe A. Tsegenidis T. Karamanos N.K. Anticancer Res. 2003; 23: 3303-3309PubMed Google Scholar, 30Yeaman C. Rapraeger A.C. J. Cell Biol. 1993; 122: 941-950Crossref PubMed Scopus (79) Google Scholar). In contrast, the absence of SG caused a major reduction (∼65–80%) in the amount of [35S]sulfate-labeled PGs recovered from conditioned media, both from peritoneal macrophages and adherent spleen cells (Table 1). The latter findings clearly suggest that SG constitutes the dominating PG species that is secreted by macrophages. The effect of the SG knock-out on the amount of secreted PGs was reproduced in independent experiments, although the total amount of incorporated radioactivity showed some variation between the different experiments (Table 1).TABLE 1Comparison of incorporation of [35S]sulfate into GAGs of in vitro cultured peritoneal macrophages and adherent spleen cells from SG+/+ and SG–/– miceSource of [35S]sulfate-labeled GAGsSG+/+SG-/-Ratio SG+/+/SG-/-cpm/106 cellsPeritoneal macrophagesExperiment 1aPapain-treated material.Medium fraction104,00023,0004.5Cell fraction9,0009,0001.0Experiment 2bNaOH-treated material.Medium fraction320,000110,0002.9Adherent spleen cellsExperiment 1aPapain-treated material.Medium fraction5,500 ± 1,200cAverage value of three individual mice ± S.D.1,400 ± 100cAverage value of three individual mice ± S.D.3.6Cell fraction600 ± 200cAverage value of three individual mice ± S.D.500 ± 150cAverage value of three individual mice ± S.D.1.2Experiment 2bNaOH-treated material.Medium fraction6,0002,0003.0a Papain-treated material.b NaOH-treated material.c Average value of three individual mice ± S.D. Open table in a new tab To address whether the absence of SG affects the type of GAG chains expressed, [35S]GAGs from peritoneal macrophages were subjected to HNO2 treatment to degrade HS and cABC to depolymerize CS, followed by gel chromatography on Superose 6 to quantify the degradation products produced by the respective treatments (Table 2). These analyses showed that ∼50% of the GAGs secreted by peritoneal macrophages from SG+/+ mice were HS, with the remainder being CS. In contrast, the HS content of the PGs secreted by SG–/– cells was lower, with only ∼25–40% of the GAGs being HS. Thus, there appears to be a preference of HS to attach to the SG core protein in murine macrophages.TABLE 2Comparison of amounts of HS and CS in conditioned media from in vitro cultured peritoneal macrophages from SG+/+ and SG–/– miceGAG typeSG+/+SG-/-%Experiment 1aPapain-treated material.HS5326CS4573Experiment 2bNaOH-treated material.HS5239CS5259a Papain-treated material.b NaOH-treated material. Open table in a new tab To analyze if the targeting of SG would affect the polyanionic properties of [35S]GAGs from peritoneal macrophages, medium and cell derived GAGs (after papain treatment) were analyzed by DEAE anion exchange chromatography together with internal standards of CS and heparin. As shown in Fig. 1, [35S]GAGs derived from conditioned medium of both SG+/+ and SG–/– macrophages eluted before the internal CS standard, indicating a relatively low anionic charge density, whereas the cell-derived [35S]GAGs were eluted later in the salt gradient. However, there was no difference in elution profiles between material from SG+/+ and SG–/– mice, suggesting that GAGs attached to the SG core protein had similar polyanionic properties as those attached to non-SG species. To analyze the macromolecular properties of the macrophage PGs, intact PGs were analyzed by Superose 6 chromatography. In addition, PGs were analyzed after liberating the GAG chains by treatment with NaOH. As shown in Fig. 2A, there were marked differences in the elution profiles of 35S-labeled PGs from SG+/+ and SG–/– mice. A major portion of the PGs secreted by SG+/+ macrophages eluted at Kav ∼0.3. The corresponding peak in the material from SG–/– cells was markedly lower; instead a major peak at Kav ∼0.75 was observed. After NaOH treatment essentially all of the material both from SG+/+ and SG–/– cells eluted with peaks at closely similar Kav values (∼0.75), indicating that the latter elution positions corresponded to free GAG chains and that the GAG chains attached to SG and to other PG species had approximately the same chain length (Fig. 