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


     
 

The Journal of Immunology, 2008, 180, 2777-2785
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
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 arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shah, V. B.
Right arrow Articles by Keshvara, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shah, V. B.
Right arrow Articles by Keshvara, L.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH

β-Glucan Activates Microglia without Inducing Cytokine Production in Dectin-1-Dependent Manner

Vaibhav B. Shah*, Yongcheng Huang*, Rohan Keshwara*, Tammy Ozment-Skelton{dagger}, David L. Williams{dagger} and Lakhu Keshvara1,*

* Division of Pharmacology, College of Pharmacy, Ohio State University, Columbus, OH 43210; and {dagger} Department of Surgery, College of Medicine, East Tennessee State University, Johnson City, TN 37614


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive responses within the CNS. Like other immune cells, microglia recognize and are activated by various pathogen-associated molecular patterns. β-glucans are pathogen-associated molecular patterns present within fungal cell walls that are known to trigger protective responses in a number of immune cells. In an effort to better understand microglial responses to β-glucans and the underlying response pathways, we sought to determine whether Dectin-1, a major β-glucan receptor recently identified in leukocytes, plays a similar role in β-glucan-induced activation in microglia. In this study, we report that Dectin-1 is indeed expressed on the surface of murine primary microglia, and engagement of the receptor with particulate β-glucan resulted in an increase in tyrosine phosphorylation of spleen tyrosine kinase, a hallmark feature of the Dectin-1 signaling pathway. Moreover, phagocytosis of β-glucan particles and subsequent intracellular production of reactive oxygen species were also mediated by Dectin-1. However, unlike in macrophages and dendritic cells, β-glucan-mediated microglial activation did not result in significant production of cytokines or chemokines; thus, the interaction of microglial Dectin-1 with glucan elicits a unique response. Our results suggest that the Dectin-1 pathway may play an important role in antifungal immunity in the CNS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the CNS, microglia are the primary immune effector cells that are required for both innate and adaptive immune responses (1, 2). Like other innate immune cells, microglia recognize pathogen-associated molecular patterns (PAMPs)2 through pattern recognition receptors, such as the TLRs (3, 4, 5). Upon exposure to pathogens, microglia undergo rapid activation, characterized by increased phagocytic activity and production of a multitude of proinflammatory molecules, including cytokines, chemokines, and reactive oxygen species (ROS). Unfortunately, microglial activation is a double-edged sword, because reactive microglia also play a central role in the pathogenesis of neurodegenerative diseases as well as in the development of postinfection brain abscess (6, 7). Thus, in this context, it is important to understand the signaling pathways that govern microglial responses to various pathogens.

Recently, a number of investigations have focused on the molecular aspects of microglial responses to bacterial pathogens, and TLRs in particular were shown to play an important role in microglial-mediated neuroinflammation and CNS injury following bacterial infection (8, 9, 10). However, to date, relatively little is known about the receptors and signaling pathways involved in microglial recognition of fungal pathogens. Components that are commonly found in various fungal cell walls include β-glucans, chitin, mannans, and glycoproteins (11). Although all of these components have varying degrees of activating effects on immune cells, β-glucans are thought to be the major PAMPs involved in host-pathogen interactions (12, 13, 14). Interestingly, β-glucans are also widely recognized for their immunomodulatory properties, because they have been shown to possess antitumor (15, 16) and anti-infective properties against bacterial (17), viral (18), fungal (19), and protozoal (20) infections. Thus, in this regard, β-glucans represent a class of PAMPs that modify the behavior of immune cells to the point that their responses to other stimuli are also altered.

Recently, Dectin-1 has emerged as the major receptor mediating β-glucan activity on leukocytes, especially macrophages (21, 22) and dendritic cells (23, 24). Dectin-1, a NK cell receptor-like C-type lectin, was originally identified as a dendritic cell-specific receptor (25). However, it is now known to be expressed by several cell types, including macrophages, monocytes, neutrophils, and a subset of T cells (26). Dectin-1 recognizes soluble and particulate β (1->3)- and/or β (1->6)-linked glucans (27), and it has been shown to mediate cellular recognition of a variety of fungal species, including Aspergillus (28), Coccidioides (29), Pneumocystis (30), and Candida (13). The crucial role of Dectin-1 in fungal recognition was recently demonstrated by genetic studies showing that mice deficient in Dectin-1 are more susceptible to infections with Candida albicans (31) and Pneumocystis carinii (32). At the molecular level, Dectin-1 mediates intracellular signaling through an ITAM sequence present within its cytoplasmic tail (24, 33, 34). Although the mechanistic details are still lacking, Dectin-1 ITAM is thought to be phosphorylated by Src family kinases (SFK) upon receptor engagement, and the phosphorylated receptor subsequently provides a docking site for the spleen tyrosine kinase (Syk), which undergoes tyrosine phosphorylation and activation upon binding to the receptor tail. In this regard, Syk is required for β-glucan-induced production of ROS and cytokines/chemokines. Interestingly, in macrophages, activation responses induced by fungal particles involve close collaboration between Dectin-1 and TLRs (23, 35, 36). Thus, fungal particles are thought to simultaneously engage multiple cell surface receptors, resulting in a variety of cellular responses, making it difficult to delineate signaling pathways specifically activated by β-glucan.

To date, most efforts to understand response pathways elicited by β-glucan have been conducted primarily in peripheral immune cells, and very little is known about the β-glucan-specific responses in microglia. Because microglia are of the myeloid cell lineage (37, 38) and exhibit cellular responses that are highly similar to those observed in macrophages (1), we sought to determine whether Dectin-1 is present in microglia and whether it can mediate microglial responses to β-glucan. In this study, we report for the first time that Dectin-1 is expressed in brain microglia as well as in the microglial cell line BV-2, where it serves as a receptor for β-glucan particles within a signaling pathway that involves Src family and Syk tyrosine kinases. β-glucan particles were phagocytosed by microglia in Dectin-1-dependent manner, and Dectin-1 was also required for subsequent ROS production. Remarkably, however, unlike in leukocytes, engagement of Dectin-1 by β-glucan did not result in any significant cytokine/chemokine production in microglia. Thus, our results indicate that Dectin-1-mediated signaling in microglia represents a new pathway of interest in neuroimmunology and neuroinflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

Unconjugated and PE-conjugated anti-Dectin-1 Abs were purchased from R&D Systems and Serotec, respectively. Anti-Syk Abs were from Santa Cruz Biotechnology. The anti-phosphotyrosine Ab 4G10 was purchased from Upstate Biotechnology. Piceatannol, PP2, cytochalasin D, and PMA were from Calbiochem. Zymosan, laminarin, and NBT were purchased from Sigma-Aldrich, whereas Pam3CSK4 was from InvivoGen.

Preparation of β-glucans

Particulate glucan was isolated and characterized, as described by Muller et al. (39) and Lowman et al. (40). Glucan phosphate was prepared and characterized, as described by Williams et al. (41) and Muller et al. (42). Glucan phosphate was selected for this study because it is bound by Dectin-1 with high affinity (35). The primary structures of the glucans were confirmed by nuclear magnetic resonance (40). Fluorescent labeled glucans were prepared, as described by Ozment-Skelton et al. (43) and Rice et al. (44).

