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Originally published In Press as doi:10.1074/jbc.M512889200 on June 28, 2006

J. Biol. Chem., Vol. 281, Issue 37, 26792-26801, September 15, 2006
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Serglycin Is the Major Secreted Proteoglycan in Macrophages and Has a Role in the Regulation of Macrophage Tumor Necrosis Factor-{alpha} Secretion in Response to Lipopolysaccharide*

Lillian Zernichow{ddagger}1, Magnus Åbrink§, Jenny Hallgren§2, Mirjana Grujic§3, Gunnar Pejler§, and Svein O. Kolset{ddagger}4

From the {ddagger}Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Box 1046 Blindern, 0316 Oslo, Norway and the §Department of Molecular Biosciences, Biomedical Centre, Swedish University of Agricultural Sciences, Box 575, 75123 Uppsala, Sweden

Received for publication, December 2, 2005 , and in revised form, June 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{alpha} 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-{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteoglycans (PGs)5 are multifunctional molecules, residing both on cell surfaces, in the extracellular matrix, and in intracellular compartments (1–4). 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 (5). However, several functions have been reported to reside in the protein cores of PGs, as shown e.g. for aggrecan (6) and perlecan (7). 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 (8, 9). For example, PGs are known to interact with such diverse molecules as mast cell chymases (10), antithrombin (11), cytokines (12), lipoprotein lipase (13), and fibroblast growth factor (14).

PGs in intracellular granules and vesicles are receiving increasing attention (2). 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 (15, 16). 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 (17). 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. 2), 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 (18). 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-{alpha} (TNF-{alpha}).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Wild type, SG–/–, and NDST-2 knock-out (NDST-2–/–) mice on C57BL/6J genetic background were as described previously (15, 17). 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 x 106 viable peritoneal cells). Isolated cells were washed three times in PBS by centrifugation at 300 x 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 x 106 cells/500 µl of culture medium in 24-well plates, 1 x 106 cells/ml culture medium in 6-well plates, or 8 x 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 (19, 20). 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 (21), 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 x 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 x 107 viable cells. The cells were seeded at a concentration 4 x 106 cells/500 µl culture medium in 24-well plates or 1 x 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 (17). 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 (17). 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 (17). 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 (22) to detect the internal standards.

GAGs released from the PG protein cores (~10,000 cpm), using either NaOH (23) or papain, were subjected to HNO2 (pH 1.5) or chondroitinase ABC (cABC) treatment, as previously described (24). 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 x 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 x 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 x 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 (25, 26) and MMP-9 (27, 28) 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 x g for 3 min. Macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), interleukin-1{alpha} (IL-1{alpha}), and TNF-{alpha} levels in conditioned media were assessed by ELISA (R&D Systems, Abingdon, UK), as described by the manufacturer. TNF-{alpha} 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-{alpha} 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 CompleteTM protease inhibitor mixture (Roche Applied Science, Mannheim, Germany). Cell debris was pelleted by centrifugation, and the supernatants were collected and subjected to TNF-{alpha} 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (29). 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 (7, 30). 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).


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  		TABLE 1
Comparison of incorporation of [35S]sulfate into GAGs of in vitro cultured peritoneal macrophages and adherent spleen cells from SG+/+ and SG–/– mice

Cells were radiolabeled with [35S]sulfate for 24 h. Conditioned media and cell fractions were recovered, and GAGs were released from the PG protein cores using either papain or NaOH. Isolation of the GAGs was performed using DEAE Sephacel anion exchange chromatography, followed by quantification of incorporated radioactivity using liquid scintillation counting.

 


Figure 1
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  		FIGURE 1.
Characterization of [35S]sulfate-labeled GAG chains from SG mice by anion exchange chromatography. Peritoneal macrophages from SG+/+ and SG–/– mice were cultured in vitro and radiolabeled with [35S]sulfate for 24 h. The PGs were isolated as described under "Experimental Procedures," and treated with papain. Papain-treated PGs from cell fractions (~7000 cpm) and from conditioned media (~10,000 cpm) were mixed with unlabeled internal standards of CS and heparin, and applied to a DEAE Sephacel anion exchange column. Samples were eluted with a LiCl gradient, and collected fractions were analyzed for 35S radioactivity (solid line), and for the internal standard CS and heparin as detected by the carbazole reaction (dashed line). A, cell fraction from SG+/+ mice. B, cell fraction from SG–/– mice. C, medium fraction from SG+/+ mice. D, medium fraction from SG–/– mice.

 


Figure 2
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  		FIGURE 2.
Macromolecular properties of [35S]sulfate-labeled PGs and GAG chains from SG mice. Peritoneal macrophages from SG+/+ (solidline) and SG–/– (dashed line) mice were cultured in vitro and radiolabeled with [35S]sulfate. The PGs and GAG chains in conditioned media were isolated as described under "Experimental Procedures." Samples (~7000 cpm) were applied to a Superose 6 gel filtration column and collected fractions were analyzed for 35S radioactivity. A, untreated. B, NaOH-treated. C, papain-treated.

 
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.


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  		TABLE 2
Comparison of amounts of HS and CS in conditioned media from in vitro cultured peritoneal macrophages from SG+/+ and SG–/– mice

Cells were radiolabeled with [35S]sulfate for 24 h. Conditioned media were recovered, and GAGs were released from the PG protein cores using either papain or NaOH. Isolation of the GAGs was performed using DEAE Sephacel anion exchange chromatography. The amounts of HS and CS, degraded by HNO2 and depolymerized by cABC treatment, respectively, were calculated from the proportions of degradation products using Superose 6 gel chromatography and liquid scintillation counting.

 
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 (31). 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.


Figure 3
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  		FIGURE 3.
Pulse-chase of [35S]PGs. Peritoneal macrophages from SG+/– and SG–/– mice were cultured in vitro (5 x 105 cells per well) and pulsed with 500 µCi/ml of [35S]sulfate for 30 min. After washing, the cells and media were chased for 0, 0.5, 1, 2, 3, and 24 h. Samples were applied to a Superose 6 gel filtration column and collected fractions were analyzed for 35S radioactivity. A, cell fractions from SG+/– mice. B, cell fractions from SG–/– mice. C, medium fractions from SG+/– mice. D, medium fractions from SG–/– mice.

 
Effect of the SG Knock-out on Secretion—Macrophages have previously been shown to secrete a multitude of compounds in response to inflammatory stimuli (32), and many of these products have been shown to interact with SG (33). 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 (34, 35). 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 (36, 37). 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 experiments conditioned media were analyzed for the presence of MIP-1{alpha} and IL-1{alpha}. However, no differences in the levels of these cytokines were observed in medium derived from SG+/+ and SG–/– peritoneal macrophages or adherent spleen cells, neither in the absence nor presence of LPS (not shown). In contrast, the levels of TNF-{alpha} were markedly higher in conditioned medium from LPS-stimulated SG–/– peritoneal macrophages as compared with medium obtained from SG+/+ cells, with medium conditioned by SG+/– peritoneal macrophages having intermediate levels of secreted TNF-{alpha} (Fig. 5A). Medium obtained from non-stimulated cells contained only low levels of TNF-{alpha}, with no differences in TNF-{alpha} levels seen between the different genotypes. Increased levels of secreted TNF-{alpha} were also seen in conditioned medium taken from LPS-stimulated SG–/– adherent spleen cells, as compared with medium from SG+/+ counterparts (Fig. 5B).

To examine whether SG–/– macrophages synthesize more TNF-{alpha} in total than SG+/+ macrophages in response to LPS, or whether they simply secrete a greater portion of the total, we measured the levels of TNF-{alpha} in both cell fractions and conditioned media. The total amount of TNF-{alpha} was approximately similar in peritoneal macrophages from SG+/+ and SG–/– mice (not shown). This indicates that the higher amount of TNF-{alpha} found in the medium of SG–/– mice after stimulation is caused by increased secretion rather than increased synthesis.

It has previously been shown that TACE is up-regulated in macrophages stimulated with LPS (38). To investigate if the loss of SG could affect TNF-{alpha} secretion through mechanisms involving TACE, the cells were incubated in the presence of 20 nM of the TACE inhibitor TIMP-3. Neither the level of cell-associated nor medium TNF-{alpha} was affected by this treatment in both cell types (not shown).


Figure 4
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  		FIGURE 4.
Secretion of lysozyme and MMP from SG mice. Lysozyme activities in conditioned media recovered from in vitro cultured A, unstimulated and B, LPS-stimulated peritoneal macrophages was measured as described under "Experimental Procedures." Cell culture medium was used as negative control. Gelatin zymography of serum-free-conditioned media from C, peritoneal macrophages and D, adherent spleen cells. Cells were cultured for 24 h under serum-free conditions and further analyzed as described under "Experimental Procedures." Note that the MMP-9 band in the zymograms has a somewhat higher molecular mass than the human counterpart (92 kDa). This is consistent with other studies (54, 55).

 
A possible explanation for the increased levels of TNF-{alpha} in medium from SG–/– macrophages could be that contaminating, strongly SG-dependent mast cell proteases, such as mast cell protease 4 (see Ref. 39), were present in the cell cultures and that such proteases could cause degradation of TNF-{alpha} in SG+/+ cultures, leading to lower levels of extracellular TNF-{alpha}. To investigate this we measured the secretion of TNF-{alpha} in peritoneal cell cultures obtained from mice lacking NDST-2. Importantly, the lack of NDST-2 will specifically interfere with mast cell heparin PG with an accompanying dramatic loss of stored of mast cell proteases (15, 16), without having any effect on macrophages. As depicted in Fig. 6A, the levels of secreted TNF-{alpha} were similar in cultures from NDST-2+/+ and NDST-2–/– cells, indicating that the effect of the SG knock-out on TNF-{alpha} secretion was not explained by the absence or presence of contaminating mast cell proteases. It was also important to identify the exact cellular source of the secreted TNF-{alpha}. It is well established that mast cells store large amounts of TNF-{alpha} in their secretory granules (40, 41), and it could therefore not be ruled out that contaminating mast cells release TNF-{alpha} following LPS stimulation. To address this possibility, mast cells were purified from total peritoneal cell populations by density gradient centrifugation (~1–2% of the total peritoneal cells are mast cells). Mast cells were subsequently cultured alone or in combination with the mast cell-depleted peritoneal cells, followed by measurement of secreted TNF-{alpha} in response to LPS. This experiment clearly showed that mast cells alone did not release measurable levels of TNF-{alpha} and that the co-culture of mast cells with macrophages, in ratios corresponding to the normal ratio of mast cells/macrophages in the peritoneum, did not affect the levels of secreted TNF-{alpha} in response to LPS.

Morphology—The absence of SG did not cause any noticeable morphological effects on macrophage morphology as observed by light microscopy after staining with May Grünwald/Giemsa (not shown). In order to assess whether the SG knock-out caused morphological effects at the ultrastructural level, peritoneal macrophages were analyzed by transmission electron microscopy (Fig. 7). Analyses of both SG+/– and SG–/– macrophages revealed normal ultrastructure of membranes, mitochondria and other features, indicating optimal culturing conditions and a successful preparation procedure. However, there were no obvious morphological effects that were caused by the absence of SG expression. In contrast, the absence of SG caused dramatic effects on the secretory granule of peritoneal mast cells, well in line with the reported major mast cell storage defects caused by the knock-out of SG (17).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The SG core protein was first cloned from rat L2 yolk sac tumor cells (42), and subsequent work has identified SG as a major PG in both normal (17) and transformed mast cells (43). However, SG core protein expression is not restricted to mast cells. In particular, SG expression has also been detected in other cells of hematopoietic origin, including macrophages, neutrophils, cytotoxic T lymphocytes and platelets, but SG has also been identified in non-hematopoietic cells such as endothelial cells (44) and pancreatic acinar cells (45). The biological function of SG in the various cell types has not been clear, although it has been widely believed that SG mediates storage of various basic compounds in secretory granules. Indeed, the recent targeting of the SG gene gave strong support for an essential role of SG in the storage of granule compounds in mast cells (17). The latter notion is also supported by an earlier report, in which the sulfation of the heparin side chains of the SG expressed by connective tissue type mast cells were affected through the targeting of NDST-2 (15). In a more recent report it was shown that SG, in addition, is essential for the storage of granzyme B in CTLs and for the dense core formation in the CTL lytic granules (18). However, the storage of granzyme A was not affected in the CTLs, indicating that SG selectively promotes the storage of certain granule constituents, whereas others are independent of SG for storage.


Figure 5
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  		FIGURE 5.
TNF-{alpha} levels in conditioned media from SG mice. A, peritoneal cells from SG+/+, SG+/–, and SG–/– mice were cultured in vitro (1 x 106 cells per well). Conditioned media were collected 1, 4, and 24 h after the removal of non-adherent cells and addition of fresh culture medium, both without (black bars) and with (open bars) the addition of LPS (1 µg/ml). Results are given per 1 x 106 cells. Data are mean ± S.D. of measurement on material from three separate wells. One-way ANOVA post hoc: *, different from SG+/+, p < 0.05. #, different from SG+/–, p < 0.05. B, spleen cells from SG+/+ and SG–/– mice were cultured in vitro (4 x 106 cells per well). Conditioned media were collected 1 and 24 h after the removal of non-adherent cells and addition of fresh culture medium, both without (black bars) and with (open bars) the addition of LPS (1 µg/ml). Results are given per 1 x 106 cells. Data are mean ± range of measurement on material from one well from three SG+/+ and two SG–/– mice. The results shown are from one experiment, but are representative for three independent experiments. One-way ANOVA: *, different from SG+/+, p < 0.05.

 


Figure 6
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  		FIGURE 6.
TNF-{alpha} levels in conditioned media from NDST-2–/– mice. A, peritoneal cells from NDST-2+/+ and NDST-2–/– mice were cultured in vitro in the absence (hatched bars) or presence (open bars) of LPS (1 µg/ml), followed by the measurement of secreted TNF-{alpha} at the time points indicated. Results are given ± S.D. (n = 3). B, peritoneal cells were depleted from mast cells. Mast cell-depleted peritoneal cells (1 x 106 cells per well) were either cultured alone (open circles) or in the presence of 5000 (filled squares) or 15,000 (filled circles) mast cells. Mast cells (15,000 cells) were also cultured alone (open squares). LPS (1 µg/ml) was added to all cell cultures and the levels of TNF-{alpha} in the conditioned media were measured at the time points indicated.

 
In the present study we specifically studied the role of SG expressed by macrophages. We show that the targeting of the SG core protein led to a major reduction in the synthesis of sulfated PGs in both peritoneal macrophages and adherent spleen cells, indicating that SG is the dominant PG species in this cell type. However, the lack of SG did not result in a total loss of PG expression, which is in support of the previous findings that macrophages and monocytes express other PGs, such as syndecan-1, syndecan-4, and versican (7, 30). A striking finding was that the SG inactivation only led to decreased levels of secreted PGs whereas the levels of cell-associated PGs not seemed to be affected. This clearly indicates that macrophages predominantly secrete their SG instead of sorting it into intracellular compartments. This is thus in sharp contrast to mast cells, in which the targeting of SG resulted in the reduction of intracellularly stored PGs without affecting the level of secreted species. Our findings are supported by previous studies comparing content and distribution of PGs (46) and sulfur-containing macromolecules (47) in mast cells and macrophages or monocytes. In line with these findings, we did not see any major observable effects at the ultrastructural level on the morphology of intracellular vesicles in macrophages because of the lack of SG, whereas mast cell secretory granules were dramatically affected. These findings strongly suggest that the macrophage SG, in contrast to mast cell and CTL SG, exerts its biological effects extracellularly.


Figure 7
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  		FIGURE 7.
Transmission electron microscopy of peritoneal macrophages and mast cells from SG+/– and SG–/– mice. Original magnification x8000.

 
Interestingly, we show that SG+/+ macrophages secrete a large portion of PGs that are protease-resistant, whereas PGs secreted from SG–/– macrophages were to a large extent degraded. This indicates that macrophage SG is resistant to protease digestion and this is clearly in line with the previous observation that mast cell SG is resistant to similar treatment, presumably because of the dense substitution of the Ser-Gly repeats in the GAG attachment region in a fashion that sterically hinders the access of proteolytic enzymes (48). Although the functional consequence of this property is not clear, we may speculate that functions carried out by the macrophage SG may be dependent on that the PG is intact, i.e. that the corresponding free GAG chains are not functionally equivalent.

Another interesting finding was that the macrophage SG contains a high proportion of HS side chains. This is in contrast to CTLs (18) and bone marrow-derived mast cells (49, 50), in which CS chains are by far the dominating GAG species attached to the SG core protein. It is also clear that the degree of sulfation of the respective GAGs attached to the SG core protein shows a large degree of variability. For example, CS chains attached to the SG core protein in CTLs are predominantly of low charge density (18, 30) whereas the SG-associated CS chains in bone marrow-derived mast cells are mainly of CS-E type, i.e. highly sulfated (17). Furthermore, whereas this report shows that HS chains attached to SG in macrophages are of relatively low charge density, the corresponding chains in SG of connective tissue type mast cells are of high charge density, i.e. heparin type (31). Hence, depending on the specific cell type in which SG is expressed, the type and degree of sulfation of the respective GAG attached to it is highly variable. However, the mechanisms for selecting type of GAG to be attached to the SG core protein remain to be elucidated.

As noted above, our results point to a predominantly extracellular role of macrophage SG. A potential role of the secreted macrophage SG could be to act as a carrier for one or several of the macrophage secretory products. Indeed, the latter notion is supported by the findings that many macrophage secretory products display affinity for PGs (33). In order to test this hypothesis we measured the levels of various macrophage secretory products, both without and after LPS stimulation of the cells. However, we were not able to see any effects of the SG targeting on the secretion of lysozyme, MMP-9, MIP-1{alpha}, or IL-1{alpha}, although we have previously shown that lysozyme and MIP-1{alpha} bind to immobilized SG (33). This indicates that the secretion/production of these major macrophage products is not dependent on SG. In contrast, we show here that the extracellular levels of TNF-{alpha} were significantly higher in cultures of SG–/– macrophages than in wild-type counterparts after LPS stimulation. This is to our knowledge the first report providing a link between TNF-{alpha} secretion and SG, although it has been reported previously that TNF-{alpha} shows affinity for GAGs (51, 52). The mechanism behind the effect of the SG-deficiency on TNF-{alpha} secretion is at present not clear.

One possibility would be that SG normally controls TNF-{alpha} secretion to prevent excessive amounts of the cytokine being released, for example by targeting excess TNF-{alpha} to degradation by the lysosomes. In line with such a notion it has been reported that SG may modulate the intracellular trafficking of myeloperoxidase (53). It has been shown in macrophages that the primary pool of biological active TNF-{alpha} in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to pathogens or tumor cells (54). SG may be involved in such processes. Results from that study also show that the active form of TNF-{alpha} can be found in the Golgi compartment of macrophage, supporting our finding that TIMP-3 did not affect the levels of TNF-{alpha}.

Another possibility would be that in the presence of SG, TNF-{alpha} is controlled extracellularly by SG-dependent mechanisms. For example, mast cells contain a spectrum of proteases that are strictly dependent on SG for storage and activity, and it could not be excluded that contaminating mast cell proteases were present in the macrophage cultures and were able to degrade TNF-{alpha}. However, our results show that the elevated levels of TNF-{alpha} were not explained by effects mediated by SG-dependent mast cell proteases. Considering the major importance of TNF-{alpha} in a variety of immune and pathological mechanisms, it is clear that a dependence of this cytokine on SG may have a large impact on our understanding of the biology of TNF-{alpha}.


   FOOTNOTES
 
* This work was supported in part by Grants from the Norwegian Cancer Society (Grant A88367 [GenBank] ), the AgriFunGen program at the Swedish University of Agricultural Sciences and the Swedish Research Council, King Gustaf V's 80th Anniversary Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Fellow of Norwegian Cancer Society. Back

2 Present address: Harvard Medical School, Div. of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA 02115. Back

3 Present address: Inst. of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. Back

4 To whom correspondence should be addressed: Dept. of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Box 1046 Blindern, 0316 Oslo, Norway. Tel.: 47-22851383; Fax: 47-22851398; E-mail: s.o.kolset{at}medisin.uio.no.

5 The abbreviations used are: PG, proteoglycan; CS, chondroitin sulfate; cABC, chondroitinase ABC; CTL, cytotoxic T lymphocyte; DS, dermatan sulfate; GAG, glycosaminoglycan; HS, heparan sulfate; IL-1{alpha}, interleukin-1{alpha}; LPS, lipopolysaccharide; MIP-1{alpha}, macrophage inflammatory protein-1{alpha}; MMP, matrix metalloproteinase; NK, natural killer; NDST-2, N-deacetylase/N-sulfotransferase-2; SG, serglycin; TACE, TNF-{alpha}-converting enzyme; TNF-{alpha}, tumor necrosis factor-{alpha}; PBS, phosphate-buffered saline; ANOVA, analysis of variance. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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