Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M411954200 on March 4, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20879-20886, May 27, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/21/20879    most recent
M411954200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, H.
Right arrow Articles by Asari, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, H.
Right arrow Articles by Asari, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Keratan Sulfate Disaccharide Gal(6S03) {beta}1,4-GlcNAc(6S03) Modulates Interleukin 12 Production by Macrophages in Murine Thy-1 Type Autoimmune Disease*

Heping Xu{ddagger}, Hitoshi Kurihara§, Tomomi Ito§, Hiroshi Kikuchi§, Keiichi Yoshida¶, Hiroko Yamanokuchi§, and Akira Asari§||

From the §Seikagaku Corporation, 1-5, Nihonbashi-honcho 2-chome, Chuo-ku, Tokyo 103-0023, Japan, the {ddagger}Department of Ophthalmology, University of Aberdeen Medical School, Foresterhill, Aberdeen AB25 2ZD, United Kingdom, and Mizutani Foundation for Glycoscience, Suite 5F, Sen-i Kaikan, 3-1-11 Nihonbashi-honcho, Chuo-ku, Tokyo 103-0023, Japan

Received for publication, October 21, 2004 , and in revised form, March 2, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO3)-GlcNAc(6-SO3) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-{gamma}. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Keratan sulfate (KS)1 is an extracellular matrix component in cornea, cartilage, and other tissues (1–3). In these tissues, KS is a sulfated glycosaminoglycan (GAG) side chain of proteoglycans and is composed of repeating units of galactose and N-acetyl-D-glucosamine (1–3). It has been reported that KS proteoglycans are important for transparency of corneal tissue (4–6). However, the functional properties of KS in cartilage are still unknown.

It has been reported that disaccharides of heparin or heparan sulfate suppress tumor necrosis factor {alpha} production in macrophages and IL-8 and IL-1{beta} secretion by intestinal epithelial cells (7, 8). These events suggest that GAG oligosaccharides have effects on cytokine expression. Moreover, it has been shown that high molecular weight hyaluronan (HA) inhibits NF-{kappa}B activation (9), whereas low molecular weight HA induces NF-{kappa}B activation (10). This suggests that the cell biological activities of GAGs are dictated in part by their molecular size.

MRL-lpr/lpr mice, which develop lymphoaccumulation (lymphoadenopathy), hypergammaglobulinemia, serum autoantibodies, and a generalized autoimmune disease, including glomerulonephritis and arthritis, have been used as a model for the study of systemic lupus erythematosus (11). This lymphoaccumulation disorder, i.e. a considerable increase in double negative (CD4-CD8-) T cells in lpr mice, is explained by defects in Fas, which mediates apoptosis (12, 13).

IL-12 is induced in macrophages, dendritic cells, and other cell types by bacterial infections. IL-12p70 is a heterodimeric cytokine composed of two subunits, p35 and p40, and acts to promote NK cells, NKT cells, and CD8+ T cell activity (14). IL-12p70 enhances cytolytic lymphocyte activity and induces production of interferon-{gamma} (IFN-{gamma}) (15). IL-12p40 monomer and IL-12p40 homodimer are antagonists of IL-12p70 (16–18). IL-12p40 homodimer, however, stimulates CD8+ Th1 development and acts as a chemotactic molecule to activate macrophages, CD4+ T cells, CD8+ T cells, and NK cells (19). It has been shown that the precursors of double negative T cells are CD8+ T cells in MRL-lpr/lpr mice (20). Double negative T cells as well as CD8+ T cells can produce IFN-{gamma} (21), which stimulates IL-12 production in macrophages and dendritic cells. In our previous study, we showed that in MRL-lpr/lpr mice, plasma IL-12p40 concentrations as well as lymph node weights increase with age, and IL-12 enhances lymphoaccumulation by suppressing cell death of T cells (22).

We present here that KS disaccharide (L4) treatment suppresses the following: 1) IL-12 production in macrophages followed by induction of apoptosis in the lymph nodes of the MRL-lpr/lpr mice, and 2) Th-1 type autoimmune disease, including IL-12 production, lymphoaccumulation, hypergammaglobulinemia, and glomerulonephritis in the MRL-lpr/lpr mice.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1.
Analysis by capillary electrophoresis. The following samples were analyzed: a, standard oligosaccharides; b, a sample that was recovered from the keratanase II-digest solution of KS by ethanol fractionation; and c, purified L4.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—KS, N-acetyl-lactosamine (LacNAc), and unsaturated disaccharides of hyaluronan ({Delta}diHA), chondroitin ({Delta}diC-0S), chondroitin 6-sulfate ({Delta}diC-6S), chondroitin 4-sulfate ({Delta}diC-4S), and of heparan sulfate/heparin (HS2: mixture of {Delta}diHS-0S, {Delta}diHS-NS, {Delta}diHS-6S, {Delta}diHS-di(6,N)S, {Delta}diHS-di(U,N)S, {Delta}diHS-tri(U,6,N)S) were obtained from Seikagaku Corp. (Tokyo, Japan). Other reagents and chemicals were obtained from commercial sources as indicated.