2B). Hence, a major portion of the 35S-labeled material released by SG–/– cells corresponded to free GAG chains. Papain digestion of PGs derived from SG–/– cells also gave an essentially complete conversion of the intact PGs into material eluting at the position of free GAG chains, indicating that the non-SG species secreted by the macrophages were sensitive to protease treatment (Fig. 2C). In contrast, a major part of the PGs secreted by SG+/+ cells was resistant to papain, a property that previously has been ascribed also to SG purified from mast cells (31Yurt R.W. Leid Jr., R.W. Austen K.F. J. Biol. Chem. 1977; 252: 518-521Abstract Full Text PDF PubMed Google Scholar). It is evident from Fig. 2B that the liberated GAG chains from SG+/+ and SG–/– cells eluted at similar positions on the Superose 6 column, indicating that the GAGs attached to SG and to other PG species had approximately the same chain length. To follow synthesis and secretion of PGs, we performed pulsechase experiments. As depicted in Fig. 3, A and B, two cell-associated populations of 35S-labeled PGs were recovered both from SG+/– and SG–/– cells, with one PG population eluting at Kav ∼0.2 and one eluting at Kav ∼0.4. It is also apparent that the SG+/– cells contained proportionally higher levels of the Kav ∼0.4 PG than the SG–/– cells. This strongly suggests that SG is eluting at the Kav ∼0.4 position and that the Kav ∼0.2 peak mostly contains PGs of non-SG species. During the chase period it was further evident that the Kav ∼0.4 PG component was released to the medium to a much larger extent by cells from SG+/– mice (Fig. 3C) than by corresponding cells from SG–/– mice (Fig. 3D). Again, these results are in agreement with SG being a major secretory PG in peritoneal macrophages. Effect of the SG Knock-out on Secretion—Macrophages have previously been shown to secrete a multitude of compounds in response to inflammatory stimuli (32Nathan C.F. J. Clin. Investig. 1987; 79: 319-326Crossref PubMed Scopus (2164) Google Scholar), and many of these products have been shown to interact with SG (33Kolset S.O. Mann D.M. Uhlin-Hansen L. Winberg J.O. Ruoslahti E. J. Leukoc. Biol. 1996; 59: 545-554Crossref PubMed Scopus (66) Google Scholar). Further experiments were therefore performed to investigate if the absence of SG would affect secretion of such molecules. Peritoneal macrophages from SG+/+, SG+/–, and SG–/– mice were cultured in vitro in the absence or presence of LPS (1 μg/ml), a commonly used reagent to trigger cytokine release by macrophages (34Cavaillon J.M. Biomed. Pharmacother. 1994; 48: 445-453Crossref PubMed Scopus (337) Google Scholar, 35Uhlin-Hansen L. Kolset S.O. J. Biol. Chem. 1988; 263: 2526-2531Abstract Full Text PDF PubMed Google Scholar). Conditioned media were recovered and subjected to further analyses. First, the level of secreted lysozyme was investigated. As shown in Fig. 4A, it is clear that the peritoneal macrophages secreted lysozyme and that the level of lysozyme was actually decreased after LPS stimulation (Fig. 4B), the latter being in agreement with previous reports (36Ohno N. Takada K. Kurasawa T. Liang A. Yadomae T. Prog. Clin. Biol. Res. 1998; 397: 179-190PubMed Google Scholar, 37Warfel A.H. Zucker-Franklin D. J. Immunol. 1986; 137: 651-655PubMed Google Scholar). However, the level of activity was not influenced by the absence of SG, indicating that lysozyme secretion is not SG-dependent. Conditioned media were also analyzed for the presence of MMPs, in particular MMP-2 and MMP-9, by gelatin zymography. Although MMP-9 was detected in the conditioned media, no consistent differences in levels or degree of activation of MMP-9 were seen between SG+/+ and SG–/– cultures (Fig. 4, C and D). In a series of expe" @default.
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• W2029383635 creator A5078126056 @default.
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• W2029383635 date "2006-09-01" @default.
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• W2029383635 title "Serglycin Is the Major Secreted Proteoglycan in Macrophages and Has a Role in the Regulation of Macrophage Tumor Necrosis Factor-α Secretion in Response to Lipopolysaccharide" @default.
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