Microglial cultures

C57BL/6 mice were purchased from The Jackson Laboratory. TLR2 knockout mice, generated and described by S. Akira (Osaka, Japan) (45), were bred on C3H/HeN background and provided by W. Lafuse (Ohio State University, Columbus, OH). Animals were housed, bred, and euthanized in accordance with protocols reviewed and approved by the Ohio State University Institutional Animal Care and Use Committee. Primary mixed glial cultures were prepared from postnatal day 1–3 mice. Brains from individual mice were removed and placed into ice-cold DMEM. After the removal of meninges, the cortices were dissected and pooled together in DMEM. The cortices were then dissociated by repeated passages through wide to fine bore pipettes. Dissociated cells were then passed through a 70-µm nylon filter and resuspended in DMEM-F12 containing 10% FBS before culturing in 60-mm dishes. These mixed glial cells were cultured for 18 days, changing medium every 4–5 days. Microglial cells were isolated from the mixed culture using mild trypsinization method, as described previously (46). The isolated microglial cells were then reseeded in 24-well plates or 60-mm dishes and cultured in astrocyte-conditioned medium for 5–7 days to allow sufficient time for them to become quiescent. This method yielded highly pure microglial cultures, as evident from the morphology and absence of astrocytic and neuronal markers. For cell stimulation, zymosan or β-glucan particles were added directly to the cells for various lengths of time before cell lysis. When indicated, cells were pretreated with various inhibitors for 30 min before stimulation.

BV-2 cells

The BV-2 mouse microglial cell line was a gift from G. Landreth (Case Western Reserve University, Cleveland, OH). The cells were maintained in DMEM supplemented with 5% FBS. For stimulation, either zymosan (100 µg/ml) or β-glucan particles (100 µg/ml) were directly added to cells in 60-mm dishes and incubated at 37°C for various lengths of time. When indicated, appropriate inhibitors were added to the dishes 30 min to 1 h before adding the particles. After appropriate treatments, cells were immediately lysed. For phagocytosis experiments, the cells were seeded at a density of 2.5 x 104 cells on 12-mm coverslips 1 day before the experiment.

Flow cytometry

Primary microglia or BV-2 cells, previously plated on 60-mm dishes, were scrapped and suspended in PBS containing 0.1% sodium azide and 2% BSA. The suspended cells were then incubated at 4°C for 30 min. PE-conjugated anti-Dectin-1 Ab or an isotype control Ab was added to the cells, followed by incubation at 4°C for 40 min. Cells were then washed three times with PBS containing 0.1% sodium azide and 0.2% BSA, and then fixed in 2% formaldehyde. Flow cytometry was conducted using FACSCalibur, and data were analyzed using CellQuest software. In some instances, cells were pretreated with laminarin or glucan phosphate for 20 min at 37°C before staining.

RT-PCR

Mouse BV-2 microglia and mouse primary microglia were lysed in 1 ml of TRIzol reagent (Invitrogen Life Technologies), and total RNA was isolated, as per manufacturer’s protocol. Reverse transcription was performed with 1 µg of total RNA as template using oligo(dT) primer and Omniscript Reverse Transcriptase enzyme (Qiagen). An aliquot of the resulting first-strand cDNA was then subjected to subsequent PCR using TaqDNA polymerase enzyme (Invitrogen Life Technologies) along with the following primers: Dectin-1, sense, 5'-AGGCCCTATGAAGAACTACAGACA-3' and antisense, 5'-TGGCCAGACAGCATAAGGAAAC-3'; GAPDH, sense, 5'-TGTCAGCAATGCATCCTGCA-3' and antisense, 5'-TGCTGTAGCCGTATTCATTG-3'.

Immunoprecipitations and Western blotting

For anti-Syk immunoprecipitations, clarified cell lysates were incubated with anti-Syk Abs (1/100 dilution) at 4°C for 2 h. Protein G-Sepharose beads (Invitrogen Life Technologies) were then added, and further incubation for 1 h at 4°C was conducted to precipitate the immune complexes. The beads were washed in lysis buffer, and the immunoprecipitated proteins were eluted by boiling with Laemmli buffer and then resolved by SDS-PAGE. For Western blotting analysis, proteins were electrotransferred to nitrocellulose membranes, and the membranes were then blocked with 5% nonfat milk. Primary Abs were diluted in 1% milk solution, and the dilutions used are as follows: anti-Syk (1/2000), 4G10 (1/400), and anti-Dectin-1 (1/3000). After incubations with primary Abs for 2 h, the membranes were washed and incubated with appropriate secondary Abs. The membranes were extensively washed, and proteins were detected by chemiluminescence using ECL (Amersham) or SuperSignal West Dura Extended (Pierce). Signals were detected by autoradiography, and multiple film exposures were conducted to capture an image within a linear range.

Phagocytosis

One day before the experiment, cells were seeded at the density of ~2.5 x 104 cells per 12-mm coverslip. Cells were then left untreated or pretreated with glucan phosphate (100 µg/ml), cytochalasin D (5 µM), PP2 (10 µM), or piceatannol (25 µM) for 40 min and then cooled to 4°C before incubation with Alexa Fluor 488-conjugated particulate β-glucan (10 particles/cell). The cells were then washed thoroughly with cold DMEM-containing serum to remove unbound particles and further incubated at 37°C for 2 h in the presence or absence of the aforementioned inhibitors to allow phagocytosis. The cells were transferred again to 4°C, washed with PBS, and fixed using 4% formaldehyde. Fixed cells were examined under fluorescence microscope, and the percentage of cells internalizing β-glucan particles was determined by taking the ratio of cells that internalized the particles to the total number of cells counted.

Reactive oxygen species

Intracellular ROS was measured using the NBT reduction assay. Superoxide generation was monitored by formation of insoluble purple/black particles of formazan. Briefly, primary microglia were plated in 24-well plates in astrocyte-conditioned medium. On the day of the experiment, the cells were left untreated or were pretreated with indicated inhibitors for 30 min at 37°C. After the pretreatment, the supernatant was removed and replaced with fresh astrocyte-conditioned medium containing NBT (1 mg/ml). The cells were then stimulated with particulate β-glucan for 1 h at 37°C in the presence or absence of respective inhibitors. PMA (500 nM) was used as a positive control for superoxide generation. Cells were then washed in PBS thrice before fixing them in 2% formaldehyde. At least three random fields of cells (>200 cells) were counted under an inverted microscope by a blind observer.

Cytokine measurements

Primary microglia were stimulated with particulate β-glucan (100 µg/ml) or zymosan (100 µg/ml) for 16 h, and the supernatants were harvested and stored at –80°C. The supernatants were then submitted to Pierce Biotechnology for cytokine profile analysis. Additional in-depth analyses of TNF-{alpha} were carried using appropriate ELISA kits purchased from BioLegend. For experiments involving the use of inhibitors, cells were treated with laminarin (500 µg/ml), glucan phosphate (100 µg/ml), or PP2 (10 µM) for 30 min before stimulation, and the inhibitors remained present in the cell medium throughout the 16-h incubation period.

