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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
-Glucan Administration
Departments of Surgery (T.R.O.-S., M.P.G., D.L.W.) and Pharmacology (D.L.W.), James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee; Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom (S.G.); and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa (G.D.B.)
Received February 3, 2006; accepted April 20, 2006.
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Abstract |
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33-85%) in peripheral leukocyte membrane-associated Dectin-1 positivity that lasted for up to 7 days. The loss of leukocyte membrane-associated Dectin-1 after GP administration was primarily due to decreased levels of Dectin-1 on neutrophil and monocyte membranes with no significant changes in the percentage of neutrophils or monocytes circulating in the blood. Administration of control carbohydrate polymers, i.e., mannan or pullulan, which are not ligands for Dectin-1, did not decrease Dectin-1 leukocyte positivity, indicating that the effect on Dectin-1 is specific to glucans. In fact, mannan administration increased leukocyte Dectin-1 positivity, thus demonstrating a differential effect on leukocyte Dectin-1, compared with GP. We conclude that systemic administration of GP has a specific and prolonged effect on loss of leukocyte membrane Dectin-1 positivity. These data may have important implications for developing dosing regimens for immunomodulatory carbohydrates.
). In purified form, glucans have been demonstrated to stimulate innate immunity (Williams, 1997). Pharmaceutical grade glucans are currently being evaluated as agents to increase resistance to infections (Williams et al., 1996), facilitate wound repair (Wei et al., 2002), prevent myocardial ischemia/reperfusion injury (Li et al., 2003), and as adjuvants for antitumor responses (Hong et al., 2004). The underlying cellular and molecular mechanisms responsible for the in vivo activities of glucans are only now coming to light. The first step in the modulation of cellular function by glucans is binding of the glucan by membrane-associated pattern recognition receptors (Mueller et al., 1996, 2000; Rice et al., 2002). Brown and Gordon (2001) identified Dectin-1 as a glucan-specific pattern recognition receptor. Ariizumi et al. (2000) had previously isolated Dectin-1 from a dendritic cell cDNA subtraction library and showed that Dectin-1 recognized an endogenous ligand on T cells. The fact that Dectin-1 can serve as a pattern recognition receptor for glucans and as a dendritic cell receptor that interacts with T cells suggests that Dectin-1 may be an important link between the innate and adaptive immune responses. It may also explain why glucans have been reported to modulate both innate and adaptive immunity (Brown and Gordon, 2003).
Dectin-1 is a type II transmembrane receptor that contains a single non-C-type lectin-like carbohydrate recognition domain extracellularly and a tyrosine-based activation motif in the cytoplasmic tail (Ariizumi et al., 2000; Brown and Gordon, 2001; Brown et al., 2002, 2003; Taylor et al., 2002). Dectin-1 is expressed at high levels on blood and splenic monocytes, neutrophils, and alveolar and inflammatory macrophages and at lower levels on dendritic cells and subpopulations of T cells (Ariizumi et al., 2000; Brown et al., 2002; Taylor et al., 2002). Dectin-1 will bind free glucans as well as whole Candida albicans and Saccharomyces cerevisiae cells in a glucan-dependent manner, and upon binding, the Dectin-1/glucan complex is rapidly internalized (Ariizumi et al., 2000; Brown and Gordon, 2001; Brown et al., 2002, 2003; Taylor et al., 2002; Herre et al., 2004). Dectin-1 mediates the response to glucans, in part, by activating intracellular signaling pathways, including nuclear factor-
B (Brown et al., 2003). In addition, glucan-Dectin-1 complexes can signal through both toll-like receptor 2-dependent or -independent mechanisms (Brown et al., 2003; Gantner et al., 2003). Recent evidence also indicates that ligation of glucan by Dectin-1 stimulates production of the anti-inflammatory cytokine interleukin-10 by recruiting the nonreceptor tyrosine kinase syk (Rogers et al., 2005). These findings have dramatically increased our knowledge of the cellular biology of glucan responses.