Preparation of KS Oligosaccharides—KS oligosaccharides, Gal(6-SO3)-GlcNAc(6-SO3) (L4), Gal(6-SO3)-GlcNAc(6-SO3)-Gal(6-SO3)-GlcNAc(6-SO3) (L4L4), and NeuAc-Gal-GlcNAc(6-SO3)-Gal(6-SO3)-GlcNAc(6-SO3) (SL2L4) were isolated from the keratanase II digest of shark fin KS (Seikagaku Corp.) by sequential steps of anion-exchange and gel-permeation chromatographies. Each oligosaccharide was identified by capillary electrophoresis (Fig. 1) and by mass spectrometry (23).

Capillary electrophoresis was done on a Quanta 4000 capillary electrophoresis system equipped with an ultraviolet detector (Waters). The capillary electrophoresis system was operated in normal polarity by applying the sample at the anode. Running buffer was 50 mM sodium tetraborate (pH 9.0). Each sample was separated and analyzed using a fused silica (externally coated except where the tube passed through the detector) capillary tube (75 µm inner diameter, 60 cm long) from Waters. Before introduction of the sample, the capillary tube was manually rinsed with 0.5 M sodium hydroxide, distilled water, and running buffer. Samples were loaded by hydrostatic pressure using a 10-s injection period. Each experiment was conducted at constant voltage (12 kV). The eluent was monitored at 185 nm. The analyses of data were done using the software Millenium 32 (version 3.06.01) from Waters.

All saccharides were checked for endotoxin by Limulus amebocyte lysate assays using a Toxicolor LS Set (Seikagaku Corp.). Both L4 and L4L4 contain 0.03 pg/mg or less of endotoxin. The concentration of endotoxin was below 0.6 pg/ml in each working solution (1 µg/ml) of LacNAc, {Delta}diHA, {Delta}diC-0S, {Delta}diC-6S, {Delta}diC-4S, and HS2 in the studies in vitro. SL2L4 could not be used for the studies in vitro or in vivo, because the amount of SL2L4 that we obtained was very small.

Mice—Four-week-old female MRL-lpr/lpr and C57/BL-6 mice were purchased from Charles River Breeding Laboratories (Yokohama, Japan) and maintained in our animal facility. All animal protocols were approved by the Animal Experiment Committee in Central Research Laboratories of Seikagaku Corp.

In Vitro Study—Peritoneal macrophages were obtained from C57BL/6 or MRL-lpr/lpr mice after injection of thioglycolate. The macrophages were treated with L4, L4L4, KS, other GAG disaccharides, as well as a PKC inhibitor (bisindolylmaleimide; Calbiochem) in the presence or absence of lipopolysaccharide (LPS, 100 ng/ml) and IFN-{gamma} (1 unit/ml). Interleukin-10 (IL-10; 10 ng/ml; Genzyme, Cambridge, MA) was used as a positive control to suppress IL-12 production (24). IL-12p70 and IL-12p40 in the culture media were detected by ELISA.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2.
Effect of GAGs on IL-12 production in macrophages in vitro. Macrophages derived from C57BL/6 mice were treated with L4, L4L4, KPS, {Delta}diHA, {Delta}diC-0S, {Delta}diC-4S, {Delta}diC-6S, and HS2 simultaneously with stimulation by LPS plus IFN-{gamma}. IL-10 was used as a positive control to suppress IL-12p70 production. The IL-12p70 level released from macrophages cultured in the absence of GAGs but in the presence of LPS plus IFN-{gamma} was taken to be 100% to compare with the effects of the other reagents on IL-12p70 production. *, p < 0.05 (versus 0 ng/ml); #, not detected.

 
Immunohistochemistry showed that L4 treatment suppresses pPKC and PI3K expression in macrophages of lymph nodes of MRL-lpr/lpr mice. It is very difficult to get sufficient numbers of peritoneal macrophages from mice for Western blotting. Therefore, we used J774.1 cells, mouse macrophage cell line, for the detection of pPKC and PI3K by Western blotting. The J774.1 macrophages were treated with L4 in the presence of LPS and IFN-{gamma} as described above. The concentration of IL-12 was measured in culture media of the J774.1 macrophages 24 h after treatment with L4. Phospho-PKC and PI3K expressions were examined 30 min and 1 h after the L4 treatment by Western blotting using anti-pPKC and anti-PI3K antisera (Upstate Biotechnology, Inc.)

To detect cell death in the L4-treated macrophages from C57BL/6 mice, the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) reaction was applied to the macrophages according to the method of Gavrieli et al. (25). Moreover to examine viability of the L4-treated macrophages from C57BL/6 mice, MTT assays were done in the macrophages. In these experiments, 10 µg/ml of camptothecin (Calbiochem) was used as a positive control. The mean ± S.E. was calculated from triplicate values. Each experiment was repeated at least three times with consistent results.

In Vivo Study—Female MRL-lpr/lpr mice were randomly divided into three groups as follows: 1) phosphate-buffered saline (PBS)-treated group, 2) dexamethasone (0.1 mg/kg/week)-treated group, and 3) L4 (5 mg/kg/day)-treated group.