Statistical analysis

Statistical significance was determined by one-way ANOVA, and the Tukey-Kramer multiple comparison test was used to determine p values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Dectin-1 is present in microglia

Dectin-1 has recently emerged as the major β-glucan receptor in macrophages and other hemopoietic cells. Given the myeloid lineage of microglia, we hypothesized that Dectin-1 would play a similar role in microglial responses to β-glucan. To determine whether Dectin-1 is present in microglia, we conducted RT-PCR and Western blotting analysis in primary microglia as well as in the BV-2 microglial cell line. As shown in Fig. 1A, RT-PCR using Dectin-1-specific primers yielded a single product of predicted size (447 bp) when total RNAs from primary microglia and BV-2 cells were used, but significantly reduced levels were observed when RNA from a whole mouse brain was used. This PCR product was absent when reverse transcriptase was omitted from the reactions (data not shown). Expression of Dectin-1 in microglia and BV-2 cells was further confirmed by Western blotting the protein extracts from these cells using anti-Dectin-1 Abs. As shown in Fig. 1B, a 28-kDa band representing Dectin-1 was present in both BV-2 and primary microglia, but not in HEK293T cells. In contrast, the loading control β-actin was present in approximately equal levels in all extracts. Thus, these results indicate that Dectin-1 is expressed in primary microglia as well as in the microglial cell line BV-2.


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 1. Dectin-1 is expressed in microglia. A, RT-PCR analysis of P0 mouse brain, BV-2 cells, and primary microglia using primers specific for Dectin-1 and GAPDH. B, Whole cell extracts were prepared from BV-2 cells, primary microglia, and 293T cells, and Western blots were conducted using anti-Dectin-1 and anti-β-actin Abs.

 
Dectin-1 expression at the cell surface is reduced by soluble glucans

Dectin-1 is a transmembrane protein with an extracellular carbohydrate recognition domain and an intracellular ITAM-containing signaling tail. To determine whether Dectin-1 is present at the cell surface of microglia, we conducted flow cytometric analysis of the cells using PE-conjugated anti-Dectin-1 Abs. Despite background autofluorescence associated with primary microglia, our data show that ~50% of the gated primary microglial cells exhibited surface staining with anti-Dectin-1 Abs (Fig. 2A). Similarly, 52% of the BV-2 cells also exhibited cell surface staining with the PE-conjugated anti-Dectin-1 Abs (Fig. 2B). Previously, it was shown that incubation of cells with the soluble glucans, such as glucan phosphate or laminarin, results in a decreased expression of Dectin-1 at the cell surface due to forced internalization of the receptor (34, 43). Thus, a reliable test for determining the specificity of the Ab used in the surface-labeling experiments is to pretreat the cells with such soluble glucans before Ab staining. Consistent with these published reports, we observed that a preincubation of primary microglia and BV-2 cells with glucan phosphate or laminarin significantly reduced the cell surface staining by the Ab (Fig. 2), indicating that the Ab specifically reacted to Dectin-1 on the cell surface. Thus, our data strongly indicate that Dectin-1 is expressed by microglia at the cell surface, where it is poised to recognize and respond to β-glucan.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2. Dectin-1 is expressed on the surface of microglial cells. A, Primary microglial cells cultured in 60-mm dishes were suspended in PBS plus 0.1% Na azide and 2% BSA and incubated at 4°C for 30 min, followed by incubation with either PE-conjugated anti-Dectin-1 Abs or an isotype control Ab for 40 min. Cells were then fixed using 2% formaldehyde solution and subjected to flow cytometry using FACSCalibur, and data were analyzed using CellQuest software. Some cells were pretreated with glucan phosphate (GP) (100 µg/ml) for 20 min at 37°C before incubation with PE-conjugated anti-Dectin-1 Ab. The percentages indicated in the lower right quadrant refer to the percentage of gated (Dectin-1-positive) events. The data shown are a representative of three independent experiments. B, BV-2 cells (~2 x 105) were plated on 60-mm dishes. Next day, cells were suspended and treated essentially in the same way as in A. Cells within one subset were pretreated with laminarin (500 µg/ml) for 20 min at 37°C before incubation with PE-conjugated anti-Dectin-1 Ab. The percentages indicated in the lower quadrant refer to the percentage of gated (Dectin-1-positive) events.

 
β-glucan induces Syk phosphorylation in microglia

Dectin-1 contains an ITAM sequence within its cytoplasmic tail, and at least one tyrosine residue within this motif is phosphorylated in response to receptor engagement (24). Phosphorylated ITAM is then thought to recruit Syk, which itself undergoes phosphorylation and activation as a result. Indeed, a hallmark feature of Dectin-1 activation is induction of Syk phosphorylation, and Syk is critical for various Dectin-1-mediated cellular responses in macrophages and dendritic cells (24, 47). Thus, in an effort to determine whether Dectin-1 is linked to intracellular signaling in microglia, we investigated the phosphorylation status of Syk upon β-glucan stimulation. Zymosan, a β-glucan-rich particulate component of Saccharomyces cerevisiae that has been widely used as a ligand for Dectin-1, was used to stimulate primary microglia as well as BV-2 cells for various lengths of time, and a combination of immunoprecipitation and Western blotting analysis was used to evaluate Syk phosphorylation. Consistent with observations in leukocytes, we observed a sharp increase in tyrosine phosphorylation of Syk in microglia upon zymosan stimulation. In both primary microglia (Fig. 3A) as well as in BV-2 cells (Fig. 3B), Syk phosphorylation was apparent within 10 min of zymosan treatment and peaked at ~30 min. Although phosphorylation in primary microglia gradually returned to basal levels within 60 min of stimulation (Fig. 3A), in BV-2 cells Syk phosphorylation persisted for much longer (Fig. 3B).


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 3. Zymosan and particulate β-glucan-induced Syk phosphorylation are dependent on both Dectin-1 and Src family kinases. Primary microglial cells (A, C, D, and E) and BV-2 cells (B) were stimulated with zymosan (100 µg/ml) (A–D) or particulate β-glucan (100 µg/ml) (C–E) at 37°C for indicated times or for 30 min when not indicated. In some experiments, microglia were preincubated with glucan phosphate (GP; 100 µg/ml) (D), PP2 (10 µM) (E), or PP3 (10 µM) (E) for 30 min at 37°C before stimulation with zymosan or particulate β-glucan. After appropriate stimulation, cells were lysed and Syk was immunoprecipitated from cell lysates using anti-Syk Abs. Phosphotyrosine content of Syk was then determined by Western blotting the immunoprecipitates with anti-phosphotyrosine Abs (4G10). Equal levels of Syk were confirmed by stripping and reprobing the membrane using anti-Syk Abs. Data are representative of at least three independent experiments.