Despite these significant advances in our knowledge of glucan-Dectin interactions, virtually all glucan-Dectin-1 studies reported to date have been conducted with in vitrocultured cell lines and/or recombinant proteins. There are virtually no data available on the in vivo effect of glucans on Dectin-1 levels. Specifically, we do not know whether systemically administered glucans are up-taken by Dectin-1-positive circulating leukocytes, nor are there any data available on leukocyte Dectin-1 membrane expression following administration of a pharmaceutical grade soluble glucan. In the present study, we administered a highly purified, water-soluble glucan and studied the changes in circulating leukocyte Dectin-1 expression in mice. We found that glucans are internalized by circulating leukocytes and that there is a significant loss of leukocyte Dectin-1 positivity for up to 7 days after a single injection of a biologically active glucan.
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Materials and Methods |
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Carbohydrates. We selected glucan phosphate (GP) for this study because it is a highly purified, pharmaceutical-grade glucan polymer that is bound by Dectin-1 (Williams et al., 1991; Mueller et al., 2000). At the dose employed in this study, GP has been shown to enhance wound repair (Wei et al., 2002), suppress tumor growth (Williams et al., 1987), and increase survival in various models of sepsis (Williams et al., 1999, 2004; Rice et al., 2005). In addition, the in vivo pharmacokinetics of GP following i.v. administration have been defined (Rice et al., 2004). Water-soluble GP was prepared and chemically characterized as described previously (Williams et al., 1991; Ensley et al., 1994; Mueller et al., 1995). The final product was stored (-80°C) as a lyophilized powder. It was dissolved in 5% (w/v) dextrose (Baxter, Toronto, ON, Canada), filter sterilized (0.45 µm), and screened for endotoxin contamination with the Endospecy assay (Seikagaku, Tokyo), which is specific for endotoxin but does not respond to (1
3)-
-D-glucans (Kambayashi et al., 1991). Mannan (Sigma, St. Louis, MO) and pullulan (Pfanstiehl, Waukegan, IL) were employed as control carbohydrate polymers because they are not recognized by Dectin-1 (Brown et al., 2002). Mannan or pullulan was dissolved in 5% (w/v) dextrose, incubated overnight in prewashed polymyxin-coated agarose beads (Bio-Rad, Hercules, CA) to remove endotoxin, and filter sterilized before use.
Preparation of Fluorescent-Labeled GP. A diaminopropane (DAP) moiety was added to the reducing terminus of GP as described previously (Kougias et al., 2001; Rice et al., 2005). In brief, the DAP was attached to the reducing terminus of the carbohydrate polymer by sodium borohydride reduction (Kougias et al., 2001). The reaction mixture was dialyzed against 18-M
ultrapure pyrogen-free water and lyophilized to dryness. The GP was stored at -20°C. Aliquots of the DAP-glucan derivatives were analyzed by gel permeation chromatography/multiangle laser light scattering and 1H and 13C NMR to confirm that the molecular weight, polydispersity, primary structure, and solution conformation were not altered by the derivatization. The carbohydrates were labeled with Alexa Fluor 488 succinimidyl ester (catalog no. A-20000; Molecular Probes, Eugene, OR). Specifically, DAP-GP (10 mg) was dissolved into sodium borate buffer (0.1 M, pH 8.5) at a concentration of 10 µg/µl in a total volume of 1 ml. Alexa Fluor 488 dye (1 mg) was dissolved in 35 µl of dimethyl sulfoxide. The dye solution was added to the DAP-GP and incubated overnight at ambient temperature on a reciprocating shaker at slow speed. The samples were incubated in foil covered microfuge tubes to prevent exposure to light. The excess dye was removed by dialyzing (10,000 molecular weight cut-off) against ultrapure water overnight at room temperature. Conjugation and binding activity were confirmed by incubation of the fluorescent-labeled GP with Dectin-1 transfected cells for 30 min at 4°C, washing three times with phosphate-buffered saline (PBS), and analyzing by flow cytometry.
