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(Journal of Leukocyte Biology. 2002;71:632-640.)
© 2002 by Society for Leukocyte Biology

Inhibitory actions of glucosamine, a therapeutic agent for osteoarthritis, on the functions of neutrophils

Jian Hua*, Koji Sakamoto{dagger} and Isao Nagaoka*

* Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan; and
{dagger} Koyo Chemical Co., Ltd., Tokyo, Japan

Correspondence: Isao Nagaoka, Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: nagaokai{at}med.juntendo.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucosamine, an amino monosaccharide naturally occurring in the connective and cartilage tissues, contributes to maintaining the strength, flexibility, and elasticity of these tissues. In recent years, glucosamine has been used widely to treat osteoarthritis in humans and animal models. Neutrophils, which usually function as the primary defenders in bacterial infections, are also implicated in the destructive, inflammatory responses in arthritis. In this study, we have evaluated the effects of glucosamine on neutrophil functions using human peripheral blood neutrophils. Glucosamine (0.01–1 mM) dose-dependently suppressed the superoxide anion generation induced by formyl-Met-Leu-Phe (fMLP) or complement-opsonized zymosan and inhibited the phagocytosis of complement-opsonized zymosan or IgG-opsonized latex particles. Furthermore, glucosamine inhibited the release of granule enzyme lysozyme from phagocytosing neutrophils and suppressed neutrophil chemotaxis toward zymosan-activated serum. In addition, glucosamine inhibited fMLP-induced up-regulation of CD11b significantly, polymerization of actin, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). In contrast, N-acetyl-glucosamine, an analogue of glucosamine, did not affect these neutrophil functions (superoxide generation, phagocytosis, granule enzyme release, chemotaxis, CD11b expression, actin polymerization, and p38 MAPK phosphorylation) at the concentrations examined (1–10 mM). Together these observations likely suggest that glucosamine suppresses the neutrophil functions, thereby possibly exhibiting anti-inflammatory actions in arthritis.

Key Words: superoxide • phagocytosis • chemotaxis • F-actin • p38 mitogen-activated protein kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arthritis is a group of chronic inflammatory diseases characterized by destruction of joint cartilage and is the principal cause of pain and disability in the elderly [1 ]. Nonsteroidal anti-inflammatory drugs (NSAID) are used widely in the pharmacological management of arthritis [2 ]. Although these medications are effective in relieving pain and limitation of functions, they cannot cure arthritis and can cause serious side effects [3 ]. Actually, there is a concern that NSAID may be toxic to the articular cartilage and accelerate the course of osteoarthritis [4 , 5 ]. In recent years, glucosamine, a naturally occurring amino monosaccharide, which is present in the connective and cartilage tissues and contributes to maintaining the strength, flexibility, and elasticity of these tissues, has been used widely to treat osteoarthritis in humans and animal models [6 , 7 ]. Several short- and long-term clinical trials in osteoarthritis have shown the significant symptom-modifying effect of glucosamine and its good safety profile [8 , 9 ]. Glucosamine, as a constituent of glycosaminoglycans, can increase proteoglycan synthesis, thereby exhibiting the therapeutic efficacy in arthritis [10 ]. Recently, glucosamine has been shown to inhibit the expression of inducible nitric oxide synthase (iNOS), thereby suppressing the excess production of NO that is implicated in the pathogenesis of arthritis [11 ]. In addition, glucosamine has been shown to act on chondrocytes to prevent the interleukin (IL)-1ß-mediated cellular responses, such as production of NO and prostaglandin E2 and suppression of galactose-ß-1,3-glucuronosyltransferase, a key enzyme for glycosaminoglycan synthesis [12 ]. Moreover, glucosamine has been suggested to exhibit protective actions on carrageenin-induced inflammation and inflammatory bowel disorders [13 , 14 ].

Neutrophils, which usually function as the primary defenders in bacterial infections, have also been implicated as the effector cells in several inflammatory conditions characterized by destructive reactions [15 ]. In response to the stimuli, neutrophils migrate to the inflammatory or infected sites and produce superoxide anion [15 ]. Although superoxide and its oxidant derivatives serve a microbicidal function normally, excessive or inappropriate release of these products contributes to the inflammatory tissue injuries [15 ]. It is interesting that previous studies suggested that superoxide and its derivatives cause tissue destruction in arthritis [16 , 17 ]. Furthermore, inhibition of the production of reaction oxygen species has been shown to reduce the arthritic symptoms profoundly [18 ]. Based on these observations, we hypothesized that glucosamine, which has a protective action on osteoarthritis, may suppress the neutrophil functions such as superoxide anion generation, thereby exhibiting the anti-inflammatory actions in arthritis.

Therefore, in this study, we have evaluated the effects of glucosamine on the functions of neutrophils in vitro using human peripheral blood neutrophils. The results obtained have suggested that glucosamine can suppress superoxide anion generation, phagocytosis, chemotaxis, release of granule enzyme, up-regulation of CD11b, actin polymerization, and phosphorylation of p38 mitogen-activated protein kinase (MAPK), and N-acetyl-glucosamine, an analogue of glucosamine, hardly affects these neutrophil functions at the concentrations examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
N-formyl-Met-Leu-Phe (fMLP), zymosan, latex beads (3 µm), N-cyclohexyl-N'- 2-(4-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate, cytochrome c, superoxide dismutase, cytochalasin B, and p-nitrophenyl phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). D-Glucosamine hydrochloride was obtained from Koyo Chemical Co. (Tokyo, Japan). N-Acetyl-D-glucosamine was purchased from Wako Pure Chemicals (Osaka, Japan).

Cell preparation
Human neutrophils were isolated from heparin-anticoagulated peripheral blood of healthy donors by PolymorphprepTM (Nycomed Pharma AS, Oslo, Norway) centrifugation as described previously [19 ]. After washing with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), isolated neutrophils were suspended at 2 x 107 cells/ml in PBS. Differential cell counts with May/Grünwald/Giemsa stain showed that >98% of the cells were neutrophils.

Opsonization of zymosan particles and latex beads
Zymosan particles were opsonized with complement C3bi by incubating at 8 mg/ml in human fresh serum at 37°C for 30 min, as described previously [20 ]. Latex beads were opsonized with immunoglobulin G (IgG) [21 ]. Briefly, 1% latex beads suspended in 0.1 M glycine-NaOH buffer (pH 8.2) were added with bovine serum albumin (BSA; 10 mg/ml), and the mixture was stirred for 30 min at room temperature. Then, BSA-coated latex beads were washed twice with PBS and mixed with human IgG (1 mg/ml; Sigma Chemical Co.) in PBS. Further, twofold volume of N-cyclohexyl-N'-2-(4-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (50 mg/ml) to cross-link IgG covalently with BSA was added to the mixture and stirred for 3 h at room temperature. Then, IgG-opsonized latex beads were washed twice with PBS.

Measurement of superoxide generation
Superoxide anion production by neutrophils was measured on the basis of superoxide dismutase-inhibitable cytochrome c reduction [22 ]. Neutrophils (106 cells) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine and then were stimulated with 0.1 µM fMLP or complement-opsonized zymosan particles (cell-to-particle ratio of 1:6) at 37°C for 30 min in a total volume of 200 µl PBS containing 1 mM CaCl2 and 1 mM MgCl2 and 60 µM cytochrome c with or without 20 µg/ml superoxide dismutase. After incubation, cell suspensions were centrifuged, and the absorbance of the supernatants was measured. Cytochrome c reduction was calculated by the absorbance difference at 550 nm using an absorption coefficient of 21,000 M-1cm-1.

Measurement of phagocytosis
Neutrophil phagocytosis was assayed, as described previously [20 ]. In brief, neutrophils (106 cells) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine and then were incubated with complement-opsonized zymosan particles or IgG-opsonized latex beads (cell-to-particle ratio of 1:6) at 37°C for 30 min in a total volume of 200 µl PBS containing 1 mM CaCl2 and 1 mM MgCl2. Aliquots of the cell suspension were used to determine phagocytic indices under a phase-contrast microscopy. Phagocytic index was defined as percent-positive ingestion multiplied by average number of ingested particles per cell.

In some experiments, attachment of complement-opsonized zymosan particles or IgG-opsonized latex beads to neutrophils was measured [20 ]. Neutrophils (106 cells) were preincubated at 37°C with 10 µg/ml cytochalasin B in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine for 20 min and then were incubated with complement-opsonized zymosan particles or IgG-opsonized latex beads (cell-to-particle ratio of 1:6) at 37°C for 30 min in a total volume of 200 µl PBS containing 1 mM CaCl2 and 1 mM MgCl2. Attachment index was defined as percent-positive attachment multiplied by average number of attached particles per cell and was determined under a phase-contrast microscope.

Measurement of enzyme release from phagocytosing neutrophils
Neutrophils (107 cells) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine and then were incubated with complement-opsonized zymosan particles (cell-to-particle ratio of 1:6) at 37°C for 30 min in a total volume of 1 ml PBS containing 1 mM CaCl2 and 1 mM MgCl2. After incubation, each tube was placed in an ice bath and centrifuged at 800 g for 5 min. The supernatants were used for measuring enzyme activities. Lysozyme and lactate-dehydrogenase activities were determined as described previously [23 ].

Assay for neutrophil migration
Neutrophil migration was determined by using a modified Boyden chamber with cellulose-nitrate filter (pore size, 3 µm), as described previously [21 , 24 ]. PBS containing 1 mM CaCl2 and 1 mM MgCl2 or zymosan-activated serum was placed in the lower compartment. Neutrophils (3x106 cells/ml) suspended in PBS or zymosan-activated serum were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine and then were introduced into the upper compartment. After incubation at 37°C for 60 min, the filters were fixed, stained, and cleared with xylene. The distance from the focal plane on the top of the filter to the farthest two cells in one focal plane was measured with the micrometer on the fine focus of a microscope. Because there are some differences in the migration distance among fields on the filter, the distances were measured in 10 random fields per filter and averaged.

Zymosan-activated serum was prepared by incubating human fresh serum with zymosan particles (8 mg/ml) at 37°C for 30 min. After incubation, the suspension was centrifuged, and serum was diluted fourfold with PBS containing 1 mM CaCl2 and 1 mM MgCl2.

Flow cytometrical analysis of CD11b expression
Neutrophils (106 cells) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine and then were incubated with 0.1 µM fMLP at 37°C for 5 min in a total volume of 1 ml PBS containing 1 mM CaCl2 and 1 mM MgCl2. The cells were then incubated with 2 µg/ml phycoerythrin (PE)-labeled anti-CD11b ICRF44 monoclonal antibody (mAb) on ice for 30 min (BD PharMingen, San Diego, CA). After washing twice in PBS, the cells were analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA) using Cell Quest software (Becton Dickinson). CD11b expression was determined by measuring the mean fluorescence intensity (MFI) and compared between resting and fMLP-stimulated cells without or with glucosamine- or N-acetyl-glucosamine treatment.

Flow cytometrical measurement of F-actin contents
Neutrophils (106 cells) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine and then were incubated with 0.1 µM fMLP at 37°C for 1 min in a total volume of 100 µl PBS containing 1 mM CaCl2 and 1 mM MgCl2. The reactions were stopped by incubating with 1/10 volume of 37% formaldehyde at 37°C for 10 min. Then, the cells were permeabilized and stained by incubating with 1/10 volume of 1 mg/ml lysophosphatidylcholine (Sigma Chemical Co.) without or with 1 µM fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma Chemical Co.) for 10 min [25 ]. After washing twice in PBS, the cells were analyzed by flow cytometry using Cell Quest software. The relative F-actin content was expressed as the MFI and compared between resting and fMLP-stimulated cells without or with glucosamine- or N-acetyl-glucosamine treatment.

Analysis of the phosphorylation of p38 MAPK
Neutrophils (2x106 cells) were preincubated at 37°C for 20 min in the absence or presence of 1 mM glucosamine or N-acetyl-glucosamine and then were incubated with 0.01 µM fMLP at 37°C for 5 min in a total volume of 0.5 ml PBS containing 1 mM CaCl2 and 1 mM MgCl2. To stop the reaction, the cell suspension was added with 0.5 ml ice-cold PBS containing 2 mM Na3VO4 and 5 mM ethylenediaminetetraacetate (EDTA). After centrifugation, the pellets were lysed in 70 µl lysis buffer [1% Triton X-100, 0.5% Nonidet P-40, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 20 mM Na3VO4, 10 µM p-nitrophenyl phosphate, and 1 mM diisopropyl fluorophosphates] containing 1/25 v/v CompleteTM (Roche Molecular Biochemicals, Mannheim, Germany). The lysates were mixed with 70 µl sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue, 5% ß-mercaptoethanol), disrupted on ice by sonication (Tomy Ultrasonic Disrupter UD-201, Tominaga Works, Tokyo, Japan), and denatured at 100°C for 2 min. Then, 50 µl aliquots were subjected to 10% SDS-PAGE. The separated proteins were transferred electrophoretically to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). The membranes were then blocked in BlockAce (Dainippon Pharmaceutical Co., Tokyo, Japan) and probed with mouse antiphosphorylated p38 MAPK (1:500) mAb (D-8) or goat anti-p38 MAPK (1:200) polyclonal antibody (E-20; Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membranes were probed further with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG/IgM (Chemicon International, Temecula, CA) or HRP-conjugated rabbit anti-goat IgG (Santa Cruz Biotechnology), and the proteins were detected with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The detected bands were quantified by densitometry using Scion Image PC software (Frederick, MD).

Measurement of neutrophil-bactericidal activity
Bactericidal activity of neutrophils was assayed, as described previously [26 ]. In brief, neutrophils (5x106 cells/ml) were preincubated at 37°C for 20 min in the absence or presence of 1 mM glucosamine or N-acetyl-glucosamine and then were incubated with Staphylococcus aureus (NIHJ JC-1) or Escherichia coli (NIHJ JC-2; 5x105 colony-forming unit/ml) at 37°C for up to 70 min in a total volume of 0.5 ml RPMI 1640 (pH 7.4; Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% heat-inactivated, human, normal serum. At 20, 45, and 70 min, 0.05 ml aliquots were diluted 100-fold in 0.1% Triton X-100 in saline and vortexed to lyse neutrophils and disrupt bacterial aggregates. The suspension was diluted further (fivefold) in saline, and 0.1 ml aliquots were spread onto heart-infusion agar. The number of viable bacteria was measured by counting colonies after aerobic, overnight incubation at 37°C. Neutrophil bactericidal activity was expressed as killing (%) using the following formula: killing (%) = [1-(number of colonies in presence of neutrophil/number of colonies in absence of neutrophil)] x 100 [27 ].

Statistical analysis
Statistical analysis was performed using the one-way analysis of variance with a multiple comparison test (StatView®, Abacus Concept, Berkely, CA), and P < 0.05 was considered to be significant. The results are shown as mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of glucosamine on superoxide anion generation
To evaluate the effect of glucosamine on superoxide generation by neutrophils, we stimulated human neutrophils with fMLP or complement-opsonized zymosan in the absence or presence of glucosamine and compared the superoxide-generating activities. As shown in Figure 1A and 1B , glucosamine inhibited the fMLP and opsonized zymosan-induced superoxide generation dose-dependently by neutrophils (P<0.05 at 0.1 and 1 mM glucosamine). In contrast, N-acetyl-glucosamine could not inhibit the fMLP and opsonized zymosan-induced superoxide generation by neutrophils at 1 mM. Thus, glucosamine could suppress the superoxide anion generation by neutrophils in response to fMLP or opsonized zymosan-induced stimulation.



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  		Figure 1. Effect of glucosamine on superoxide anion generation by neutrophils. Neutrophils (5x106 cells/ml) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine (NAG) and were then stimulated with 0.1 µM fMLP (A) or complement-opsonized zymosan (cell-to-particle ratio of 1:6; B) for 30 min. After incubation, superoxide dismutase-inhibitable cytochrome c reduction was calculated. Glucosamine or NAG treatment did not affect the superoxide generation by resting neutrophils incubated without fMLP or complement-opsonized zymosan (unpublished results). Data represent the means ± SD of three separate experiments. Values were compared between fMLP or complement-opsonized zymosan-stimulated neutrophils without (Control) and with glucosamine or NAG treatment. *, P < 0.05; **, P < 0.01.

 
Effect of glucosamine on phagocytosis
Next, we examined the effect of glucosamine on neutrophil phagocytosis using complement- or IgG-opsonized particles. Figure 2A and 2B shows the effect of glucosamine on phagocytosis of complement-opsonized zymosan and IgG-opsonized latex beads by neutrophils. Glucosamine treatment suppressed the uptake of complement-opsonized zymosan and IgG-opsonized latex beads in a dose-dependent manner (P<0.01 at 0.1 and 1 mM glucosamine). However, it is interesting that glucosamine did not affect the attachment of opsonized zymosan and latex beads to neutrophils. In contrast, N-acetyl-glucosamine did not inhibit uptake or attachment of opsonized zymosan and latex particles at a concentration of 1 mM. Thus, glucosamine suppressed the ingestion of opsonized particles by neutrophils but did not affect the binding of opsonized particles to their respective receptors.



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  		Figure 2. Effect of glucosamine on neutrophil phagocytosis. Neutrophils (5x106 cells/ml) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM of N-acetyl-glucosamine (NAG) and were then incubated with complement-opsonized zymosan particles (A) or IgG-opsonized latex beads (cell-to-particle ratio of 1:6; B) at 37°C for 30 min. The aliquots of cell suspension were used to determine phagocytic indices under a phase-contrast microscopy. Furthermore, to determine the attachment of phagocytic particles to neutrophils, neutrophils (5x106 cells/ml) were preincubated at 37°C for 20 min with 10 µg/ml cytochalasin B in the absence or presence of 0.01–1 mM glucosamine or 1 mM NAG and then were incubated with complement-opsonized zymosan particles (A) or IgG-opsonized latex beads (B) at 37°C for 30 min. Data represent the means ± SD of three separate experiments. Values were compared without (Control) and with glucosamine or NAG treatment. **, P < 0.01; ***, P < 0.001.

 
Effect of glucosamine on enzyme release from phagocytosing neutrophils
To examine whether glucosamine affects the extracellular release of granule enzyme from phagocytosing neutrophils, we measured the activity of lysozyme (a specific granule constituent) in the extracellular supernatant after stimulation with complement-opsonized zymosan. As shown in Figure 3 , glucosamine dose-dependently inhibited the lysozyme release from phagocytosing neutrophils (P<0.05 at 0.1 and 1 mM glucosamine), whereas N-acetyl-glucosamine did not inhibit the lysozyme release at 1 mM. The release of lactate dehydrogenase (a cytoplasmic constituent) was always less than 10% under the condition examined (resting and phagocytosing neutrophils without or with glucosamine or N-acetyl-glucosamine treatment; unpublished results).



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  		Figure 3. Effect of glucosamine on enzyme release from phagocytosing neutrophils. Neutrophils (107 cells/ml) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1mM glucosamine or 1 mM N-acetyl-glucosamine (NAG) and then were incubated with complement-opsonized zymosan particles (cell-to-particle ratio of 1:6) at 37°C for 30 min. After incubation, the supernatants of cell suspensions were used for the assay of lysozyme and lactate-dehydrogenase activities. The release of lactate dehydrogenase was less than 10% under the condition examined (resting and phagocytosing neutrophils with or without glucosamine or NAG treatment). Glucosamine or NAG treatment did not affect the enzyme release from resting neutrophils incubated without complement-opsonized zymosan particles (unpublished results). Data represent the means ± SD of three separate experiments. Values were compared between phagocytosing neutrophils without (Control) and with glucosamine or NAG treatment. *, P < 0.05; **, P < 0.01.

 
Effect of glucosamine on neutrophil migration
Next, we examined the effect of glucosamine on neutrophil migration. As shown in Figure 4 , glucosamine dose-dependently inhibited chemotaxis (P<0.01 at 0.01, 0.1, and 1 mM glucosamine) toward zymosan-activated serum. Furthermore, glucosamine inhibited the random migration for zymosan-activated serum and PBS in a dose-dependent manner (P<0.05 at 0.01, 0.1, and 1 mM glucosamine). In contrast, N-acetyl-glucosamine did not suppress the neutrophil migration (chemotaxis and random migration) at 1 mM.



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  		Figure 4. Effect of glucosamine on neutrophil migration. PBS containing 1 mM CaCl2 and 1 mM MgCl2 or zymosan-activated serum was placed in the lower compartment. Neutrophils (3x106 cells/ml) suspended in PBS or zymosan-activated serum were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine or 1 mM N-acetyl-glucosamine (NAG) and then were introduced into the upper compartment. After incubation at 37°C for 60 min, the filters were fixed and stained, and the migration distance was determined. Data represent the means ± SD of three separate experiments. Values were compared without (Control) and with glucosamine or NAG treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Effect of glucosamine on CD11b expression
Neutrophil stimulation is known to increase the expression of adhesion molecules, such as CD11b, a component of ß2 integrin, which is involved in neutrophil adhesion and phagocytosis of complement-opsonized particles [28 ]. To determine whether glucosamine affects CD11b expression, we analyzed the expression of CD11b by flow cytometry using neutrophils stimulated with fMLP. As shown in Figure 5A and 5B , glucosamine inhibited the fMLP-induced up-regulation of CD11b expression significantly at 1 mM (P<0.05), whereas 1 mM N-acetyl-glucosamine did not affect the CD11b expression.



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  		Figure 5. Effect of glucosamine on CD11b expression by neutrophils. Neutrophils (106 cells/ml) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine (GS) or 1 mM N-acetyl-glucosamine (NAG) and then were stimulated with 0.1 µM fMLP at 37°C for 5 min. The cells were then incubated with PE-labeled anti-CD11b mAb. After washing, CD11b expression was analyzed by flow cytometry (A). The MFIs were measured for resting and fMLP-stimulated cells without or with glucosamine or NAG treatment, and the up-regulation of CD11b expression was expressed as the percentage of that of fMLP-stimulated cells without glucosamine or NAG treatment (Control; B). Glucosamine or NAG treatment did not affect the fluorescence intensity of resting cells incubated without fMLP (unpublished results). Data represent the means ± SD of three separate experiments. Values were compared between fMLP-stimulated cells without (Control) and with glucosamine or NAG treatment. *, P < 0.05.

 
Effect of glucosamine on the F-actin formation
The above results indicated that glucosamine inhibited neutrophil functions such as superoxide generation, phagocytosis, granule enzyme release, migration, and up-regulation of adhesion molecules (CD11b). It is well-established that actin, a major component of the cytoskeletal system, is transferred rapidly and transiently from a globular (G-actin) to a filamentous (F-actin) form in neutrophils after treatment with chemotactic as well as phagocytic stimuli [29 ]. To determine whether the inhibitory actions of glucosamine on neutrophil functions are related to the inhibition of actin polymerization, we measured the contents of F-actin in human neutrophils stimulated with fMLP. As shown in Figure 6A and B , glucosamine inhibited the fMLP-induced increase of F-actin contents in neutrophils dose-dependently (P<0.01 at 0.1 and 1 mM glucosamine). On the contrary, N-acetyl-glucosamine (1 mM) did not suppress actin polymerization in fMLP-stimulated neutrophils.



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  		Figure 6. Effect of glucosamine on F-actin contents in neutrophils. Neutrophils (107 cells/ml) were preincubated at 37°C for 20 min in the absence or presence of 0.01–1 mM glucosamine (GS) or 1 mM N-acetyl-glucosamine (NAG) and then were incubated with 0.1 µM fMLP at 37°C for 1 min. The reactions were stopped by incubating with 3.7% formaldehyde, and then the cells were permeabilized and stained by incubating with 0.1 mg/ml lysophosphatidylcholine and 0.1 µM FITC-labeled phalloidin. After washing, the content of F-actin was analyzed by flow cytometry (A). The MFIs were measured for resting and fMLP-stimulated cells without or with glucosamine or NAG treatment, and the increase of F-actin content was expressed as the percentage of that of fMLP-stimulated cells without glucosamine or NAG treatment (Control; B). Glucosamine or NAG treatment did not affect the F-actin contents in resting cells incubated without fMLP (unpublished results). Data represent the means ± SD of three separate experiments. Values were compared between fMLP-stimulated cells without (Control) and with glucosamine or NAG treatment. **, P < 0.01; ***, P < 0.001.

 
Effect of glucosamine on phosphorylation of p38 MAPK
p38 MAPK has been demonstrated to play a crucial role in neutrophil functions [30 31 32 33 34 ]. To determine whether the inhibitory actions of glucosamine on neutrophil functions are mediated by the suppression of p38 MAPK pathway, we stimulated neutrophils with fMLP in the presence of glucosamine and evaluated its effect on the phosphorylation of p38 MAPK. As shown in Figure 7A and 7B , treatment with 1 mM glucosamine but not N-acetyl-glucosamine suppressed the fMLP-induced phosphorylation of p38 MAPK significantly (P<0.001), although almost the same amounts of p38 MAPK protein were recovered in each sample.



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  		Figure 7. Effect of glucosamine on the phosphorylation of p38 MAPK. Neutrophils (4x106 cells/ml) were preincubated at 37°C for 20 min in the absence (Control) or presence of 1 mM glucosamine (GS) or N-acetyl-glucosamine (NAG) and then were stimulated with 0.01 µM fMLP for 5 min. After the incubation, the cell lysates were subjected to 10% SDS-PAGE. The separated proteins were blotted to a polyvinylidene-difluoride membrane, and the membrane was probed with mouse antiphosphorylated p38 MAPK mAb or goat anti-p38 MAPK polyclonal antibody. The membranes were probed further with HRP-conjugated goat anti-mouse IgG/IgM or HRP-conjugated rabbit anti-goat IgG, and the phosphorylated p38 MAPK (pp38 MAPK) and p38 MAPK proteins (p38 MAPK) were detected (A). The detected bands were quantified by densitometry, and the phosphorylation of p38 MAPK was expressed as the percentage of that of fMLP-stimulated cells without glucosamine or NAG treatment (Control; B). Glucosamine or NAG treatment did not affect the phosphorylation of p38 MAPK in resting cells incubated without fMLP (unpublished results). Data represent the means ± SD of three separate experiments. Values were compared between fMLP-stimulated cells without (Control) and with glucosamine or NAG treatment. ***, P < 0.001.

 
In separate experiments, we examined the effects of higher concentrations of N-acetyl-glucosamine on neutrophil functions. N-acetyl-glucosamine could not substantially affect the neutrophil functions investigated even at 10 mM (superoxide generation, phagocytosis, granule enzyme release, chemotaxis, CD11b up-regulation, actin polymerization, and p38 MAPK phosphorylation; unpublished results).

Effect of glucosamine on bactericidal activity of neutrophils
It is known that neutrophils serve in the primary defense against microbial pathogens. From the inhibitory actions of glucosamine on several neutrophil functions described above, it could be suggested that glucosamine may hinder the antibacterial activity of neutrophils. To examine the effect of glucosamine on neutrophil-bactericidal activity, neutrophils were incubated with target bacteria (S. aureus and E. coli) in the absence or presence of glucosamine, and the antibacterial activity was determined. As shown in Figure 8 , 1 mM glucosamine did not affect the bactericidal activity of neutrophils significantly, although it inhibited neutrophil functions substantially, such as superoxide generation and phagocytosis, which are important for the neutrophil antibacterial activity. This observation is unexpected and likely suggests that the residual activities of neutrophil functions after glucosamine treatment may be sufficient for the expression of bactericidal activity by neutrophils. In contrast to the action of glucosamine, N-acetyl-glucosamine did not affect bactericidal activity or other neutrophil functions investigated.



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  		Figure 8. Effect of glucosamine on bactericidal activity of neutrophils. Neutrophils were preincubated at 37°C for 20 min in the absence (Control) or presence of 1 mM glucosamine (GS) or N-acetyl-glucosamine (NAG) and then were incubated with S. aureus (A) or E. coli (B) at 37°C for up to 70 min in a total volume of 0.5 ml RPMI 1640 containing 10% heat-inactivated human normal serum. At 20, 45, and 70 min, aliquots (0.05 ml) were withdrawn and diluted, and the number of viable bacteria was measured by colony counting. Neutrophil bactericidal activity was expressed as killing (%), using the following formula: killing (%) = [1-(number of colonies in presence of neutrophil /number of colonies in absence of neutrophil)] x 100. Data represent the means ± SD of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glucosamine, a constituent of articular glycosaminoglycans, is being marketed extensively as a therapeutic agent for osteoarthritis. Several short- and long-term clinical trials in osteoarthritis have shown the significant symptom-modifying effects of glucosamine [8 , 9 ]. As a mechanism for the chondroprotective action, glucosamine is assumed to increase proteoglycan synthesis, thereby exhibiting the therapeutic efficacy in arthritis [10 ]. Of interest, superoxide and its derivatives have been shown to play a pivotal role in pathogenesis of arthritis, and the inhibition of reaction oxygen production is shown to strongly relieve the arthritic symptoms [16 17 18 ]. To clarify the possible effect of glucosamine on neutrophils that extracellularly release reactive oxygen products and granular enzymes at inflammatory sites and that are implicated as the effector cells in destructive tissue injuries [15 ], in this study, we evaluated the actions of glucosamine on neutrophil functions in vitro. The results indicated that glucosamine suppressed the neutrophil functions including superoxide generation, phagocytosis, granule enzyme release, and chemotaxis and also repressed the up-regulation of CD11b, actin polymerization, and phosphorylation of p38 MAPK associated with neutrophil activation. In contrast, glucosamine did not affect the bactericidal activity of neutrophils.

p38 MAPK is a Ser/Thr kinase belonging to the family of MAPKs, and its importance in neutrophil functions has been demonstrated [30 31 32 33 34 ]. p38 MAPK is activated (phosphorylated) via the stimulation of the receptor for the Fc region of IgG (Fc{gamma}R); the complement receptor type 3, C3bi receptor (CR3); and the N-formyl peptide receptor on neutrophils. It is also involved in the up-regulation of CD11b, activation of reduced nicotinamide adenine dinucleotide phosphate oxidase, and actin polymerization (reorganization) necessary for the cellular movement, including phagocytosis and cell migration (random and chemotactic movement), in response to soluble agonists and IgG- or complement-opsonized targets [32 33 34 ]. In this study, we revealed that phosphorylation of p38 MAPK, as well as superoxide generation, phagocytosis, phagocytosis-associated granule enzyme release, neutrophil migration, up-regulation of CD11b, and actin polymerization, was suppressed by glucosamine. Thus, the suppression of p38 MAPK activation is assumed to mediate inhibitory action of glucosamine on these neutrophil functions.

Recently, it has been demonstrated that the receptors are functionally linked on neutrophil surfaces and act cooperatively to stimulate the intracellular signaling [35 36 37 38 39 ]. For example, Fc{gamma}R and formyl peptide receptor are assumed to act in concert to mediate neutrophil chemotaxis, because a mAb against Fc{gamma}R inhibits neutrophil migration toward formyl peptides [36 ]. Moreover, CR3 and Fc{gamma}R are shown to be physically associated (cocapped) on the cell surfaces to mediate neutrophil functions, because N-acetyl-glucosamine, which interferes with the lectin-carbohydrate interactions of CR3 and Fc{gamma}R, prevents the cocapping of two receptors and dramatically inhibits neutrophil functions such as cytosolic calcium mobilization and superoxide production in response to immune complexes [35 , 37 , 38 ]. Of interest, we noticed in the present study that glucosamine inhibited the phagocytosis of complement-opsonized zymosan or IgG-opsonized latex beads by neutrophils but did not affect the binding of particles to their respective receptors (CR3 and Fc{gamma}R), suggesting that glucosamine suppresses neutrophil functions without affecting the ligand-receptor binding. Thus, it is tempting to speculate that glucosamine, like N-acetyl-glucosamine could interfere with the lectin-saccharide interactions and inhibit the functional association of cell-surface receptors, thereby suppressing the intracellular signaling in neutrophils. However, we found that glucosamine at 1 mM could substantially inhibit, but even at 10 mM, N-acetyl-glucosamine could not affect(unpublished results), neutrophil functions. Consistent with our observations, it has been shown that very high concentrations of N-acetyl-glucosamine (50–150 mM) are required for the inhibition of receptor cocapping and receptor-mediated neutrophil activation [35 , 37 , 38 ]. Thus, the mechanism whereby glucosamine suppresses neutrophil functions remains speculative and may be different from that of N-acetyl-glucosamine. Further experimental development will be needed for elucidation of the inhibitory actions of glucosamine on neutrophils.

In this study, we have suggested that glucosamine can inhibit several neutrophil functions possibly via the suppression of p38 MAPK, a major signal-transduction molecule. At inflammatory sites, neutrophils accumulate and extracellularly release reactive oxygen products and granular enzymes that are implicated in the destructive tissue injuries [15 16 17 ]. Of importance, plasma concentrations of glucosamine are estimated to reach 0.12–0.29 mM after intravenous administration of 0.5 mmol (108 mg) glucosamine hydrochloride/kg into rats [11 ]. For humans, usually 1.5 or 3 g/day of glucosamine sulfate or hydrochloride is administered for treatment of osteoarthiris [7 , 40 , 41 ]. We have observed that serum levels of glucosamine range from 0.02 to 0.03 mM after oral administration of 1.5 g glucosamine hydrochloride into humans (three males and one female, weighing 46–85 kg; unpublished results). Noticeably, in this study, the inhibitory actions of glucosamine on neutrophil functions were detected at concentrations higher than 0.01 mM (especially for chemotaxis). Thus, glucosamine is expected to suppress neutrophil functions (such as chemotaxis, phagocytosis, superoxide generation, and granule enzyme release) in vivo, thereby possibly exhibiting anti-inflammatory actions.

Long-term clinical trials in osteoarthritis have indicated that several symptoms including increased blood pressure, abdominal pain, diarrhea, and fatigue are observed as adverse events during glucosamine therapy [9 ]. In contrast, accelerated wound healing, relief from chronic vascular headaches, and symptomatic improvement in inflammatory bowel diseases are recorded as interesting side effects of glucosamine [41 ]. The present study has revealed that glucosamine inhibits neutrophil functions such as superoxide generation and phagocytosis, which are closely related to the bactericidal activity. Thus, it is possible that glucosamine may exhibit detrimental effects on the host-defense mechanism against bacterial infection in vivo by hindering essential neutrophil functions. However, bactericidal activity of neutrophils was not affected significantly by glucosamine in vitro under the conditions used. Whatever the case may be, glucosamine should be administered cautiously in vivo, considering its suppressive effects on neutrophil functions. The issue whether glucosamine could be detrimental to a de novo or ongoing bacterial infection/colonization remains unclear and needs further experimental development in the future.

In conclusion, glucosamine could be an attractive candidate for adjunctive therapy in arthritis by exhibiting not only chondroprotective actions but also anti-inflammatory actions via the suppression of neutrophil functions.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Atopy (Allergy) Research Center, Juntendo, University and the Foundation for Total Health Promotion, Japan. We are grateful to Dr. Kazuhisa Iwabuchi for his kind advice in performing the analysis of p38 MAPK phosphorylation.

Received July 23, 2001; revised December 6, 2001; accepted December 15, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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