 
Although zymosan is rich in β-glucan, it also contains other components, such as mannans, which have various stimulatory activities on immune cells (48). For example, in macrophages, zymosan was shown to simultaneously activate both Dectin-1- and TLR2-mediated pathways and cellular responses (23). Therefore, to determine the specific role of Dectin-1 in Syk phosphorylation in microglia, we chose to use a purified particulate form of β-glucan (39, 40) devoid of other receptor activities for cell activation. Furthermore, to confirm that β-glucan-induced Syk phosphorylation is Dectin-1 dependent, a subset of cells was treated with glucan phosphate to decrease the surface expression of Dectin-1 before stimulation with particulate glucan. Indeed, we observed that particulate β-glucan was as effective as zymosan in inducing Syk phosphorylation (Fig. 3C), suggesting that the effects of zymosan are β-glucan specific. Moreover, because β-glucan-induced Syk phosphorylation was drastically reduced in cells pretreated with glucan phosphate (Fig. 3D), the phosphorylation response was most likely mediated by Dectin-1.

Based on what is known about Syk activation in other immune receptor systems, such as the BCR, it is thought that the cytoplasmic ITAM tyrosine of Dectin-1 is phosphorylated by SFK and the phosphotyrosines within the ITAM serve as docking sites for Syk (49). Receptor-bound Syk then undergoes activation, and the subsequent increase in its tyrosine content is a result of both autophosphorylation and transphosphorylation by SFK (50). Therefore, we asked whether SFK inhibition can block Syk phosphorylation. Indeed, preincubation of primary microglia with the Src family inhibitor PP2 prevented Syk phosphorylation upon β-glucan treatment (Fig. 3E). In contrast, PP3, an inactive analog of PP2, did not inhibit β-glucan-induced Syk phosphorylation. Thus, taken together, these results strongly suggest that the Dectin-1-Syk pathway is intact in microglia and is likely to play a major role in microglial responses to β-glucan stimulation.

Phagocytosis of particulate β-glucan is dependent on Dectin-1

Phagocytosis of pathogens is an integral component of the innate immune response. Recently, it was shown that Dectin-1 is required for phagocytosis of fungal particles by macrophages (34). Microglia also display antifungal activity, in part due to their ability to phagocytose fungal particles (51). However, the importance of β-glucan recognition in fungal phagocytosis by microglia and possible involvement of Dectin-1 in this process remain unclear. To address this question, we first examined the ability of microglia to phagocytose particulate β-glucans. As shown in Fig. 4, BV-2 microglial cells actively phagocytosed fluorescent labeled β-glucan particles, and approximately one-half of the cells contained three or more particles. However, phagocytosis was almost completely suppressed in cells pretreated with glucan phosphate, and the inhibitory effect of glucan phosphate was comparable to cytochalasin D, which inhibits the phagocytic machinery by disrupting actin polymerization. In a separate experiment, we observed that anti-Dectin-1 Abs also inhibited phagocytosis (data not shown). Thus, phagocytosis of particulate β-glucan is likely to involve a direct interaction with Dectin-1 at the cell surface. Interestingly, treatment of microglia with PP2 consistently resulted in a slight inhibition in phagocytosis, but this effect was not statistically significant (Fig. 4). Similarly, preincubation with the Syk inhibitor piceatannol also did not inhibit phagocytosis to any significant extent. Thus, SFK/Syk tyrosine kinase activities do not appear to be critical for the phagocytic process. Our results are consistent with previous studies showing that in macrophages, Dectin-1 is essential and sufficient for phagocytosis of zymosan, whereas SFK and Syk activities were dispensable (34). Thus, in this aspect, the phagocytic machinery for internalizing β-glucan particles in microglia appears to be the same as in macrophages.


Figure 4
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 4. Dectin-1 mediates phagocytosis of particulate β-glucan by microglia. BV-2 cells were fed Alexa Fluor 488-conjugated β-glucan particles (100 µg/ml) in the presence or absence of glucan phosphate (GP; 100 µg/ml), PP2 (10 µM), PP3 (10 µM), piceatannol (PICE, 25 µM), or cytochalasin D (Cyto D; 5 µM) and incubated at 4°C for 1 h, followed by an incubation at 37°C for 2 h. The cells were then visualized under fluorescent microscope. Four random fields (>100 cells) were counted, and data were quantified as percentage of total cells internalizing particulate β-glucan. The values shown are mean ± SEM, n = 6. *, p < 0.05 compared with β-glucan alone.

 
β-glucan induces ROS production in microglia

Microglia, like other phagocytic cells, are known to produce ROS as part of the respiratory burst that occurs during phagocytosis (52). Therefore, we analyzed intracellular superoxide production in primary microglia following stimulation with particulate β-glucan using the NBT reduction technique. Upon treatment with PMA, which was used as a positive control, ~80% of the cells displayed NBT reduction (Fig. 5). Strikingly, treatment with particulate β-glucan caused NBT reduction in roughly the same proportion of the cells counted. However, the number of NBT-positive cells was reduced by ~50% when primary microglia were preincubated with glucan phosphate before β-glucan stimulation, suggesting that surface expression of Dectin-1 is required for β-glucan-induced ROS production. Because a similar reduction in NBT-positive cells was also observed in microglia pretreated with PP2 or piceatannol, SFK and Syk are also likely to play a role in β-glucan-induced ROS generation. These results are consistent with the results by Underhill et al. (47), showing that in macrophages, zymosan-induced ROS production requires Dectin-1 as well as both SFK and Syk catalytic activities.


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 5. β-glucan induces ROS production in Dectin-1-dependent manner. Primary microglia obtained from wild-type mice were cultured in conditioned medium (described in Materials and Methods) containing NBT, followed by stimulation with particulate β-glucan (100 µg/ml) or PMA (500 nM) for 1 h. In a subset of experiments, cells were pretreated with glucan phosphate (GP; 100 µg/ml), PP2 (10 µM), PP3 (10 µM), or piceatannol (PICE; 25 µM) for 30 min before stimulation with particulate β-glucan at 37°C for 1 h. The cells were then rinsed with PBS and fixed with formaldehyde. Superoxide anion generation was monitored by visualization of insoluble formazan particles under an inverted microscope. At least three random fields of cells (>200 cells) were counted by a blind observer. The graph represents the mean ± SEM of three independent experiments. *, p < 0.05 compared with β-glucan. **, p < 0.05 compared with control.

 
β-glucan does not induce cytokine/chemokine production

In macrophages, Dectin-1 was shown to cooperate with TLR by enhancing zymosan-induced production of inflammatory cytokines, such as TNF-{alpha} and IL-12, and this response was inhibited by the soluble glucan laminarin (23). Similarly in dendritic cells, both Dectin-1 and Syk were required for optimal production of IL-2, IL-10, and IL-12 in response to zymosan stimulation (24). Thus, Dectin-1 is thought to cooperate with TLRs through an undefined mechanism, whereby engagement of Dectin-1 by particulate β-glucan enhances the production of cytokines and chemokines. Consistent with their innate function, activated microglia are known to secrete a number of cytokines and chemokines in response to bacterial pathogens, and the role of TLRs in this response has been particularly well addressed by Kielian (4). However, it is not clear whether β-glucans can induce cytokine/chemokine production in microglia in the absence of simultaneous TLR stimulation. Therefore, we sought to determine whether particulate β-glucan could induce cytokine/chemokine production in microglia and whether Dectin-1 is required for this response.