Experimental Protocol. To study the uptake and internalization of circulating glucans, mice were injected i.v. or i.p. at time 0 with 5 mg/mouse fluorescent-labeled GP. Control mice received unlabeled GP or isovolumetric dextrose. Peripheral leukocytes and peritoneal leukocytes were harvested at 5 h postinjection. To examine the effect of glucan on leukocyte membrane Dectin-1 levels, mice were injected at time 0 with 1 mg/mouse GP, 1 mg/mouse mannan, or 1 mg/mouse pullulan. Control mice received isovolumetric dextrose 5% (w/v) i.v. or i.p. Mice were sacrificed at 0, 3, 6, and 12 h and 1, 3, 5, 7, and 10 days postinjection. Peripheral blood leukocytes, peritoneal cells, bone marrow cells, and splenocytes were obtained for flow cytometric analysis of Dectin-1. Peritoneal cells were obtained by peritoneal lavage. Bone marrow cells were obtained by flushing the femoral marrow cavities with sterile PBS. Splenocytes were isolated by teasing apart the spleens and separation of the splenic stroma by sedimentation. The peritoneal cells, bone marrow cells, and splenocytes were suspended in Pharmingen Stain Buffer (San Diego, CA) before staining and analysis. Whole blood was collected into EDTA microtainer tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ).
Flow Cytometry. Erythrocytes were lysed with PharmLyse buffer (Pharmingen) according to the manufacturer's directions. Leukocytes were blocked with 5% rabbit serum, 0.5% bovine serum albumin, and 5 mM EDTA with anti-murine CD16/32 (Pharmingen) before staining. Cells were stained with biotin conjugated rat anti-murine Dectin-1 (Brown et al., 2003), fluorescein isothiocyanate-conjugated anti-neutrophil (clone 7/4) (Serotec, Oxford, UK), allophycocyanin-conjugated anti-F4/80 (Caltag, Burlingame, CA), and peridinin chlorophyll-A protein-conjugated anti-CD3 (Pharmingen) or their isotype control antibodies. Staining was performed according to the protocol described by the manufacturer. Biotinylated antibodies were detected by streptavidin-phycoerythrin (Pharmingen). Cells were suspended in Pharmingen Stain buffer and analyzed using a FACScalibur flow cytometer with CellQuest software (BD Biosciences, Mountain View, CA).
Confocal Microscopy. Resident peritoneal cells, peripheral leukocytes, and splenocytes were harvested from mice at 3 and 5 h after injection with fluorescent-labeled GP. The cells were prepared as described for flow cytometry, except that after lysis of red blood cells, the cells were fixed in 4% (w/v) paraformaldehyde at ambient temperature for 10 min. The cellular nuclei were counterstained by incubating the cells with 1 µg/ml propidium iodide (Molecular Probes) for 10 min and then washed three times with PBS. The cells were mounted onto glass slides and coverslipped using Prolong Anti-Fade mounting medium (Molecular Probes). All procedures were performed in the dark. The slides were evaluated on a Leica DM IRBE inverted confocal microscope with the TC2 SP2 microscope system (Leica, Exton, PA).
Colocalization of GP and Dectin-1. Elicited macrophages were harvested by peritoneal lavage 72 h after i.p. injection of thioglycollate (2 ml). Cells were cultured in four-chamber slides (Lab-Tek; Nalge Nunc International, Naperville, IL) at 100,000 cells per well in RPMI-1640 medium (Sigma) supplemented with 10% serum and antibiotics. Cells were incubated with 100 µg/ml fluorescent-labeled GP in serum-free medium at 4°C for 3 h, then at 37°C for 10 min. The slides were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100. The cells were blocked (1 h) with 10% normal horse serum and then incubated overnight with goat anti-Dectin-1 antibody (R&D Systems, Minneapolis, MN) at 4°C. Following three washes with PBS, the cells were stained with tetramethylrhodamine B isothiocyanate-conjugated anti-goat antibody (Jackson ImmunoResearch, West Grove, PA) diluted in Sytox green nuclear counterstain (Molecular Probes). The slides were washed in PBS, followed by water, and then coverslips were mounted using Prolong Anti-Fade. The slides were evaluated on a Leica DM IRBE inverted confocal microscope with the TC2 SP2 microscope system (Leica). Images were evaluated using multicolor/two-dimensional cytofluorogram software from Leica Microsystems. The software quantifies the extent of colocalization by creation of a binary mask of the image data in the cytofluorogram. The binary mask is created by masking all of the pixels that are double positive for both the GP fluorescence and for the tetramethylrhodamine B isothiocyanate-labeled Dectin-1 fluorescence. Colocalization was then assessed using the mask intensity rate for the colocalized GP versus the overall intensities of the GP in the image. A mask intensity rate of 
50% was used to confirm colocalization.
Statistics. Dectin-1 data were summarized by the mean and SEM. Group mean responses were compared by analysis of variance and pair-wise multiple comparison testing (the least significant difference procedure or Tukey's procedure for cases where analysis of variance was not significant). Flow cytometry data were normalized to the mean percent positive cells for the untreated control for each time interval. Control values were set to 100. Probability levels of 0.05 or smaller were considered significant.
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Results |
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; Herre et al., 2004). However, it is not known whether GP was up-taken by leukocytes following in vivo administration. To address this issue, blood and splenic leukocytes were harvested 5 h after administration of fluorescent-labeled GP. Five hours after i.v. administration, fluorescent-labeled GP was found within 1.86% of peripheral blood leukocytes, 93.67% of which were polymorphonuclear cells (Fig. 1A). Fluorescent-labeled GP was also detected in 1.91% splenic leukocytes at 5 h postinjection (Fig. 1A). Five hours after i.p. administration, fluorescent-labeled GP could be detected in 86.20% of peritoneal leukocytes (Fig. 1A). Fluorescent-labeled GP was also visible in the peripheral blood leukocytes and splenocytes after i.p. administration of fluorescent-labeled GP (data not shown). This indicates that systemically administered glucans are up-taken by circulating and tissue-residing leukocytes.
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GP and Dectin-1 Colocalize in Primary Murine Macrophage Cultures. Herre et al. (2004) have reported that macrophages internalize Dectin-1 in response to GP treatment; however, there is no evidence to indicate whether the GP and Dectin-1 are internalized and colocalized within the leukocyte. Incubation of murine peritoneal macrophages with fluorescent-labeled GP resulted in uptake of the GP (Fig. 1B, green). In the cells that do not contain glucan, Dectin-1 is primarily membrane-associated (Fig. 1B, red). In the cell containing GP, the GP colocalized with Dectin-1 in an intracellular vesicle (Fig. 1B, yellow). Colocalization with Dectin-1 was found in 69.23% of cells containing GP (data not shown). We interpret these data to mean that glucans are internalized and colocalized with Dectin-1 in leukocytes.
Parenteral Administration of GP Induces a Prolonged Decrease in the Percentage of Dectin-1-Expressing Peripheral Blood Leukocytes. During the course of these experiments, three populations of peripheral blood Dectin-1-expressing leukocytes were identified in normal mice: negative for Dectin-1, low levels of Dectin-1, and high levels of Dectin-1. A portion (60%) of the total peripheral leukocytes expressed little or no Dectin-1 (Fig. 2A). The majority of peripheral blood leukocytes from control mice that do express Dectin-1 show high levels of Dectin-1 expression (Fig. 2A). Following GP administration (i.v. or i.p.), there is a dramatic and significant decrease in membrane Dectin-1 levels (Fig. 2A). However, Dectin-1 expression was not completely lost from all leukocytes in the presence of GP treatment. Low levels of Dectin-1 were detectable on leukocyte membranes after either i.v. or i.p. GP administration (Fig. 2A). Thus, there is a shift from high to low levels of Dectin-1 expression following GP administration.
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Intraperitoneal administration of GP resulted in a decrease in Dectin-1-positive peritoneal leukocytes, but this decrease was of less magnitude and shorter duration than observed in peripheral blood leukocytes (Fig. 2C). Membrane Dectin-1 levels on peritoneal leukocytes were decreased by 63.1, 61.9, and 47.8% at 3, 6, and 12 h after i.p. injection and returned to normal levels by 24 h compared with control mice (p < 0.05) (Fig. 2C). GP administration also decreased the percentage of Dectin-1-positive splenic leukocytes; however, this decrement (18.1%, p < 0.05) was only observed on day 1 postinjection and following i.p. GP administration (data not shown).
Administration of GP Induces a Prolonged Loss of Dectin-1 Positivity in Peripheral Blood Neutrophil and Monocyte Dectin-1. Dectin-1 expression varies on different blood leukocyte cell types. For this reason, cells were stained for phenotype to determine whether the changes observed were due to changes in peripheral leukocyte population dynamics or due to changes in Dectin-1 expression on individual leukocyte cell types. Although there was a slight trend toward neutrophilia in GP-injected mice, this increase was only significant at 3 h following i.v. GP administration (data not shown). Neither i.p. nor i.v. administration of GP had an effect on the percentage of monocytes in the overall blood leukocyte population at any time interval compared with control (data not shown). Therefore, the modest changes in peripheral blood leukocyte numbers could not account for the dramatic changes observed in membrane Dectin-1 levels following GP administration.
When Dectin-1 levels were examined in peripheral leukocyte phenotypes, peripheral blood neutrophils and monocytes, which normally express high levels of Dectin-1, showed dramatic and significant loss of membrane Dectin-1 following GP administration (Fig. 2D). Neutrophil membrane Dectin-1 levels were decreased (p < 0.05) by 49.5, 82.5, 64.3, and 49.0% at 1, 3, 5, and 7 days after i.v. GP administration, respectively (Fig. 2E). Monocyte membrane Dectin-1 levels were decreased (p < 0.05) by 39.0, 74.5, and 33.4% at 1, 3, and 5 days after i.v. GP administration, respectively (Fig. 2F). Intraperitoneal GP administration resulted in 84.9, 57.3, and 64.6% decreases in neutrophil Dectin-1 levels at 3 h and 3 and 5 days postinjection (Fig. 2E). Monocyte membrane Dectin-1 levels were decreased (p < 0.05) by 36.4, 34.6, and 48.2% at 3 h and 1 and 3 days following i.p. GP administration, respectively (Fig. 2F), compared with the controls. Thus, the changes in peripheral leukocyte Dectin-1 levels following GP administration are primarily due to loss of membrane Dectin-1 positivity on peripheral blood neutrophils and monocytes.
Administration of GP Does Not Alter Membrane Dectin-1 Levels in Leukocyte Bone Marrow Precursors. The data shown above demonstrate that GP will decrease neutrophil and monocyte membrane Dectin-1 levels for up to 7 days following a single injection (Fig. 2). However, neutrophils and monocytes have a circulating half-life of hours to a few days. In addition, Rice et al. (2004) have reported that parenterally administered GP has a plasma elimination half-life of 3.8 h. Therefore, an important question was how a single injection of GP could result in such prolonged loss of leukocyte membrane Dectin-1? One possibility is that GP may also act at the level of leukocyte bone marrow precursors. To answer this question, bone marrow cells were isolated from mice treated with GP at the same time intervals (3 h to 10 days) that were examined in Fig. 2. GP administration had no effect on bone marrow cell Dectin-1 levels compared with control animals at any time interval studied (data not shown). Therefore, Dectin-1 levels do not appear to be decreased in bone marrow precursors before their entry into the systemic circulation.
The Effect of Glucan on Loss of Leukocyte Dectin-1 Levels Is Specific and Not Observed with Nonglucan Carbohydrate Polymers. To examine the specificity of glucan on leukocyte membrane Dectin-1 expression, mice were injected with the non-Dectin-1-interacting ligands mannan, a fungal cell wall mannose polymer (Chauhan et al., 2002), or pullulan, an 
-linked glucose polymer (Mueller et al., 1995) before harvesting peripheral leukocytes on days 1 and 5. These time intervals were selected based on the results obtained in Fig. 2. These carbohydrates were compared with GP. Blood leukocytes were assessed for phenotype and Dectin-1 expression by flow cytometry. As expected, GP resulted in significantly lower levels of leukocyte Dectin-1 compared with the controls (Fig. 3A). Surprisingly, mannan administration increased (p < 0.05) peripheral blood leukocyte Dectin-1 levels by 35.0 and 55.4% on days 1 and 5 postinjection, compared with control animals (Fig. 3A) (p < 0.05). Pullulan had no effect on peripheral leukocyte Dectin-1 levels (Fig. 3A).
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Changes in peripheral leukocyte numbers in response to mannan, pullulan, and GP were also observed. Mannan resulted in a neutrophilic and monocytic leukocytosis 5 days postinjection (data not shown). Thus, the increase in neutrophil and monocyte Dectin-1 levels induced by mannan may be attributable to an increased population of these cell types. GP or pullulan had no significant effect on peripheral leukocyte numbers at either time interval (data not shown).
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Discussion |
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; Herre et al., 2004). In the present study, we have extended these observations to demonstrate that systemically administered glucans are up-taken from the systemic circulation by peripheral leukocytes, and the glucan-Dectin-1 complex is internalized and colocalized within the leukocyte. Thus, we conclude that the loss of Dectin-1 from the leukocyte membrane is due to internalization of the glucan-Dectin-1 complex, following receptor ligand interaction. We also observed that a single injection of GP resulted in decreased peripheral leukocyte Dectin-1 positivity for up to 7 days. Splenic and peritoneal leukocytes also showed a decrement in surface Dectin-1 following glucan administration, but the magnitude and duration was less than that observed in peripheral leukocytes. Although Dectin-1 levels were significantly decreased on circulating leukocytes in response to glucan, we did not observe a complete loss of leukocyte membrane Dectin-1. The maximum decrement was approximately 85%, even in the presence of a high-loading dose of GP. The loss of Dectin from the cell membrane was primarily observed in peripheral neutrophils and monocytes, although no dramatic changes were found in overall leukocyte numbers. Therefore, systemic administration of glucan results in a preferential decrease in neutrophil and monocyte Dectin-1 levels due to internalization of the glucan-Dectin complex but does not change the peripheral leukocyte population dynamics.
We also compared and contrasted the effect of the nonglucan carbohydrate polymers on leukocyte membrane-associated leukocyte Dectin-1 levels. We employed mannan and pullulan as control polymers for several reasons. First, they are non--linked carbohydrate polymers, and they are not ligands for Dectin-1 (Brown et al., 2002). In addition, mannan is a mannose polymer that is found in association with glucan in the fungal cell wall (Stone and Clarke, 1992). The mannan employed in this study was isolated from the same fungal source as the GP (Peat et al., 1961). Although pullulan had no effect on membrane Dectin-1 levels, we observed a differential effect of GP and mannan on leukocyte Dectin-1 positivity. Although GP resulted in an overall loss of leukocyte Dectin-1 positivity, mannan resulted in a modest, but significant, increase in leukocyte Dectin-1 levels. Mannan also induced a neutrophilic and monocytic leukocytosis. Thus, the loss of leukocyte membrane-associated Dectin-1 is specific for glucan polymers and not a nonspecific effect of carbohydrate polymers in general.
The prolonged duration of effect of GP on Dectin-1 levels was an unexpected finding for several reasons. First, the effect of glucan on Dectin-1 was primarily observed in neutrophils and monocytes. These leukocytes are present in the systemic circulation for relatively short time periods, i.e., hours or days. Second, Rice et al. (2004) have reported that GP has a distribution half-life of 4.3 ± 0.7 min, an elimination half-life of 3.8 ± 0.8 h, a volume of distribution of 350 ± 88 ml/kg, and a clearance rate of 42 ± 6 ml/kg/h from the plasma following i.v. administration. Thus, the effect of glucan on leukocyte membrane Dectin-1 levels lasts beyond the expected half-life of neutrophils and monocytes and exceeds the anticipated clearance of the drug from the systemic circulation. Because glucans have been reported to modulate leukocyte bone marrow precursors (Patchen and Lotzova, 1980), one possible explanation for the prolonged effect was that glucan administration altered Dectin-1 membrane expression at the level of monocyte and/or neutrophil precursors in the bone marrow. However, we found that glucan did not alter Dectin-1 levels in bone marrow cells. Another potential explanation for the prolonged effect of glucan administration on Dectin-1 cell surface expression may be recycling of the glucan. Recent studies by Hong et al. (2004) suggest that orally administered water-insoluble glucans are up-taken by macrophages and transported to the spleen, lymph nodes, and bone marrow. The authors speculated that the glucan was degraded and released into the systemic circulation, where it was subsequently internalized by granulocytes (Hong et al., 2004). However, mammalian cells do not have the enzymes necessary to specifically catabolize glucans (Stone and Clarke, 1992); therefore, a more reasonable explanation is that leukocytes that have bound and internalized glucan may release the glucan at the end of their life cycle, and it is then internalized by leukocytes that have recently entered the systemic circulation. In support of this concept, Monari et al. (2003) have reported that neutrophils rapidly ingest certain carbohydrates and then subsequently release the carbohydrates. This effect may not be limited to leukocytes. Receptors for glucans have been identified on vascular endothelial cells, epithelial cells, and fibroblasts (Ahren et al., 2001; Kougias et al., 2001; Lowe et al., 2002). Thus, it is possible that glucans are up-taken from the systemic circulation by a variety of cells and tissues and are then slowly released over time to be internalized by newly released circulating leukocytes. We also noted that membrane Dectin-1 levels normalized by day 7 postinjection. We know that Dectin-1 receptors do not recycle to the cell surface once they are complexed and internalized with a biologically active glucan, such as glucan phosphate (Herre et al., 2004). New receptor must be synthesized and expressed (Brown et al., 2002, 2003). Thus, the reappearance of leukocyte membrane Dectin-1 in our study may relate to prolonged elimination of the glucan from the body as well as synthesis of new receptor in some leukocytes. Further studies will be necessary to determine which mechanism(s) are primarily involved in the prolonged response to parenterally administered soluble glucans.
Numerous studies have shown that systemic administration of pharmaceutical grade glucans will stimulate innate immunity (Williams, 1997), increase resistance to infectious challenge (Sener et al., 2005), decrease myocardial injury following ischemia/reperfusion injury (Li et al., 2003), suppress the growth of transplanted tumors (Cheung et al., 2002; Hong et al., 2004), and facilitate wound repair (Wei et al., 2002). The cellular and molecular mechanisms by which glucans mediate these effects are currently the subject of intense investigation (Williams et al., 1999, 2004). Several investigators speculate that Dectin mediates the biological effects of glucans (Brown et al., 2003). Indeed, it has been reported that ligation of Dectin-1 is sufficient to transduce an activating signal into macrophages and that internalization of the ligand-receptor complex is not required for induction of biological activity (Brown et al., 2003; McCann et al., 2005). However, those experiments were performed with cultured cells, the stimulus was zymosan, a glucan-mannan cell wall extract, and the endpoint was cytokine production, not modification of disease (Brown et al., 2003). In addition, we have shown that a single injection of glucan, either i.v. or i.p., can induce biological effects lasting up to 7 days (Williams, 1997). This coincides with the period when leukocyte Dectin levels are decreased. This may indicate that binding and internalization of glucans by Dectin are crucial to expression of in vivo biological activity. Because the Dectin-1 responses to both i.v. and i.p. administration of GP are not significantly different at most time points, these data also support previous findings that both the i.v. and i.p. routes of administration are equally effective in modulating immune responses (Williams et al., 1999). These data may have significant implications for glucan dosing regimens.
In conclusion, this is the first report of the effect of systemic administration of glucan on Dectin-1 expression in vivo. We have shown that glucan decreases the cell surface levels of the pattern recognition receptor Dectin-1 on neutrophils and monocytes and that this decrease persists well beyond the clearance half-life of glucan and longer than the normal circulating lifespan of neutrophils and monocytes. These data provide important insights into in vivo modulation of Dectin-1 by glucans. These results may also shed additional light on the mechanisms by which glucans modulate innate immunity.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GP, glucan phosphate; DAP, diaminopropane; PBS, phosphate-buffered saline.
Address correspondence to: Dr. David L. Williams, Department of Surgery, P.O. Box 70575, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614. E-mail: williamd{at}etsu.edu
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, p < 0.05 i.p. glucan compared with control.