Mice of the L4 and PBS groups were injected intramuscularly 5 times a week for 4 weeks from age 20 to 24 weeks. Mice of the dexamethasone group were injected intramuscularly in the legs once a week during the same period. One day after the last administration, all animals were sacrificed by exsanguination under general anesthesia. To elucidate the effects of L4 on apoptosis of T cells as well as on serum IL-12 concentrations, the following parameters were analyzed: serum IL-12 concentration (ELISA), serum creatinine concentration (Autoanalyzer, Roche Applied Science), serum IgG and IgM concentrations (ELISA), weights of mesenteric, inguinal, and axillary lymph nodes, incidence of apoptotic cells in mesenteric lymph nodes (in situ nick-end labeling), hematoxylin and eosin staining in mesenteric lymph node tissues to detect chromatin condensation, and electron microscopy of mesenteric lymph node tissues to observe apoptotic morphological changes. The effect of oral administration of L4 (80 mg/kg) for 9 weeks was also examined by the same methods.

ELISA—We used an OptEIA Mouse IL-12 (p40) set (Pharmingen) and Endogen mouse interleukin-12 (p70) ELISA (Endogen, MA) for the detection of IL-12p40 monomer and homodimer and of IL-12p70, respectively. For the detection of IgG and IgM, anti-mouse IgG and anti-mouse IgM antibodies (The Jackson Laboratory) were used.

Histopathological Examination—MRL-lpr/lpr mice were sacrificed 24 h after the last administration of L4. Mesenteric lymph nodes were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin sections were prepared and stained with hematoxylin and eosin. Morphological changes in the lymphocytes were evaluated as brightness per unit area (820 x 550 µm2) in the hematoxylin and eosin-stained tissues using an image analyzer (Pias, Osaka, Japan).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3.
Effect of L4 on IL-12 production in macrophages. Macrophages derived from C57BL/6 (a) and MRL-lpr/lpr (b) mice were treated with L4 simultaneously with stimulation by LPS plus IFN-{gamma}. Macrophages derived from MRL-lpr/lpr mice were treated with L4 for 24 h before stimulation by LPS plus IFN-{gamma} (c). Macrophages derived from C57BL/6 mice were treated with N-acetyl-lactosamine simultaneously with stimulation by LPS plus IFN-{gamma}. d, J774.1 macrophages were treated with L4 for 24 h simultaneously with LPS plus IFN-{gamma} (e). *, p < 0.05 (versus 0 ng/ml); **, p < 0.01 (versus 0 ng/ml).

 



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 4.
MTT assay (a) and TUNEL staining (b) in the L4-treated macrophages that were stimulated with LPS plus IFN-{gamma}. Treatment with camptothecin for 24 h reduced viability (a) and induced apoptosis (b) in macrophages. L4 did not elicit any changes (a and b). Control, culture media. Magnification, x23.

 
To detect DNA fragmentation, the TUNEL reaction was applied to the paraffin sections according to the method of Gavrieli et al. (25). The sections were then treated with peroxidase-conjugated streptavidin solution followed by 0.3% H2O2. The numerical density of TUNEL-positive cells was determined in areas excluding blood vessels. Data were expressed as number per area (820 x 550 µm2).

Electron Microscopy—Mesenteric lymph node tissues were fixed with 2% paraformaldehyde, 2% glutaraldehyde and postfixed with OsO4. They were dehydrated with graded concentrations of ethanol and embedded in Epon 812. Thin sections were cut with an ultramicrotome (Ultracut N, Reichert-Nissei, Tokyo, Japan) and observed with an Hitachi H-7000 electron microscope (Tokyo, Japan) after staining with uranyl acetate and lead citrate.

Immunohistochemistry—Mesenteric lymph node tissues were embedded in OTC compound and cut into frozen sections. The frozen sections were fixed with 10% formaldehyde. After blocking with 10% donkey serum, sections were incubated with either rabbit anti-PI3K, anti-pPKC (Upstate Biotechnology, Inc.), anti-p38, anti-pERK, or anti-pJNK antibodies (Cell Signaling Technology, Inc.) followed in each case by donkey anti-rabbit IgG conjugated with Texas Red (Jackson ImmunoResearch). The sections were then incubated with goat anti-CD14 antibody (Santa Cruz Biotechnology) and rabbit anti-goat IgG conjugated with FITC (Jackson ImmunoResearch). The numbers of pPKC, PI3K, pp38, pERK or pJNK-positive cells in CD14-positive cells were counted using a confocal laser scan microscope (Leica, Wetzlar, Germany).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 5.
In vivo effects of L4 on increase in serum IL-12p40 concentration (a) and mesenteric lymph node weights (b) in MRL-lpr/lpr mice. Plasma IL-12p40 levels and weights of mesenteric lymph nodes of the control group were taken to be 100% to compare with the effect of L4 and dexamethasone. IL-12p40 levels and weights of mesenteric lymph nodes of the control group were 1000–3000 pg/ml and 900–1500 mg, respectively. i.m., intramuscular administration; p.o., oral administration; *, p < 0.05 (versus control), **, p < 0.01 (versus control).

 



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 6.
Effect of L4 on enlargement of mesenteric lymph nodes (upper panel) and spleen (lower panel) in MRL-lpr/lpr mice.

 
Statistical Analysis—Statistical significance was determined by Williams multiple comparison test or Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of GAGs on Macrophage IL-12 Production in Vitro— Treatment with L4 suppressed IL-12p70 production in macrophages from C57BL/6 mice (Fig. 2). Although treatment with IL-10 completely suppressed IL-12 production, GAGs other than L4 had little effect (Fig. 2).

When L4 and LPS plus IFN-{gamma} were simultaneously added to peritoneal macrophages from both MRL-lpr/lpr (Fig. 3b) and control C57BL/6 mice (Fig. 3a), IL-12p70 production was reduced. L4 treatment also suppressed IL-12p40 monomer and homodimer production in the peritoneal macrophages (data not shown).

When peritoneal macrophages derived from MRL-lpr/lpr mice were treated with L4 prior to treatment with LPS-IFN-{gamma}, the production of IL-12p70 was more strongly suppressed (Fig. 3c). Treatment of C57BL/6 mice macrophages with LacNAc did not change IL-12p70 production in the presence of LPS and IFN-{gamma} (Fig. 3d). Treatment of J774.1 macrophages with L4 suppressed IL-12p40 production in the presence of LPS plus IFN-{gamma} (Fig. 3e). IL-12p70 was not produced in the J774.1 macrophages by the treatment with LPS plus IFN-{gamma} (data not shown).

The L4 treatment suppressed pPKC expression in macrophages in vivo as well as in vitro as described below. Therefore, we confirmed that treatment with bisindolylmaleimide, a PKC inhibitor, suppressed IL-12p70 production in macrophages from C57BL/6 mice in the presence of LPS plus IFN-{gamma} (data not shown).

Effect of L4 on Macrophage Viability in Vitro—MTT and TUNEL assays showed that the treatment with L4 did not elicit any change in viability and cell death of the macrophages of C57BL6 mice (Fig. 4).

Effects of L4 on MRL-lpr/lpr Mice—Intramuscular injection of L4 suppressed the increase in serum contents of IL-12p40 monomer and homodimer in MRL-lpr/lpr mice to the same or greater extent as dexamethasone (Fig. 5a). IL-12p70 was not detected in the serum of any MRL-lpr/lpr mice (data not shown). Treatment with L4 also suppressed the increase in weight of mesenteric (Fig. 5b), submandibular, inguinal and axillary lymph nodes, and spleen (data not shown). Oral administration of L4 also decreased serum IL-12p40 levels (Fig. 5a) and suppressed the enlargement of lymph nodes (Figs. 5b and 6) and spleen (Fig. 6).

The incidence of apoptotic cells demonstrated by TUNEL was higher in the lymph nodes of L4-treated MRL-lpr/lpr mice than in lymph nodes of PBS-treated MRL-lpr/lpr mice (Fig. 7, a–c). Moreover, treatment with L4 reduced the brightness of hematoxylin and eosin staining of lymph node tissues (Fig. 8, a–d). Chromatin condensation and reduction of cytoplasm were observed in many lymphocytes in the lymph nodes in L4-treated groups (Fig. 8, d and f). Electron microscopy showed that discrete clumps of condensed chromatin were abutted against the nuclear membrane, and more clumps were present in the lymphocytes in the lymph nodes of L4-treated mice (Fig. 8f). In dexamethasone-treated mice, such ultrastructural features were not observed, but lymphocytes that showed characteristic chromatin condensation of apoptosis were scattered in the lymph node tissues (data not shown). The L4 treatment suppressed increases in serum creatinine, IgG, and IgM (Fig. 9).

Effect of L4 on Expression of PI3K, PKC, and MAPKs (pp38, pERK, and pJNK) in Macrophages in Vivo—The numbers of macrophages (CD14+) expressing pPKC and PI3K in mesenteric lymph node tissues of MRL-lpr/lpr mice were decreased by treatment with L4 (Fig. 10). On the other hand, treatment with L4 did not elicit any changes in the number of pMAPKs positive macrophages (Fig. 10).

Effect of L4 on Expression of pPKC and PI3K in J774 Macrophages—Western blotting showed that the treatment with L4 did not elicit any change in PI3K expression but did suppress pPKC expression in J774.1 macrophages 30 min and 1 h after treatment with L4 in the presence of LPS and IFN-{gamma} (Fig. 11).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 7.
TUNEL staining (a and b) and incidence of apoptotic cells (c) in mesenteric lymph nodes of MRL-lpr/lpr mice. i.m., intramuscular administration; *, p < 0.05 (versus control). Magnification, x109. L4 increased TUNEL staining and the number of apoptotic cells to similar extents as those stimulated by dexamethasone.

 



View larger version (191K):
[in this window]
[in a new window]
  		FIG. 8.
Hematoxylin and eosin staining and electron micrographs of mesenteric lymph nodes of MRL-lpr/lpr mice. a and b, hematoxylin and eosin staining (magnification, x109); c and d, hematoxylin and eosin staining (magnification, x852); e and f, electron micrograph (magnification, x3194).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our data indicate that the disaccharide, L4, but interestingly, not the tetrasaccharide, L4L4, or the parent KS suppressed IL-12 production by macrophages. Moreover, other GAG disaccharides also did not suppress IL-12 production. The observation that LacNAc did not elicit any change of IL-12 production suggests that the sulfates in L4 are required to suppress IL-12 production. Thus, the effect of L4 is specific and implies the presence of a receptor with high specificity. A mannose receptor has been identified (26, 27). This receptor in murine macrophages is implicated in inflammation and immunity (27), and it recognizes a sulfated carbohydrate, chondroitin sulfates A and B, and sulfated oligosaccharides (28, 29). Whereas these ligands for the mannose receptor are similar to L4, they are not recognized by the putative L4 receptor. Furthermore, Kaposzta et al. (30) showed that the mannose receptor is not specifically implicated in rapid recruitment of late endosomes and lysosomes in mouse macrophages ingesting Candida albicans. We will investigate the putative receptor for L4 in the near future.

Since L4 treatment suppressed not only IL-12p70 but also IL-12p40 in cultured macrophages, the suppression of IL-12p70 production in vitro may be due at least in part to the suppression of IL-12p40 production. The treatment with LPS and IFN-{gamma} induced IL-12p40 but not IL-12p70 in J774.1 macrophages. This suggests that the sensitivity of J774.1 macrophages to LPS and IFN-{gamma} is different from that of mouse peritoneal macrophages. When using the J774.1 macrophages, 200 pg/ml of IL-12p40 was reduced by the treatment with 1 µg/ml of L4. When using mouse peritoneal macrophages, 1 µg/ml of L4 reduced 20–50 pg/ml of IL-12p70. These events indicate that the treatment with L4 significantly suppressed the IL-12p40 production even in the J774.1 macrophages.

As indicated under "Experimental Procedures," endotoxin in the L4 fraction is negligible. Moreover, we performed a standard toxicity test of L4. L4 (500 mg/kg) was intravenously injected into rats once a day for 2 weeks (data not shown). We examined the lymph nodes, spleen, thymus, heart, liver, kidney, stomach, intestine, and brain for pathology. In addition, we performed hematological and blood biochemical examinations. No changes were observed in any of these tissues in the L4-treated rats. Furthermore, TUNEL and MTT assays showed that neither cell death nor reduction of viability in macrophages is induced by the treatment with L4. These events strongly indicate that L4 did not contain a nonspecific toxic compound.

IL-12 was detected in lymph nodes, spleen, liver, kidneys, and other organs of MRL-lpr/lpr mice (data not shown). We previously showed that IL-12 enhances lymphoaccumulation by suppressing cell death of double negative T cells in MRL-lpr/lpr mice (22). In that study we found the following. 1) Ordinary cell cultures induced cell death of double negative T cells from lymph nodes, indicating the presence of humoral factors in vivo that suppress cell death of the double negative T cells. 2) The cell death was suppressed by treatment with IL-12p70 or IL-12p40. 3) Plasma IL-12p40 concentrations as well as the weights of lymph nodes increased with age. 4) Treatment of MRL-lpr/lpr mice with an antibody neutralizing IL-12 diminished lymphoaccumulation. 5) Treatment of MRL-lpr/lpr mice with either IL-12p70- or IL-12p40-encoding plasmids enhanced lymphoaccumulation (22). These results suggest that the humoral factors in vivo that suppress cell death of the double negative T cells in MRL-lpr/lpr mice are IL-12p70 or IL-12 p40. Kikawada et al. (31) showed that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation. Moreover, it is reported that IL-12 provides proliferation and survival signals to murine CD4+ T cells (32). These events strongly indicate that IL-12 enhances lymphoaccumulation by suppressing cell death of T cells in MRL-lpr/lpr mice. In the present study, treatment with L4 suppressed lymphoaccumulation as well as the increase in serum IL-12p40. The number of apoptotic lymphocytes was increased by treatment with L4. Electron microscopy confirmed the finding of hematoxylin and eosin staining that chromatin was condensed by the L4 treatment. The ultrastructure of the radial chromatin condensation in the lymph node lymphocytes of L4-treated mice differs from that of typical apoptosis induced by dexamethasone. However, it closely resembles that of the earliest stage of dexamethasone-induced apoptosis in thymocytes prior to an effect of endonuclease in the presence of zinc (33). Zinc arrests apoptotic ultrastructural changes and inhibits DNA laddering in dexamethasone-treated thymocytes (33). Thus, whereas treatment with L4 induced the earliest stage of apoptosis in most of the T cells in the lymph nodes, TUNEL-positive cells were scattered in the lymph node tissues treated with L4. The morphological change without DNA fragmentation induced by L4 indicates an early stage of apoptosis prior to an effect of endonuclease. This stage was not found in dexamethasone-treated mice. These results suggest that the pathway of apoptosis induced by treatment with L4 is different from that induced by dexamethasone. Radioisotope-labeled L4 that was orally administered was detected in blood (data not shown). This suggests that the oral as well as intramuscular treatment with L4 was effective in reducing serum IL-12 levels and in suppression of lymph node and spleen enlargement.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 9.
Serum creatinine (a), IgG (b), and IgM (c). Concentrations in MRL-lpr/lpr mice. **, p < 0.01 (versus control). i.m., intramuscular administration.