Several cytokines/chemokines, including IL-1β, IL-2, IL-6, IL-10, MIP-1{alpha}, and TNF-{alpha}, were analyzed in supernatants of microglia treated with either particulate β-glucan or zymosan. As expected, zymosan induced production of all the cytokines and chemokines examined (Fig. 6A). In contrast, particulate β-glucan did not cause any significant production of these cytokines/chemokines. In separate experiments, cells were pretreated with laminarin or PP2 before zymosan stimulation to determine whether the response to zymosan is in part mediated by the Dectin-1 pathway, but we did not see any significant decrease in cytokine/chemokine production in the presence of these inhibitors (data not shown), suggesting that the effects of zymosan were most likely independent of the β-glucan-Dectin-1 interaction. To address the possibility that Dectin-1-mediated cytokine/chemokine production occurs at doses greater than those required to induce phagocytosis and ROS production, we conducted a dose-response experiment in which microglia were treated with increasing doses of either particulate β-glucan or zymosan. However, even at the highest dose used (400 µg/ml), we did not detect any statistically significant induction of TNF-{alpha} (Fig. 6B), which was used as a marker for the general cytokine/chemokine response of microglia. In contrast, zymosan treatment resulted in maximum TNF-{alpha} levels even at the lowest dose used (50 µg/ml). To further determine whether Dectin-1 is required for this response, a subset of cells was preincubated with glucan phosphate before stimulation with particulate glucan or zymosan, but this pretreatment did not significantly antagonize the respective responses to glucan and zymosan. Rather, we consistently observed that glucan phosphate slightly enhanced the effects of zymosan, but did not cause any induction of its own. Although we cannot offer a mechanism that would explain this effect, our results suggest that zymosan-induced production of cytokines/chemokines is most likely mediated through a Dectin-1-independent pathway.


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 6. Particulate β-glucan does not induce cytokine production in microglia. A, Primary microglia were left untreated (control) or were stimulated with zymosan (100 µg/ml) or β-glucan (100 µg/ml) for 16 h, and supernatants were harvested and stored at –80°C. The supernatants were then sent to Pierce Biotechnology for cytokine array analysis and quantification. Data are presented as mean ± SEM, n = 3. *, p < 0.05 compared with control. B, Primary microglia were left untreated or were stimulated for 16 h with different concentrations of zymosan or β-glucan, as indicated. To investigate the role of Dectin-1 pathway, a subset of cells was preincubated with glucan phosphate (GP; 100 µg/ml) at 37°C for 30 min immediately before stimulation with zymosan or β-glucan. Supernatants were then collected, and TNF-{alpha} levels were measured using ELISA. Data are presented as mean ± SEM, n = 3. *, p < 0.05 compared with control.

 
Zymosan-induced cytokine response in microglia is TLR2 dependent

In macrophages, zymosan simultaneously activates Dectin-1- and TLR2-dependent pathways to induce the production of cytokines (23). Because particulate β-glucan did not induce cytokine/chemokine release, we asked whether the effects observed with zymosan were mediated through TLR2. Microglial cultures established from wild-type or TLR2-deficient mice were stimulated with zymosan or particulate β-glucan, and the cell supernatants were analyzed for the presence of TNF-{alpha}. As in wild-type microglia, stimulation with particulate β-glucan did not invoke any significant cytokine response in TLR2-deficient microglia (Fig. 7A). Interestingly, zymosan-induced TNF-{alpha} production was severely diminished in TLR2-deficient microglia. Moreover, this low level of TNF-{alpha} produced upon zymosan stimulation was not reduced any further when cells were treated with glucan phosphate, confirming that β-glucan does not induce cytokine production. In contrast to zymosan, absence of TLR2 did not have a significant impact on TNF-{alpha} production by the TLR4 agonist LPS. Although microglia are known to express all the major TLRs (53), we were able to confirm the expression of TLR2 by RT-PCR (data not shown). Together, these results provide additional support for the idea that zymosan induces cytokine/chemokine production in microglia by activating TLR2, and this induction proceeds independently of β-glucan-Dectin-1 interaction.


Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 7. Zymosan-induced cytokine production in microglia is mediated by TLR2. A, Primary microglia from wild-type (WT) or TLR2-deficient mice (TLR2 KO) were left untreated (Control) or were stimulated with particulate β-glucan (100 µg/ml), zymosan (100 µg/ml), or LPS (1 µg/ml) for 16 h at 37°C. A subset of cells was pretreated with glucan phosphate (GP; 100 µg/ml) for 20 min before stimulation with zymosan. Supernatants were collected, and TNF-{alpha} concentrations were determined using ELISA. Data are presented as mean ± SEM, n = 3. B, As above, primary microglia were left untreated (Control) or were stimulated with zymosan, particulate β-glucan, Pam3CSK4 (1 µg/ml) alone, or particulate β-glucan plus Pam3CSK4 for 16 h at 37°C. Supernatants were collected, and TNF-{alpha} concentrations were determined using ELISA. Data are mean ± SEM, n = 3. *, p < 0.05 compared with control. **, p < 0.05 compared with Pam3CSK4.

 
Previously, it was suggested that whereas TLR signaling is necessary and sufficient for cytokine/chemokine production, Dectin-1 engagement enhances this TLR-mediated response (23, 24). Therefore, to determine whether such interplay between Dectin-1 and TLR-2 occurs in microglia, we treated the cells with particulate β-glucan or Pam3CSK4 (TLR2 agonist) alone or in combination with each other and compared their effects on TNF-{alpha} production with those induced by zymosan alone. Remarkably, in contrast to our expectation, we consistently observed that particulate β-glucan caused a slight reduction in TNF-{alpha} induction in response to Pam3CSK4 (Fig. 7B). Thus, unlike in macrophages and dendritic cells, in which Dectin-1 enhances the effects of TLR2 signaling, in microglia β-glucan does not cause any significant increase in cytokines. Rather, the Dectin-1 pathway may in fact have inhibitory effects on TLR2-mediated cytokine/chemokine production. However, the mechanistic basis for this inhibition is not clear.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We describe in this study the identification and functional role of Dectin-1 in microglia. Dectin-1 has emerged as a key mediator of antifungal responses in leukocytes, and mice lacking Dectin-1 were recently shown to be more susceptible to systemic fungal infections (31, 32). Although fungal infections of the CNS are not common in the general population, opportunistic infections involving common organisms, such as Aspergillus and Candida species, often lead to life-threatening complications in immunocompromised cancer patients (54) and organ transplant recipients (55). Although microglia are known to play a critical role in the innate immune responses required to protect against such fungal pathogens, underlying molecular mechanisms that govern microglial responses remain relatively unexplored. Therefore, as a first step toward a better understanding of microglial responses to fungal PAMPs, we investigated the role of Dectin-1 in microglial recognition and response to β-glucans.

In this study, we report for the first time that Dectin-1 is expressed in primary microglia, as well as in the microglial cell line BV-2. Flow cytometric analysis showed that Dectin-1 is indeed expressed on the surface of these cells, and consistent with previous reports (22, 43), treatment of cells with glucan phosphate or laminarin resulted in a marked reduction of Dectin-1 at the cell surface. Flow cytometric analysis suggested that Dectin-1 was expressed by only ~50% of the microglial population, which is not very surprising because microglial population is thought to be highly heterogeneous (7, 56). The cytoplasmic tail of Dectin-1 contains an ITAM-like sequence that has been proposed to function in a manner analogous to the BCR tail, where tyrosine residues within the motif are phosphorylated by SFK upon receptor engagement and subsequently serve as the docking site for Syk (57). Syk, which associates with the phosphorylated receptor tail through its src homology 2 domains, is subsequently phosphorylated and activated and mediates downstream signaling. Consistent with this model, zymosan readily stimulates phosphorylation of Dectin-1 ITAM as well as Syk in leukocytes, and these phosphorylation events are thought to be required for the production of inflammatory factors. Our results showing that Syk is phosphorylated in response to particulate β-glucan, and that this phosphorylation is suppressed by pretreatment with glucan phosphate as well as by SFK inhibitor PP2, further indicate that Dectin-1 is mediating similar events in microglia. Indeed, we observed that particulate β-glucan stimulated phagocytosis as well as ROS production in a Dectin-1-dependent manner. Previously, it was shown in macrophages that deletion of the Dectin-1 tail region containing the ITAM sequence prevented phagocytosis of zymosan particles without affecting surface expression, but Syk was dispensable for the phagocytic activity (34). In contrast, ROS production subsequent to zymosan stimulation required Dectin-1 as well as both Syk and SFK activities (47). Our results with microglia are remarkably similar to these observations. We also found that phagocytosis of particulate β-glucans occurred even in the presence of SFK/Syk inhibitors, whereas ROS was dramatically inhibited in the presence of these inhibitors. Thus, the underlying mechanisms for Dectin-1-mediated phagocytosis and ROS production appear to be highly conserved in microglia.

One distinguishing feature of the microglial response to β-glucan is that, unlike in leukocytes, engagement of Dectin-1 does not cause cytokine/chemokine production. Pure particulate β-glucan did not elicit any cytokine/chemokine response in microglia. Similarly, the effects of zymosan were mostly mediated by TLR2 and were not antagonized by soluble glucans. Our results are remarkable in that they are in sharp contrast with observations in macrophages, where soluble glucans were used as antagonists to support the role of Dectin-1 in cytokine/chemokine release (23, 35). Indeed, in leukocytes, engagement of Dectin-1 serves to enhance TLR-dependent cytokine/chemokine production such that the two pathways cooperate to maximize the inflammatory response to fungal pathogens (23). Although the mechanism by which Dectin-1 activation leads to cytokine production in leukocytes remains unclear, Card9 has recently emerged as an important mediator of this response (58, 59). It was shown that a signalosome containing Card9, together with Bcl-10 and MALT1, is a critical intermediate linking Syk phosphorylation to NF-{kappa}B, leading to cytokine production independently of TLR. It is still unclear how Syk activates Card9, but one suggestion is that Syk activates phospholipase C-{gamma}2, which then leads to activation of protein kinase C and subsequent activation of the Card9/Bcl-10/MALT1 complex. Although it is not known whether the Card9-associated signaling complex is present in microglia and whether Dectin-1-mediated Syk activation results in similar downstream signaling events, the lack of cytokine release suggests that at least one molecular component in this sequence of events is most likely missing in microglia. Regardless of the mechanism involved, our results raise an interesting possibility that Dectin-1 is mainly required for internalization and clearance of fungal pathogens by microglia, whereas the TLR signaling pathways are largely responsible for the production of cytokines, which then coordinate the adaptive immune responses. Our observations are also consistent with the role of β-glucans as immunomodulators that enhance immune responses without significant proinflammatory side effects that may lead to tissue damage. In this regard, it will be interesting to see whether this property of β-glucans can be exploited to correct microglial deficiencies that are central in a number of neurodegenerative diseases.

A particular challenge associated with the use of zymosan to study β-glucan response pathways is that although it is a good model to study fungal response pathways, zymosan activates multiple receptors and signaling pathways, making it difficult to delineate β-glucan-specific responses and underlying signaling events. Therefore, in our studies, we used a more pure form of particulate β-glucan devoid of other known receptor activity. In attributing these responses to Dectin-1, we have relied on experimental approaches that were previously used to demonstrate the role of Dectin-1 in macrophages and dendritic cells. Because microglial responses to β-glucan were highly similar to Dectin-1-mediated responses in leukocytes, Dectin-1 is likely to be a major β-glucan receptor in microglia. Of course, we cannot completely rule out the possibility that microglial responses to particulate β-glucan involved an additional receptor. For example, β-glucans have been reported to also interact with lactosylceramide (60), scavenger receptors (61, 62), and complement receptor 3 (63, 64). Although it is possible one or more of these receptors are additionally involved, our data are consistent with a model in which Dectin-1-SFK/Syk is the major signaling pathway mediating the biochemical and cell biological responses to β-glucans in microglia.

In summary, we have characterized innate immune responses of microglia to particulate β-glucan, and we have presented evidence showing that these responses are mediated by Dectin-1. In particular, we have shown that β-glucan-induced microglial activation response is unique in that there is no significant production of cytokines/chemokines. Our study is the first necessary step toward a better understanding of the biochemical pathways that govern recognition of fungal pathogens and subsequent inflammatory responses by microglia.


   Acknowledgments
 
We are grateful to Dr. William Lafuse for providing us the TLR2-deficient mice and to Dr. Gary Landreth for the BV-2 cells. We thank Dr. Kari Hoyt for her help with ELISA experiments and Dr. Keli Hu for the use of microscope. We also extend our appreciation to Dr. Gordon Brown for valuable discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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.

1 Address correspondence and reprint requests to Dr. Lakhu Keshvara, Division of Pharmacology, College of Pharmacy, 500 West 12th Avenue, Columbus, OH 43210. E-mail address: Keshvara.1{at}osu.edu Back

2 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; ROS, reactive oxygen species; SFK, Src family kinase; Syk, spleen tyrosine kinase. Back

Received for publication September 27, 2007. Accepted for publication December 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Aloisi, F.. 2001. Immune function of microglia. Glia 36: 165-179. [Medline]
  2. Kreutzberg, G. W.. 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19: 312-318. [Medline]
  3. Bsibsi, M., R. Ravid, D. Gveric, J. M. van Noort. 2002. Broad expression of Toll-like receptors in the human central nervous system. J. Neuropathol. Exp. Neurol. 61: 1013-1021. [Medline]
  4. Kielian, T.. 2006. Toll-like receptors in central nervous system glial inflammation and homeostasis. J. Neurosci. Res. 83: 711-730. [Medline]
  5. Laflamme, N., G. Soucy, S. Rivest. 2001. Circulating cell wall components derived from Gram-negative, not Gram-positive, bacteria cause a profound induction of the gene-encoding Toll-like receptor 2 in the CNS. J. Neurochem. 79: 648-657. [Medline]
  6. Kielian, T.. 2004. Immunopathogenesis of brain abscess. J. Neuroinflammation 1: 16[Medline]
  7. Town, T., V. Nikolic, J. Tan. 2005. The microglial "activation" continuum: from innate to adaptive responses. J. Neuroinflammation 2: 24[Medline]
  8. Esen, N., T. Kielian. 2005. Recognition of Staphylococcus aureus-derived peptidoglycan (PGN) but not intact bacteria is mediated by CD14 in microglia. J. Neuroimmunol. 170: 93-104. [Medline]
  9. Hoffmann, O., J. S. Braun, D. Becker, A. Halle, D. Freyer, E. Dagand, S. Lehnardt, J. R. Weber. 2007. TLR2 mediates neuroinflammation and neuronal damage. J. Immunol. 178: 6476-6481. [Abstract/Free Full Text]
  10. Kielian, T., P. Mayes, M. Kielian. 2002. Characterization of microglial responses to Staphylococcus aureus: effects on cytokine, costimulatory molecule, and Toll-like receptor expression. J. Neuroimmunol. 130: 86-99. [Medline]
  11. Bowman, S. M., S. J. Free. 2006. The structure and synthesis of the fungal cell wall. BioEssays 28: 799-808. [Medline]
  12. Brown, G. D., S. Gordon. 2005. Immune recognition of fungal β-glucans. Cell Microbiol. 7: 471-479. [Medline]
  13. Netea, M. G., N. A. Gow, C. A. Munro, S. Bates, C. Collins, G. Ferwerda, R. P. Hobson, G. Bertram, H. B. Hughes, T. Jansen, et al 2006. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J. Clin. Invest. 116: 1642-1650. [Medline]
  14. Nishimura, K., S. Nishimura, N. Nishi, I. Saiki, S. Tokura, I. Azuma. 1984. Immunological activity of chitin and its derivatives. Vaccine 2: 93-99. [Medline]
  15. Seljelid, R.. 1986. A water-soluble aminated β1–3D-glucan derivative causes regression of solid tumors in mice. Biosci. Rep. 6: 845-851. [Medline]
  16. Ross, G. D., V. Vetvicka, J. Yan, Y. Xia, J. Vetvickova. 1999. Therapeutic intervention with complement and β-glucan in cancer. Immunopharmacology 42: 61-74. [Medline]
  17. Kokoshis, P. L., D. L. Williams, J. A. Cook, N. R. Di Luzio. 1978. Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan. Science 199: 1340-1342. [Abstract/Free Full Text]
  18. Itoh, W., I. Sugawara, S. Kimura, K. Tabata, A. Hirata, T. Kojima, S. Mori, K. Shimada. 1990. Immunopharmacological study of sulfated schizophyllan (SPG). I. Its action as a mitogen and anti-HIV agent. Int. J. Immunopharmacol. 12: 225-233. [Medline]
  19. Williams, D. L., J. A. Cook, E. O. Hoffmann, N. R. Di Luzio. 1978. Protective effect of glucan in experimentally induced candidiasis. J. Reticuloendothel. Soc. 23: 479-490. [Medline]
  20. Cook, J. A., T. W. Holbrook, W. J. Dougherty. 1982. Protective effect of glucan against visceral leishmaniasis in hamsters. Infect. Immun. 37: 1261-1269. [Abstract/Free Full Text]
  21. Willment, J. A., A. S. Marshall, D. M. Reid, D. L. Williams, S. Y. Wong, S. Gordon, G. D. Brown. 2005. The human β-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells. Eur. J. Immunol. 35: 1539-1547. [Medline]
  22. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams, L. Martinez-Pomares, S. Y. Wong, S. Gordon. 2002. Dectin-1 is a major β-glucan receptor on macrophages. J. Exp. Med. 196: 407-412. [Abstract/Free Full Text]
  23. Gantner, B. N., R. M. Simmons, S. J. Canavera, S. Akira, D. M. Underhill. 2003. Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J. Exp. Med. 197: 1107-1117. [Abstract/Free Full Text]
  24. Rogers, N. C., E. C. Slack, A. D. Edwards, M. A. Nolte, O. Schulz, E. Schweighoffer, D. L. Williams, S. Gordon, V. L. Tybulewicz, G. D. Brown, C. Reis e Sousa. 2005. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22: 507-517. [Medline]
  25. Ariizumi, K., G. L. Shen, S. Shikano, S. Xu, R. Ritter, III, T. Kumamoto, D. Edelbaum, A. Morita, P. R. Bergstresser, A. Takashima. 2000. Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning. J. Biol. Chem. 275: 20157-20167. [Abstract/Free Full Text]
  26. Taylor, P. R., G. D. Brown, D. M. Reid, J. A. Willment, L. Martinez-Pomares, S. Gordon, S. Y. Wong. 2002. The β-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J. Immunol. 169: 3876-3882. [Abstract/Free Full Text]
  27. Brown, G. D., S. Gordon. 2001. Immune recognition: a new receptor for β-glucans. Nature 413: 36-37. [Medline]
  28. Gersuk, G. M., D. M. Underhill, L. Zhu, K. A. Marr. 2006. Dectin-1 and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states. J. Immunol. 176: 3717-3724. [Abstract/Free Full Text]
  29. Viriyakosol, S., J. Fierer, G. D. Brown, T. N. Kirkland. 2005. Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1. Infect. Immun. 73: 1553-1560. [Abstract/Free Full Text]
  30. Steele, C., L. Marrero, S. Swain, A. G. Harmsen, M. Zheng, G. D. Brown, S. Gordon, J. E. Shellito, J. K. Kolls. 2003. Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 β-glucan receptor. J. Exp. Med. 198: 1677-1688. [Abstract/Free Full Text]
  31. Taylor, P. R., S. V. Tsoni, J. A. Willment, K. M. Dennehy, M. Rosas, H. Findon, K. Haynes, C. Steele, M. Botto, S. Gordon, G. D. Brown. 2007. Dectin-1 is required for β-glucan recognition and control of fungal infection. Nat. Immunol. 8: 31-38. [Medline]
  32. Saijo, S., N. Fujikado, T. Furuta, S. H. Chung, H. Kotaki, K. Seki, K. Sudo, S. Akira, Y. Adachi, N. Ohno, et al 2007. Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat. Immunol. 8: 39-46. [Medline]
  33. Gantner, B. N., R. M. Simmons, D. M. Underhill. 2005. Dectin-1 mediates macrophage recognition of Candida albicans yeast but not filaments. EMBO J. 24: 1277-1286. [Medline]
  34. Herre, J., A. S. Marshall, E. Caron, A. D. Edwards, D. L. Williams, E. Schweighoffer, V. Tybulewicz, C. Reis e Sousa, S. Gordon, G. D. Brown. 2004. Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages. Blood 104: 4038-4045. [Abstract/Free Full Text]
  35. Brown, G. D., J. Herre, D. L. Williams, J. A. Willment, A. S. Marshall, S. Gordon. 2003. Dectin-1 mediates the biological effects of β-glucans. J. Exp. Med. 197: 1119-1124. [Abstract/Free Full Text]
  36. Luther, K., A. Torosantucci, A. A. Brakhage, J. Heesemann, F. Ebel. 2007. Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 β-glucan receptor and Toll-like receptor 2. Cell Microbiol. 9: 368-381. [Medline]
  37. Alliot, F., I. Godin, B. Pessac. 1999. Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Brain Res. Dev. Brain Res. 117: 145-152. [Medline]
  38. Pessac, B., I. Godin, F. Alliot. 2001. [Microglia: origin and development.]. Bull. Acad. Natl. Med. 185: 346-347.
  39. Muller, A., H. Ensley, H. Pretus, R. McNamee, E. Jones, E. McLaughlin, W. Chandley, W. Browder, D. Lowman, D. Williams. 1997. The application of various protic acids in the extraction of (1–>3)-β-D-glucan from Saccharomyces cerevisiae. Carbohydr. Res. 299: 203-208. [Medline]
  40. Lowman, D. W., D. A. Ferguson, D. L. Williams. 2003. Structural characterization of (1–>3)-β-D-glucans isolated from blastospore and hyphal forms of Candida albicans. Carbohydr. Res. 338: 1491-1496. [Medline]
  41. Williams, D. L., R. B. McNamee, E. L. Jones, H. A. Pretus, H. E. Ensley, I. W. Browder, N. R. Di Luzio. 1991. A method for the solubilization of a (1–3)-β-D-glucan isolated from Saccharomyces cerevisiae. Carbohydr. Res. 219: 203-213. [Medline]
  42. Muller, A., H. A. Pretus, R. B. McNamee, E. L. Jones, I. W. Browder, D. L. Williams. 1995. Comparison of the carbohydrate biological response modifiers Krestin, schizophyllan and glucan phosphate by aqueous size exclusion chromatography with in-line argon-ion multi-angle laser light scattering photometry and differential viscometry detectors. J. Chromatogr. B Biomed. Appl. 666: 283-290. [Medline]
  43. Ozment-Skelton, T. R., M. P. Goldman, S. Gordon, G. D. Brown, D. L. Williams. 2006. Prolonged reduction of leukocyte membrane-associated Dectin-1 levels following β-glucan administration. J. Pharmacol. Exp. Ther. 318: 540-546. [Abstract/Free Full Text]
  44. Rice, P. J., E. L. Adams, T. Ozment-Skelton, A. J. Gonzalez, M. P. Goldman, B. E. Lockhart, L. A. Barker, K. F. Breuel, W. K. Deponti, J. H. Kalbfleisch, et al 2005. Oral delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge. J. Pharmacol. Exp. Ther. 314: 1079-1086. [Abstract/Free Full Text]
  45. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11: 443-451. [Medline]
  46. Saura, J., J. M. Tusell, J. Serratosa. 2003. High-yield isolation of murine microglia by mild trypsinization. Glia 44: 183-189. [Medline]
  47. Underhill, D. M., E. Rossnagle, C. A. Lowell, R. M. Simmons. 2005. Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood 106: 2543-2550. [Abstract/Free Full Text]
  48. Di Carlo, F. J., J. V. Fiore. 1958. On the composition of zymosan. Science 127: 756-757. [Free Full Text]
  49. Kurosaki, T.. 1999. Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17: 555-592. [Medline]
  50. Keshvara, L. M., C. C. Isaacson, T. M. Yankee, R. Sarac, M. L. Harrison, R. L. Geahlen. 1998. Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J. Immunol. 161: 5276-5283. [Abstract/Free Full Text]
  51. Lee, S. C., Y. Kress, D. W. Dickson, A. Casadevall. 1995. Human microglia mediate anti-Cryptococcus neoformans activity in the presence of specific antibody. J. Neuroimmunol. 62: 43-52. [Medline]
  52. Wilkinson, B., J. Koenigsknecht-Talboo, C. Grommes, C. Y. Lee, G. Landreth. 2006. Fibrillar β-amyloid-stimulated intracellular signaling cascades require Vav for induction of respiratory burst and phagocytosis in monocytes and microglia. J. Biol. Chem. 281: 20842-20850. [Abstract/Free Full Text]
  53. Olson, J. K., S. D. Miller. 2004. Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J. Immunol. 173: 3916-3924. [Abstract/Free Full Text]
  54. Rogers, L. R.. 2003. Cerebrovascular complications in cancer patients. Neurol. Clin. 21: 167-192. [Medline]
  55. Singh, N., S. Husain. 2000. Infections of the central nervous system in transplant recipients. Transpl. Infect. Dis. 2: 101-111. [Medline]
  56. Streit, W. J., N. W. Sammons, A. J. Kuhns, D. L. Sparks. 2004. Dystrophic microglia in the aging human brain. Glia 45: 208-212. [Medline]
  57. Brown, G. D.. 2006. Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat. Rev. Immunol. 6: 33-43. [Medline]
  58. Hara, H., C. Ishihara, A. Takeuchi, T. Imanishi, L. Xue, S. W. Morris, M. Inui, T. Takai, A. Shibuya, S. Saijo, et al 2007. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat. Immunol. 8: 619-629. [Medline]
  59. LeibundGut-Landmann, S., O. Gross, M. J. Robinson, F. Osorio, E. C. Slack, S. V. Tsoni, E. Schweighoffer, V. Tybulewicz, G. D. Brown, J. Ruland, C. Reis e Sousa. 2007. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17. Nat. Immunol. 8: 630-638. [Medline]
  60. Zimmerman, J. W., J. Lindermuth, P. A. Fish, G. P. Palace, T. T. Stevenson, D. E. DeMong. 1998. A novel carbohydrate-glycosphingolipid interaction between a β-(1–3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J. Biol. Chem. 273: 22014-22020. [Abstract/Free Full Text]
  61. Rice, P. J., J. L. Kelley, G. Kogan, H. E. Ensley, J. H. Kalbfleisch, I. W. Browder, D. L. Williams. 2002. Human monocyte scavenger receptors are pattern recognition receptors for (1–>3)-β-D-glucans. J. Leukocyte Biol. 72: 140-146. [Abstract/Free Full Text]
  62. Pearson, A., A. Lux, M. Krieger. 1995. Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 92: 4056-4060. [Abstract/Free Full Text]
  63. Thornton, B. P., V. Vetvicka, M. Pitman, R. C. Goldman, G. D. Ross. 1996. Analysis of the sugar specificity and molecular location of the β-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J. Immunol. 156: 1235-1246. [Abstract]
  64. Ross, G. D., J. A. Cain, P. J. Lachmann. 1985. Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin as functions as a receptor for zymosan and rabbit erythrocytes as well as a receptor for iC3b. J. Immunol. 134: 3307-3315. [Abstract]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Rosas, K. Liddiard, M. Kimberg, I. Faro-Trindade, J. U. McDonald, D. L. Williams, G. D. Brown, and P. R. Taylor
The Induction of Inflammation by Dectin-1 In Vivo Is Dependent on Myeloid Cell Programming and the Progression of Phagocytosis
J. Immunol., September 1, 2008; 181(5): 3549 - 3557.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
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 arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shah, V. B.
Right arrow Articles by Keshvara, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shah, V. B.
Right arrow Articles by Keshvara, L.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS