The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans.

Matrix metalloproteinases (MMPs) secreted from the leukemic macrophage cell-line THP-1 have been investigated. Under serum-free conditions, this cell-line synthesizes and secretes proMMP-9, which was detected in the culture medium as a monomer of 92 kDa, and in dimeric forms, including a homodimer of approximately 225 kDa. In addition, a new heterodimer complex is described, in which proMMP-9 is covalently linked to the core protein of chondroitin sulphate proteoglycan (CSPG) through one or more disulphide bridges. After SDS-PAGE electrophoresis, at least two forms of this complex were detected, a large form in the stacking gel and a smaller form with an estimated size of 300 kDa. When the CS chains were removed by chondroitin ABC lyase treatment, heterodimers of proMMP-9/CSPG core protein of approximately 145, 127 and 109 kDa were found, based on zymography and Western blots. Since as much as 10-15 % of the total proMMP-9 secreted from THP-1 cells was covalently linked to CSPG, this association may have important implications for transport, targetting and regulation of the enzyme activity.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver.

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP-1 was cultured under serum-free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and 35S-macromolecules were purified by Q-Sepharose anion-exchange chromatography and Superose 6 gel chromatography. After labeling with 125I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino-terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was coinjected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin.

Serglycin-binding proteins in activated macrophages and platelets.

The major proteoglycan in macrophages and platelets is the chondroitin sulphate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell-associated proteins from macrophages and blood platelets was examined. Affinity chromatography with serglycin-Sepharose and chondroitin sulphate-Sepharose was used to isolate proteoglycin-binding proteins from macrophages and platelets. Antibodies against human macrophage inflammatory protein-1 alpha (MIP-1 alpha) precipitated a 14-kDa 35S-methionine-labeled protein among the chondroitin sulfate binding proteins secreted from the macrophage-like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of approximately 10 and 14 kDa were precipitated by an antiserum against the murine MIP-1 alpha. Protein sequencing of fragments obtained by trypsin digestion of a 14-kDa chondroitin sulfate-binding protein from cell extracts of stimulated U937 cells revealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed high homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4-sulphate. Amino-terminal sequencing of two proteins from platelet extracts that bound to serglycin-Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PE4). Chondroitin sulfate-Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin-Sepharose indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparin sulfate in blocking the binding of [3H] chondrotin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine-labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP-1 alpha, and lysozyme play important role in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response.

C-reactive protein (CRP), a comparison of pre- and post-mortem blood levels.

During autopsy of 26 inpatients with elevated CRP levels (14-536 mg/l) blood was taken from the femoral vessels and analysed for the content of CRP. The post-mortem CRP values were compared with the results of CRP analysis performed within the last 24h before death. This showed that the post-mortem values of CRP in average were reduced with 35% compared to the ante-mortem values. The decrease in CRP levels was not significantly influenced by the time interval from death until blood was drawn for analysis, at least up to 6 days. When blood taken from healthy individuals killed in accidents was analysed, no elevated CRP values were observed, indicating that there is negligible risk for false positive results. It is also shown that frozen blood can be used for CRP analysis if an immunometric kit assay based on whole blood is applied. The results demonstrate that an elevated level of CRP in post-mortem blood is a good marker for an ongoing inflammatory process prior to death. CRP analysis may therefore be a helpful tool in post-mortem examinations, especially in non-hospitalised individuals without medical records.

Proteases in Plasma and Kidney of db/db Mice as Markers of Diabetes-Induced Nephropathy.

Db/db mice are overweight, dyslipidemic and develop diabetic complications, relevant for similar complications in human type 2 diabetes. We have used db/db and db/+ control mice to investigate alterations in proteinase expression and activity in circulation and kidneys by SDS-PAGE zymography, electron microscopy, immunohistochemistry, Western blotting, and in situ zymography. Plasma from db/db mice contained larger amounts of serine proteinases compared to db/+ mice. Kidneys from the db/db mice had a significantly larger glomerular surface area and somewhat thicker glomerular basement membranes compared to the db/+ mice. Furthermore, kidney extracts from db/+ mice contained metalloproteinases with M(r) of approximately 92000, compatible with MMP-9, not observed in db/db mice. These results indicate that higher levels of serine proteinases in plasma may serve as potential markers for kidney changes in db/db mice, whereas a decrease in MMP-9 in the kidney may be related to the glomerular changes.

Human cord blood monocytes: binding and ingesting capacity mediated by Fc and C3b receptors.

Human cord blood monocytes have been compared to monocytes from adults. Our results show that unstimulated cord blood monocytes are as effective as monocytes from adults with regard to their binding and ingesting capacity mediated by Fc and C3b receptors. Furthermore, when stimulated with PMA (phorbol-myristate-acetate), the C3b receptor-mediated phagocytosis in cord blood monocytes was enhanced to the same extent as in monocytes from adults. These findings show that Fc and C3b receptor functions in human monocytes are fully developed at birth and cannot explain the increased susceptibility to infections seen in the neonate.

Proteoglycan biosynthesis in relation to differentiation of cord blood monocytes in vitro.

Human monocytes were obtained from umbilical cord blood and cultured in vitro. By morphological criteria, the neonatal monocytes developed into macrophage-like cells in the course of 3-5 days in culture. The cells were exposed to [35S]sulphate for 24 h, either from day 0-1 or day 9-10 in vitro. The 35S-labelled macromolecules recovered were mainly associated with the medium fraction (approximately 75%) in both day 1 and day 10 cultures. These secretory macromolecules were demonstrated by the use of chondroitinase ABC-digestions to contain predominantly chondroitin sulphate proteoglycan (CSPG). [35S]galactosaminoglycan chains from day 10 cultures were more highly sulphated than the corresponding day 1 species due to the appearance of (glucuronosyl-4,6-diS-N-acetylgalactosamine) disulphated disaccharide units. The galactosaminoglycan chains in neonatal CSPG were found to increase in Mr during cultivation in vitro; from mean Mr of 20,400 to 30,200 (n = 5) in day 1 and day 10 medium proteoglycans, respectively. The corresponding Mr values for adult monocyte [35S]galactosaminoglycan chains were 21,300 and 22,800. On the basis of the concomitant changes in cellular morphology and glycosaminoglycan structure, it is concluded that neonatal monocytes, like monocytes from adults, differentiate into macrophage-like cells in vitro.

Cell density-dependent expression of chondroitin sulfate proteoglycan in cultured human monocytes.

Monocytes were isolated and established in vitro at different cell densities. The incorporation of [35S]sulfate into macromolecules in monocytes (day 1 in culture) and monocyte-derived macrophages (day 5 in culture) was found to increase with decreasing cell density in approximately the same way in both day 1 and day 5 cell cultures. [35S]Sulfate was found to be incorporated almost exclusively into chondroitin sulfate proteoglycan (CSPG) in both high and low density monocyte and monocyte-derived macrophage cultures. The molecular size of the [35S]CSPGs produced by the high and low cell density cultures were not found to differ as judged by gel chromatography elution patterns. The molecular size and the structure of the glycosaminoglycan chains were found to be almost similar in high and low density day 1 and day 5 cultures. Only a small degree of proteoglycan degradation could be observed in both high and low density cultures. Furthermore, cell density-dependent differences in CSPG biosynthesis could be observed already 2 h after the establishment of the cultures, indicating that a process of "down-regulation" in high density cultures was already in operation. The glycosaminoglycan synthesis in high cell density day 1 cultures could be increased slightly following exposure to 0.5 mM benzyl-beta-D-xyloside, but not to the same level as that observed in untreated low cell density cultures. By contrast, the expression of 35S-macromolecules by cells cultured at high cell density for 5 days could be increased by xyloside treatment almost to the same level as that observed in the low density cultures.

Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells.

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.

Differential effect of brefeldin A on the biosynthesis of heparan sulfate and chondroitin/dermatan sulfate proteoglycans in rat ovarian granulosa cells in culture.

The subcellular localization of the enzymes involved in the glycosylation of proteoglycans was studied in rat ovarian granulosa cells by interfering with the normal traffic in the Golgi apparatus using brefeldin A. Cell cultures were metabolically labeled with [35S] sulfate and [3H]glucosamine, and the radiolabeled macromolecules were analyzed by ion-exchange and gel chromatography in combination with chondroitinase or heparitinase treatment. In the absence of brefeldin A, the cells synthesized both dermatan sulfate proteoglycans (DSPGs) and heparan sulfate proteoglycans (HSPGs) which were isolated from the culture medium, the plasma membrane, and intracellular compartments. However, in the presence of brefeldin A, the synthesized proteoglycans were almost exclusively HSPGs and were found only in the intracellular compartment. Analyses of HSPGs synthesized in the presence of brefeldin A indicated that: (i) the HS chains are synthesized on the same core protein as for the normal HSPGs, (ii) the chains are two to three times the normal molecular size; and (iii) a significant proportion of the HS chains are normally sulfated. Brefeldin A induces a disassembly of the proximal part of the Golgi complex, resulting in a redistribution of cis-, medial-, and trans-Golgi resident enzymes back to the endoplasmic reticulum (ER), and blocks the transport of proteins to the trans-Golgi network. Our results indicate that the complete set of enzymes involved in the biosynthesis of HS chains are localized in the ER/proximal part of the Golgi complex, whereas the enzymes involved in the elongation/sulfation of DS chains are exclusively located in the trans-Golgi network. Furthermore, our results indicate that the enzymes involved in the biosynthesis of HS chains are specific to HS core proteins, since no DS core proteins were substituted with HS chains in the presence of brefeldin A.

Modulation of the expression of chondroitin sulfate proteoglycan in stimulated human monocytes.

Proteoglycan biosynthesis was studied in human monocytes and monocyte-derived macrophages (MDM) after exposure to typical activators of the monocyte/macrophage system: interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). By morphological examination, both monocytes and MDM were stimulated by these activators. Treatment with IFN-gamma resulted in a slight decrease in the expression of [35S]chondroitin sulfate proteoglycan (CSPG) in both monocytes and MDM, whereas LPS treatment increased the [35S]CSPG expression 1.8 and 2.2 times, respectively. PMA, in contrast, decreased the CSPG expression 0.4 times in monocytes, whereas MDM were stimulated to increase the biosynthesis 1.9 times. An increase in the sulfate density of the chondroitin sulfate chains was evident following differentiation of monocytes into MDM due to the expression of disulfated disaccharide units of the chondroitin sulfate E type (CS-E). However, monocytes exposed to PMA did also express disaccharides of the chondroitin sulfate E type. Furthermore, the expression of CS-E in MDM was increased 2 times following PMA treatment. An inactive phorbol ester, phorbol 12,13-diacetate, did not affect the expression of CS-E in either monocytes or MDM when compared with control cultures, suggesting that protein kinase C-dependent signal pathways may be involved in the regulation of sulfation of CSPG. Exposure to LPS or IFN-gamma did not lead to any changes in the sulfation of the chondroitin sulfate chains.

Antibody-mediated enhancement of BK virus infection in human monocytes and a human macrophage-like cell line.

We have monitored BK virus (BKV) antigen expression and multiplication in human monocytes and in a human macrophage (M luminal diameter)-like cell line (U937) in the presence or absence of dilution series of human or rabbit anti-BKV antisera. After infection with BKV alone, restricted expression (structural antigens and T-antigen) and multiplication was recorded in monocytes from some donors, while in U937 cells and monocytes from other donors, no signs of viral activity were detected. Monocyte cultures established from the same donor at different times demonstrated antigen expression/multiplication on two occasions but not on the third. A pronounced enhancement of BKV expression/multiplication in human monocytes and multiplication in U937 cells was seen with some dilutions of all antisera (human and rabbit) used. The pattern of enhancement and the dilution resulting in maximum viral activity was constant and seemed to be determined by the serum, but the exact level of enhancement for a given serum differed considerably in monocytes from different donors and seemed to be determined by the cells. In the latter respect, monocytes taken from the same donor some weeks apart showed variations at the same level, as did cells from different donors. PMA (phorbol-12-myristate-13-acetate) stimulation of monocytes and U937 cells resulted in stronger antibody enhancement in terms of infectivity, without affecting the number of monocytes showing antigen expression. No expression/multiplication of BKV was detected in the murine M luminal diameter-like cell line P338 DI.

Blood platelets stimulate the expression of chondroitin sulfate proteoglycan in human monocytes.

Mononuclear phagocytes synthesize chondroitin sulfate proteoglycan (CSPG), which is constitutively secreted. Because mononuclear phagocytes are known to interact with blood platelets, the effect of platelets on the release of CSPG in cultured human monocytes was investigated. After 6 days in vitro, the monocytes were supplied with fresh medium with different additions and subsequently exposed to [35S]sulfate for 24 hours before the medium fractions were harvested and analyzed for content of [35S]CSPG. Indirect evidence for the release of stimulatory factors from blood platelets was found when the addition of medium containing 50% serum made from platelet-rich plasma increased the expression of [35S]CSPG almost sevenfold compared with serum-free medium, whereas medium containing 50% serum made from platelet-depleted plasma increased the expression of [35S]CSPG about fourfold. Further, direct evidence for the stimulatory effect of platelets was found as the addition of autologous platelets to serum-free medium increased the expression of [35S]CSPG about threefold, and addition of supernatant from a corresponding number of thrombin-stimulated platelets was almost as efficient. The effect of five different platelet-derived factors (which are all present in serum) was investigated. Both platelet-derived growth factor (PDGF), platelet factor 4 (PF 4), and prostaglandin E2 (PGE2) used in physiologic concentrations were found to stimulate the expression of [35S]CSPG twofold to threefold, whereas transforming growth factor-beta had a slight inhibitory effect. 12-Hydroxyeicosatetraenoic acid had no significant effect on the expression of [35S]CSPG. Further evidence for the stimulatory effect of PDGF, PF 4, and PGE2 was found as serum depleted of these factors had significantly less stimulatory effect than control serum. The increased incorporation of [35S]sulfate into [35S]CSPG in cultures stimulated with serum or platelet-derived factors was not due to differences in molecular size or extent of sulfation of the proteoglycan molecules.

Mouse mastocytoma cells synthesize undersulfated heparin and chondroitin sulfate in the presence of brefeldin A.

In order to study the subcellular localization and organization of the enzymes involved in the glycosylation of the hybrid proteoglycan serglycin, mouse mastocytoma cells were metabolically labeled with [35S]sulfate or [3H]glucosamine in the absence or presence of brefeldin A. This drug is known to induce a disassembly of the proximal part of the Golgi complex, resulting in a redistribution of cis-, medial-, and trans-Golgi resident enzymes back to the endoplasmic reticulum, and to block the anterograde transport of proteins to the trans-Golgi network. Although the total incorporation of [3H]glucosamine into glycosaminoglycan chains was reduced to about 25% in brefeldin A-treated cells compared to control cells, both control cells and cells treated with brefeldin A synthesized heparin as well as chondroitin sulfate chains. Therefore, enzymes involved in the biosynthesis of both types of glycosaminoglycan chains seem to be present proximal to the trans-Golgi network in these cells. Chondroitin sulfate and heparin synthesized in cells exposed to brefeldin A were undersulfated, as demonstrated by ion-exchange chromatography, compositional analyses of disaccharides, as well as by a lower [35S]sulfate/[3H]glucosamine ratio compared to controls. In heparin biosynthesis, both N- and O-sulfation reactions were impaired, with a larger relative decrease in 2-O-sulfation than in 6-O-sulfation. Despite undersulfation, the heparin chains synthesized in the presence of brefeldin A were larger (30 kDa) than the heparin synthesized by control cells (20 kDa). The reduced [3H]glucosamine incorporation in brefeldin A-treated cells was partly due to decreased number of glycosaminoglycan chains synthesized, but also to the biosynthesis of chondroitin sulfate chains of smaller molecular size (8 versus 15 kDa in control cells). Brefeldin A had no effect on the glycosaminoglycan synthesis when used in a cell-free, microsomal fraction, indicating that brefeldin A does not interfere directly with the enzymes involved in the biosynthesis of glycosaminoglycans.

Serglycin expression during monocytic differentiation of U937-1 cells.

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.

Proteoglycan metabolism in normal and inflammatory human macrophages.

To study proteoglycan metabolism in inflammatory macrophages, primary cultures of human macrophages were cultured in the absence and presence of bacterial lipopolysaccharide (LPS). When exposed to [35S]sulfate, the cells incorporated the label almost exclusively into chondroitin sulfate proteoglycan (CSPG), which was recovered from the culture medium and the cell layer. Cells stimulated with LPS secreted approximately three times more [35]CSPG into the culture medium than control cells. Furthermore, cell adhesion was also found to promote proteoglycan secretion; when nonadherent monocytic cells were induced to adhere, the release of proteoglycan increased two times. The increased secretion seen in LPS-stimulated macrophages was partly due to increased biosynthesis, but was mostly due to increased sorting of CSPG to the secretory pathway. Only about 20% of the CSPG synthesized in unstimulated cells was secreted, whereas the corresponding figure in LPS-treated cells was 35%. In both cell types, the remaining [35S]CSPG was degraded, probably in the lysosomes. The degradation was a two-step process. First, the [35S]CSPG was rapidly cleaved to yield free glycosaminoglycan (GAG) chains (t1/2 = 15 to 30 minutes). Secondly, the GAG chains were completely depolymerized (t1/2 = 2 to 3 hours). Neither resting nor LPS-stimulated cells sorted CSPG to intracellular storage, as is evident in many hematopoietic cells. The LPS-treated cells synthesized [35S]CSPG of smaller molecular size than did control cells, with GAG chains of approximate molecular mass of 12 kD versus 16 kD in control cells. No difference was seen in the disaccharide composition of the GAG chains; both LPS-stimulated and unstimulated cells expressed a mixture of 80% to 90% chondroitin 4-sulfate and 10% to 20% chondroitin 4,6-disulfate. N-terminal sequence and Northern blot analysis indicate that the core protein of the CSPG secreted by human macrophages is serglycin.