 



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 10.
Double immunostaining for PI3K or pPKC and CD14 (a–h) and ratio of PI3K-, PKC-, and MAPKs (pp38, pERK, and pJNK)-positive cells in macrophages. pPKC (a and b) or PI3K (e and f) expression is observed in many macrophages (CD14+) in PBS-treated mice. In L4-treated mice, most of the macrophages (CD14+) express neither pPKC (c and d) nor PI3K (g and h). Arrows, PI3K-, pPKC-, pp38-, pERK-, or pJNK-positive macrophages (CD14+). a, c, e, g, i, k, m, o, q, and s, CD14 (Texas Red); b and d, pPKC (FITC); f and h, PI3K (FITC); j and l, pp38 (FITC); n and p, pERK (FITC); r and t, pJNK (FITC). a, b, e, f, i, j, m, n, q, and r, PBS-treated mice; c, d, g, h, k, l, o, p, s, and t, L4-treated mice. **, p < 0.01 (versus control). Magnification, x225.

 
The treatment with L4 suppressed serum creatinine concentration in MRL-lpr/lpr mice in the present study. It is reported that lupus nephritis is observed in MRL-lpr/lpr mice (11, 31). Kikawada et al. (31) showed that IL-12 deficiency in MRL-lpr/lpr mice delays nephritis. The creatinine concentration in serum was lower in the L4-treated than in negative control groups in the present study. L4 was administered in the MRL-lpr/lpr mice during 20–24 weeks of age. Kikawada et al. (31) showed that IL-12 deficiency in MRL-lpr/lpr mice delays nephritis, when they measured serum creatinine during 12–22 weeks of age. This indicates that serum creatinine concentration was suppressed by the L4 treatment in the present study and that the delay of nephritis is due to IL-12 reduction by the treatment with L4. Moreover, the treatment with L4 suppressed hypergammaglobulinemia as indicated by high levels of serum IgG and IgM. In vivo, IL-12 administration induces Th-1-associated patterns of antibody production (34). Taken together, the treatment with L4 totally suppressed Th-1 type autoimmune disease, including production of IL-12, lymphoaccumulation, hypergammaglobulinemia, and glomerulonephritis in MRL-lpr/lpr mice.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 11.
Western blotting for pPKC and PI3K in J774 macrophages 30 min and 1 h after the treatment with L4 in the presence of LPS and IFN-{gamma}.

 



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 12.
Proposed model of effects of L4 on IL-12 production and apoptosis of T cells in MRL-lpr/lpr mice. L4 treatment induces apoptosis in lymph node cells of MRL-lpr/lpr mice by reducing the production of IL-12 in macrophages, which suppresses apoptosis of T cells.

 
To investigate the molecular mechanism of suppression of IL-12 production by L4, we examined the expression of several signal transduction molecules, namely PI3K, pPKC, and pMAPKs (pERK, pJNK, and pp38), in CD14-positive cells (macrophages) in lymph nodes from MRL-lpr/lpr mice. The number of PI3K- or pPKC-positive macrophages in lymph nodes from MRL-lpr/lpr mice was notably decreased by treatment with L4. However, L4 did not change expression of pERK, pJNK, and pp38 in macrophages. Western blotting showed that pPKC, but not PI3K expression, in J774.1 macrophages was suppressed by the L4 treatment. Inhibition of PKC activation using bisindolylmaleimide, a pantothenate-PKC inhibitor, was associated with a dose-dependent decrease of LPS-induced IL-12 production (35). CD40 ligand-induced IL-12 production in dendritic cells was mediated by PI3K pathways (36). Fabio and Strominger (37) have shown that LY294002, an inhibitor of PI3K, blocked IL-12 production by human dendritic cells stimulated with LPS. PI3K expression in J774.1 macrophages was not changed by the L4 treatment, suggesting that the treatment with L4 indirectly reduces PI3K expression in macrophages in MRL-lpr/lpr mice or that the characteristics of J774.1 macrophages are different from those of MRL-lpr/lpr mice macrophages. Taken together, L4 likely suppresses IL-12 production at least by down-regulating pPKC signal pathways in MRL-lpr/lpr mice.

IL-12p70 consists of IL-12p40 and IL-12p35 (14). Since IL-12p40 as well as IL-12p70 suppresses cell death of T cells in MRL-lpr/lpr mice (22), the increase in the apoptotic cells after treatment with L4 is due at least in part to the decrease in IL-12p40. Fig. 12 presents a model of L4-mediated apoptosis in T cells of MRL-lpr/lpr mice in which the production of IL-12 in macrophages is reduced via inhibition of pPKC and PI3K signals. Further studies are required to elucidate the precise mechanism of the suppressive effect of the KS disaccharides, L4, on IL-12 production.

Relevant to the current study, Bacillus infection of the cornea produces keratanases, a mechanism to protect against host defenses. Furthermore, IL-12 and IFN-{gamma} play important roles against Bacillus infection (38). Since KS is a component of the extracellular matrix, in particular of cornea (1–3), Bacillus may produce a keratanase thereby producing L4 and reducing IL-12 production in infiltrating macrophages responding to the infection. On the other hand, L4 may be useful for the treatment of Th-1 type autoimmune disease in which IL-12 production is augmented.

Since we have shown previously (39) that tetrasaccharides of HA, but not high molecular weight HA, up-regulate heat shock protein 72 expression in K562 cells after hyperthermia treatment, we postulate that glycosaminoglycans, i.e. giant molecules in the extracellular matrix, obtain novel activities after depolymerization.


   FOOTNOTES
 
* 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

|| To whom correspondence and reprint requests should be addressed: Seikagaku Corp., 1-5, Nihonbashi-honcho 2-chome, Chuo-ku, Tokyo 103-0023, Japan. Tel.: 81-3-3270-0242; Fax: 81-3-3270-0310; E-mail: aaquira{at}hotmail.com.

1 The abbreviations used are: KS, keratan sulfate; IL, interleukin; GAG, glycosaminoglycan; IFN-{gamma}, interferon-{gamma}; {Delta}diHA, unsaturated disaccharides of hyaluronan; {Delta}diC-0S, unsaturated disaccharides of chondroitin; {Delta}diC-6S, unsaturated disaccharides of chondroitin-6-sulfate; {Delta}diC-4S, unsaturated disaccharides of chondroitin-4-sulfate, HS2 unsaturated disaccharides of heparan sulfate; pPKC, phospho-protein kinase C; PI3K, phosphoinositide 3-kinase; IL-10, interleukin-10; LPS, lipopolysaccharides; TUNEL, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick-end labeling; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; LacNAc, N-acetyl-lactosamine; p, phospho-; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase. Back


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Hideo Mochizuki, Kyoko Imai, and Tatsuya Miyazaki (Seikagaku Corp.) for technical assistance. We thank Dr. Hascall at the Cleveland Clinic Foundation and Dr. Savani at Children's Hospital of Philadelphia for review of this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Funderburgh, J. L. (2000) Glycobiology 10, 951-958[Abstract/Free Full Text]
  2. Hascall, V. C. (1982) Prog. Clin. Biol. Res. 110, 3-15
  3. Nieduszynski, I. A., Huckerby, T. N., Dickenson, J. M., Brown, G. M., Tai, G. H., and Bayliss, M. T. (1990) Biochem. Soc. Trans. 18, 792-793[Medline] [Order article via Infotrieve]
  4. Lewis, D., Davies, Y., Nieduszynski, L. A., Lawrence, F., Quantock, A. J., Bonshek, R., and Fullwood, N. J. (2000) Glycobiology 10, 305-312[Abstract/Free Full Text]
  5. Quantock, A. J., Meek, K. M., and Chakravarti, S. (2001) Investig. Ophthalmol. Vis. Sci. 42, 1750-1756[Abstract/Free Full Text]
  6. Hasegawa, N., Torii, T., Kato, T., Miyajima, H., Furuhata, A., Nakayasu, K., Kanai, A., and Habuchi, O. (2000) Investig. Ophthalmol. Vis. Sci. 41, 3670-3677[Abstract/Free Full Text]
  7. Chowers, Y., Lider, O., Schor, H., Barshack, I., Tal, R., Ariel, A., Bar-Meir, S., Cohen, I. R., and Cahalon, L. (2001) Gastroenterology 120, 449-459[CrossRef][Medline] [Order article via Infotrieve]
  8. Cahalon, L., Lider, O., Schor, H., Avron, A., Gilat, D., Hershkoviz, R., Margalit, R., Eshel, A., Shoseyev, O., and Cohen, I. R. (1997) Int. Immunol. 9, 1517-1522[Abstract/Free Full Text]
  9. Neumann, A., Schinzel, R., Palm, D., Riederer, P., and Munch, G. (1999) FEBS Lett. 453, 283-287[CrossRef][Medline] [Order article via Infotrieve]
  10. McKee, C. M., Lowenstein, C. J., Horton, M. R., Wu, J., Bao, C., Chin, B. Y., Choi, A. M., and Noble, P. W. (1997) J. Biol. Chem. 272, 8013-8018[Abstract/Free Full Text]
  11. Cohen, P. L., and Eisenberg, R. A. (1991) Annu. Rev. Immunol. 9, 243-269[CrossRef][Medline] [Order article via Infotrieve]
  12. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Nagata, S. (1992) Nature 356, 314-317[CrossRef][Medline] [Order article via Infotrieve]
  13. Wu. J., Zhou, T., Zhang, J., He, J., Gause, W. C., and Mountz, J. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2344-2348[Abstract/Free Full Text]
  14. Gately, M. K., Desai, B. B., Wolitzky, A. G., Quinn, P. M., Dwyer, C. M., Podlaski, F. L., Familletti, P. C., Sinigaglia, F., Chizonnite, R., Gubler, U., and Stern, A. S. (1991) J. Immunol. 147, 874-882[Abstract]
  15. Gately, M. K., Warrier, R. R., Honsage, S., Carvajal, D. M., Faherty, D. A., Connaughton, S. E., Anderson, T. D., Sarmiento, U., Hubbard, B. R., and Murphy, M. (1994) Int. Immunol. 6, 157-167[Abstract/Free Full Text]
  16. Piccotti, J. R., Chan, S. Y., Li, K., Eichwald, E. J., and Bishop, D. K. (1997) J. Immunol. 158, 643-648[Abstract]
  17. Kato, K., Shimozato, O., Hoshi, K., Wakimoto, H., Hamada, H., Yagita, H., and Okumura, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9085-9089[Abstract/Free Full Text]
  18. Gately, M. K., Carvajal, D. M., Connaughton, S. E., Gillessen, S., Warrier, R. R., Kolinsky, K. D., Wilkinson, V. L., Dwyer, C. M., Higgins, G. F., Jr., Podlaski, F. J., Faherty, D. A., Familletti, P. C., Stern, A. S., and Presky, D. H. (1996) Ann. N. Y. Acad. Sci. 795, 1-12[Medline] [Order article via Infotrieve]
  19. Ha, S. J., Lee, C. H., Lee, S. B., Kim, C. M., Jang, K. L., Shin, H. S., and Sung, Y. C. (1999) J. Immunol. 163, 2902-2908[Abstract/Free Full Text]
  20. Mehal, W. Z., and Crispe, I. N. (1998) J. Immunol. 161, 1686-1693[Abstract/Free Full Text]
  21. Murray, L., and Martens, C. (1989) Eur. J. Immunol. 19, 563-565[Medline] [Order article via Infotrieve]
  22. Xu, H., Kurihara, H., Ito, T., Nakajima, S., Hagiwara, E., Yamanokuchi, H., and Asari, A. (2001) J. Autoimmun. 16, 87-95[CrossRef][Medline] [Order article via Infotrieve]
  23. Kubota, M., Yoshida, K., Tawada, A., and Ohashi, M. (2000) Eur. J. Mass Spectrom. 6, 193-203
  24. Liu, T. F., and Jones, B. M. (1998) Cytokine 10, 140-147[CrossRef][Medline] [Order article via Infotrieve]
  25. Gavrieli, Y., Sherman, Y., and Ben-Saaon, S. A. (1992) J. Cell Biol. 119, 493-501[Abstract/Free Full Text]
  26. Martinez-Pomares, L., Linehan, S. A., Taylor, P. R., and Gordon, S. (2001) Immunobiology 204, 527-535[CrossRef][Medline] [Order article via Infotrieve]
  27. Linehan, S. A., Martinez-Pomares, L., da Silva, R. P., and Gordon, S. (2001) Eur. J. Immunol. 31, 1857-1866[CrossRef][Medline] [Order article via Infotrieve]
  28. Liu, Y., Chirino, A. J., Misulovin, Z., Leteux, C., Feizi, T., Nussenzweig, M. C., and Bjorkman, P. J. (2000) J. Exp. Med. 191, 1105-1116[Abstract/Free Full Text]
  29. Leteux, C., Chai, W., Loveless, R. W., Yuen, C. T., Uhlin-Hansen, L., Combarnous, Y., Jankovic, M., Maric, S. C., Misulovin, Z., Nussenzweig, M. C., and Feizi, T. (2000) J. Exp. Med. 191, 1117-1126[Abstract/Free Full Text]
  30. Kaposzta, R., Marodi, L., Hollinshead, M., Gordon, S., and da Silva, R. P. (1999) J. Cell Sci. 112, 3237-3248[Abstract]
  31. Kikawada, E., Lenda, D. M., and Kelley, V. R. (2003) J. Immunol. 170, 3915-3925[Abstract/Free Full Text]
  32. Yoo, J. K., Cho, J. H., Lee, S. W., and Sung, Y. C. (2002) J. Immunol. 169, 3637-3643[Abstract/Free Full Text]
  33. Cohen, G. M., Sun, X. M., Snowden, R. T., Ormerod, M. G., and Dinsdale, D. (1993) in Programmed Cell Death (Lavin, M., ed) pp. 123-132, Harwood Academic Publishers GmbH, Chur, Switzerland
  34. Rempel, J. D, Wang, M., and HayGlass, K. T. (1997) J. Immunol. 159, 1490-1496[Abstract]
  35. Aksoy, E., Amraoui, Z., Goriely, S., Goldman, M., and Willems, F. (2002) Eur. J. Immunol. 32, 3040-3049[CrossRef][Medline] [Order article via Infotrieve]
  36. Yu, Q., Kovacs, C., Yue, F. Y., and Ostrowski, M. A. (2004) J. Immunol. 172, 6047-6056[Abstract/Free Full Text]
  37. Fabio, R., and Strominger, J. L. (2001) J. Biol. Chem. 276, 37692-37699[Abstract/Free Full Text]
  38. Flesch, I. E., Hess, J. H., Huang, S., Aguet, M., Rothe, J., Bluethmann, H., and Kaufmann, S. H. (1995) J. Exp. Med. 181, 1615-1621[Abstract/Free Full Text]
  39. Xu, H., Ito, T., Tawada, A., Maeda, H., Yamanokuchi, H., Isahara, K., Yoshida, K., Uchiyama, Y., and Asari, A. (2002) J. Biol. Chem. 277, 17308-17314[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Am. Soc. Nephrol.Home page
D. Mahajan, Y. Wang, X. Qin, Y. Wang, G. Zheng, Y. M. Wang, S. I. Alexander, and D. C.H. Harris
CD4+CD25+ Regulatory T Cells Protect against Injury in an Innate Murine Model of Chronic Kidney Disease
J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2731 - 2741.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/21/20879    most recent
M411954200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, H.
Right arrow Articles by Asari, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, H.
Right arrow Articles by Asari, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement