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Zymosan treatment of mouse mast cells enhances dectin-1 expression and induces dectin-1-dependent reactive oxygen species (ROS) generation
ARTICLE IN PRESS
Immunobiology 214 (2009) 321–330 www.elsevier.de/imbio
Zymosan treatment of mouse mast cells enhances dectin-1 expression and induces dectin-1-dependent reactive oxygen species (ROS) generation Zhan Yangb, Jean S. Marshalla, a
Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, N.S., Canada B3H 1X5 Atlantic Baptist University, N.B., Canada
b
Received 7 March 2008; received in revised form 29 July 2008; accepted 10 September 2008
Abstract Dectin-1 is a major b-glucan receptor expressed in the innate immune cells such as macrophages, neutrophils and dendritic cells. It can mediate pro-inflammatory mediator release and other cellular responses such as phagocytosis and respiratory burst in response to pathogens. Mast cells are sentinel cells of the immune system found in greater numbers at sites of pathogen exposure such as the skin and airways than in other body sites. Dectin-1 on human mast cells has been shown to mediate the production of leukotrienes in response to yeast zymosan. In this study, using RT-PCR and FACS analysis, we examined both mRNA and protein expression of dectin-1 on bone marrow-derived cultured mast cells (BMMC) from either C57BL/6 or TLR2 deficient mice. Low levels of surface dectin-1 were detected on the mast cell surface, which could be up-regulated by zymosan activation. Neither mouse plasma nor decomplemented plasma (56 1C, 30 min) induced altered dectin-1 protein expression although zymosan-activated C57BL/6 BMMCs expressed two dectin-1 mRNA isoforms. Addition of laminarin, a well-established dectin-1 inhibitor, significantly inhibited surface expression of dectin-1 (po0.05), which further confirmed that dectin-1 surface expression was up-regulated by non-opsonized zymosan activation. Further studies showed that zymosan stimulated both C57BL/6 and TLR2(/) deficient BMMCs to generate intracellular oxidative burst. Pretreatment of BMMCs with laminarin inhibited ROS generation significantly (po0.05) after 2 h zymosan activation. Therefore, intracellular ROS generation in murine mast cells in response to zymosan is dependent on dectin-1 receptors. r 2008 Elsevier GmbH. All rights reserved. Keywords: Dectin-1; Innate immunity; Reactive oxygen species; Mast cells; Zymosan
Introduction Dectin-1, an important b-glucan surface receptor, is expressed on a broad range of innate immune cells such as monocytes/macrophages, neutrophils, NK cells, fibroblasts and dendritic cells (Ariizumi et al., 2000; Corresponding author. Tel.: +1 902 494 5118; fax: +1 902 494 5125. E-mail address: [email protected] (J.S. Marshall).
0171-2985/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2008.09.002
Kougias et al., 2001; Taylor et al., 2002; Williams, 1997). b-glucan is a major fungal cell wall component, and dectin-1 has been found to play an important role in the innate immunity against pathogenic fungi such as Coccidiodies posadasii, Pneumocystis carinii and Aspergillus fumigatus (Hohl et al., 2005; Steele et al., 2003; Viriyakosol et al., 2005). Recently, two different, functionally distinct dectin-1 isoforms were observed in murine macrophages. Either isoform can stimulate TNF production, but to different degrees (Heinsbroek et al.,
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2006). Dectin-1 is a type II transmembrane protein receptor with a C-type lectin-domain and a cytoplasmic tail with ITAM motif (Adachi et al., 2004) phosphorylation of which can stimulate pro-inflammatory mediators release and other cellular responses such as phagocytosis and respiratory burst in collaboration with TLR-2 (Brown et al., 2002; Gantner et al., 2003). However, recent studies showed that early inflammatory responses of mast cells to pathogens can often be attributed to complement receptors, not TLR-2 (Mullaly and Kubes, 2007). Several studies also showed that dectin-1 could interact with zymosan as well as yeast from Candida albicans, which stimulated phagocytosis and ROS generation in a TLR-independent manner (Rogers et al., 2005; Underhill et al., 2005). Little information is available concerning dectin-1 expression on murine mast cells and its role in regulating cellular responses to b-glucan. Zymosan derived from the cell wall of Saccharomyces cerevisiae is rich in both b-glucan and mannan (Di Carlo and Fiore, 1958). It has been widely used as a model fungal particle to study immune responses conducted by different innate and adaptive immunity cells. Zymosan not only interacts with leukocytes, such as macrophages via non-opsonic dectin-1 receptors to stimulate proinflammatory mediator or cytokine release (Brown et al., 2002; Heinsbroek et al., 2006), but also can interact with complement or TLR-2 receptors in mediating inflammatory responses, such as phagocytosis (Gantner et al., 2003; McCurdy et al., 2003; Mullaly and Kubes, 2007). Both mast cells and other leukocyte populations have been shown to have important roles in the response to zymosan in models of peritonitis (Kolaczkowska et al., 2001, 2007). Recently, we described that fungal zymosan can stimulate human mast cells to produce LTC4 in a dectin-1-dependent manner (Olynych et al., 2006). Mast cells are distributed widely throughout the body tissues, which are commonly found at submucosal surfaces of skin, airway and the intestine. Mast cells function as sentinel cells in host defense, including enhancement of both innate and specific defenses, against pathogens. Mast cells are well known to release a large amount of pro-inflammatory and inflammatory mediators, which contribute to both immediate allergic reactions and inflammation (Marshall, 2004). They are also capable of phagocytosis of invaded pathogens, which plays an important role in innate immunity (Abraham and Malaviya, 1997). Following phagocytosis, the production of reactive oxygen species (ROS) is critical for pathogen killing (Forman and Torres, 2001; Henricks and Nijkamp, 2001; Malaviya et al., 1999, 1996). There are many substances such as IgE/Ag, calcium ionophore A23187, gold compound, D-penicillamine, compound 48/80, nerve growth factor and others (Brooks et al., 1999; Niu et al., 1996; Wolfreys
and Oliveira, 1997) that can stimulate mast cells to generate intracellullar ROS. ROS generation has also been demonstrated to be frequently accompanied by degranulation (Swindle et al., 2002, 2004). Inhibition of ROS generation leads to decreased release of mediators such as histamine and LTC4 in RBL-2H3 mast cells (Suzuki et al., 2003). Several studies showed ROS such as potassium superoxide or hydrogen peroxide could induce mast cells to degranulate (Akagi et al., 1994; Peden et al., 1994). In macrophage cell lines, dectin-1 mediates zymosan-induced ROS generation (Gantner et al., 2003). It is not yet known whether zymosan can induce intracellular ROS generation in mast cells after phagocytosis and the role of dectin-1 in ROS generation has not been determined in mast cells. In this study, we examined murine mast cell expression of dectin-1 and intracellular ROS generation in response to zymosan as a model of fungal-induced ROS generation. Even though the level of dectin-1 expression on murine mast cells was far lower than that on the cell surface of macrophages, it was demonstrated that zymosan-induced intracellular ROS generation was dectin-1 dependent in mast cells. Furthermore, dectin1 expression can be up-regulated by zymosan activation alone. To our knowledge, this is the first study to show the relationship between dectin-1 and ROS production in mast cells.
Methods Reagents Yeast zymosan from S. cerevisiae and laminarin were purchased from Sigma (St. Louis, MO). Dichlorodihydrofuorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR). Zymosan suspensions were made in endotoxin free saline and vortexed immediately before use to ensure particles were equally distributed in suspension. Laminarin was also dissolved in endotoxin free saline and sonicated immediately prior to use. The primary Abs used in this study were 2A11, monoclonal rat anti-mouse dectin-1 receptors conjugated with biotin (ABD Serotec, Raleigh, NC). The secondary Abs were Streptavidin-PE obtained from BD pharmingen (San Diego, CA). IgG2b-biotin (eBioscience, San Diego, CA) was used as an isotype control.
Mice C57BL/6 (Jackson Labs, Bar Harbor, Maine) mice and TLR2-deficient mice on a C57BL/6 background (a gift from Dr. S. Akira, Osaka, Japan) were used in this study. All mice were 6–10 weeks of age and housed
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under specific pathogen-free conditions with food and water provided ad libitum. All experiments were approved by the animal research ethics board of Dalhousie University.
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modifications: (1) 50 -CAAGTGCTCTGCCTACCTAG; (2) CACCA TCTTTATATTCTCACATAC. These primers amplified A and B isoforms of mouse dectin-1. PCR products were detected by electrophoresis on a 1.5% agarose gel and ethidium bromide staining.
Cell culture Bone marrow-derived cultured mast cells (BMMC) were obtained from 4- to 8-week-old C57BL/6 and TLR2-deficient mice according to a previously published method (Tertian et al., 1981). BMMC cells were incubated in RPMI-1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin and streptomycin (Life Technologies, Grand Island, NY), 5 105 M 2-mercaptoethanol (Sigma) and 10% WEHI3-B supernatant as a source of IL-3. Cells were grown at 37 1C, 5% CO2 for 5–8 weeks, at which time the purity of mast cells was determined morphologically and by the presence of metachromatic granules following toluidine blue staining (pH 1.0). Only 495% pure mast cells preparation were used.
Cell activation BMMC were washed and resuspended at a final concentration of 1 106 cells/ml in RPMI containing 1% bovine serum albumin (Sigma), 3 ng/ml IL-3 (PeproTech Inc., Rocky Hill, NJ) and 100 mg/ml of soybean trypsin inhibitor (Sigma) for activation. The cells were incubated with media alone or 100 mg/ml zymosan for 3 and 6 h, respectively, with the addition of mouse plasma or heated (56 1C, 30 min) mouse plasma (2% v/v) at 37 1C, 5% CO2. Cells were harvested by centrifugation for RT-PCR assays. The dose of zymosan was chosen based on preliminary dose response experiments (data not shown), which indicated that this dose gave a strong response but the viability of treated cells remained over 90%, as well as on the results of previous studies examining human mast cell responses to zymosan suspensions (Olynych et al., 2006)
RT-PCR Using RNA isolation kit (Qiagen, Mississauga, ON), RNA was isolated from each cell sample (2 106) with different stimulation as previously described and diluted to the same concentration based on the optical density. RNA integrity was assessed by examining ribosomal RNA bands, following gel electrophoresis. cDNA was then obtained using RT-PCR kit (Invitrogen, Carlsbad, CA) with Oligo(dT) primers according to manufacturer’s recommended methodology. Primers used for PCR amplification of reverse-transcribed RNA samples for mouse dectin-1 were manufactured according to the previous studies (Heinsbroek et al., 2006) with some
FACS analysis C57BL/6 BMMCs were activated by zymosan as described above. In the inhibition experiment, 50 mg/ml laminarin was added at the same time as zymosan to treat cells for 3 h at 37 1C, 5% CO2. 2.5 105 cells, following each different treatment, were added to wells of a 96-well tissue culture plate (Life Technologies). Cells were washed twice with phosphate buffered saline (PBS)–bovine serum albumin (BSA) (0.1%) buffer and incubated in a Fc receptor blocking buffer (human IgG in PBS–BSA) at 4 1C for 15 min. Cells were then incubated with primary Ab, mAb211-biotin (Brown et al., 2002), for 30 min at 4 1C, in separate tubes biotinylated IgG2b, with the same concentration, was used as an isotype control. After washing three times with PBS–BSA, cells were incubated in PBS–BSA buffer with secondary Ab, Streptavidin-PE at 4 1C for 30 min. Cells were fixed in 1% formaldehyde after washing three times, as described before. Finally cells were analyzed on a FACS Calibur flow cytometer using CellQuest software.
Measurement of intracellular ROS production by flow cytometry Mast cells (5 105) suspended in PBS (Life Technologies) were incubated with 5 mM DCFH-DA for 15 min at 37 1C. Then cells were washed twice or three times and resuspended in PBS at 4 1C, which were followed with activation by 100 mg/ml zymosan at 37 1C and 5% CO2 for different time periods. In inhibition experiments, cells were pre-incubated with 50 mg/ml laminarin on ice for 30 min before zymosan stimulation. Cells stimulated with 50 ng/ml PMA for 30 min at 37 1C and 5% CO2 were used as a positive control to check for authentic intracellular ROS generation. Intracellular ROS generation was analyzed on a FACS Calibur flow cytometer using CellQuest software.
Statistics Differences in fluorescence level between anti-dectin-1 and control antibody stained cells and fluorescent staining of cells subjected to different treatments were evaluated using the method of Komogorov–Smivov. Only p values of o0.05 were considered significant.
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Results
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Expression of dectin-1 mRNA in murine mast cells Using RT-PCR analysis, we examined the expression of dectin-1 mRNA in C57BL/6 and TLR2(/) BMMC mast cells. Without the addition of mouse plasma or heated plasma, BMMCs, either at rest or following activation with zymosan for 3 and 6 h, respectively, consistently showed no evidence of dectin-1 isoform expression. Following addition of mouse plasma and zymosan activation for 3 h, expression of two dectin-1 isoforms was consistently detected in C57BL/6 BMMC. Weaker expression of Dectin-1 mRNA was consistently observed in BMMC cells with the addition of heated plasma after zymosan stimulation within the same time period (Fig. 1a). TLR2/ BMMC treated with zymosan and plasma combined for 3 h showed lower expression of dectin-1 mRNA (Fig. 1b) compared with TLR2 expressing mast cells in each of the three separate experiments (Fig. 1a). After 6 h of zymosan stimulation in the presence of plasma, mast cells from both sources showed decreased dectin-1 mRNA expression compared with the earlier 3 h time point. RNA from splenic cells was used as a positive control (Fig. 1c). Results representative of 3–4 experiments/group.
Expression of dectin-1 receptors on cell surface We examined the surface expression of dectin-1 receptors on C57BL/6 BMMCs using a dectin-1 specific antibody (mAb211). After 3 h incubation in media alone, a very low expression of dectin-1 receptor was detected on the cell surface. Cells stimulated by zymosan for 3 h in the absence of mouse plasma consistently exhibited significantly higher surface expression of dectin-1 receptors by comparing the mean fluorescence with that of untreated negative control cells (po0.05). However, the addition of mouse plasma or heated plasma alone had no further inducing effect on dectin 1 expression (Fig. 2a). With or without the presence of plasma or heated plasma, C57BL/6 BMMCs, following zymosan activation for 6 h, did not show evidence of increased dectin-1 surface expression compared with
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Fig. 1. Plasma induces expression of two dectin-1 isoforms by BMMCs. RT-PCR analysis shows dectin-1 expression in BMMCs activated by zymosan (100 mg/ml) with or without further addition of plasma or heated plasma for 3 and 6 h, respectively, (a) examines responses of BMMCs from C57BL/6 mice. (b) To examine the role of TLR2 in the response to zymosan plus mouse plasma, a similar experiment was performed using BMMC’s from TLR2-deficient (TLR2/) mice. RNA from the same cell types was used as a negative control, while RNA from splenic cells was used as a positive control (c). 700 BP and 800 BP bands are observed. P: plasma, HP: heated plasma. Representative of three similar experiments.
untreated cells and treatment with heated plasma alone appeared to further reduce the low levels of dectin-1 expression observed in control cells (Fig. 2b). Pretreatment of 3 h, zymosan-activated C57BL/6 BMMCs with 50 mg/ml laminarin reduced the observed increase in dectin-1 surface expression (Fig. 2c), while the same laminarin treatment had no effect on dectin-1 expression
Fig. 2. Expression of dectin-1 receptors on C57BL/6 BMMCs. C57BL/6 BMMC cells expressed very low level of dectin-1 receptors on membrane surface with or without the addition of plasma or heated plasma. Activation by zymosan for 3 h consistently increased dectin-1 receptor expression, which was not enhanced by the addition of mouse plasma (a). Activation for 6 h by zymosan did not lead to a sustained increase in expression (b). Laminarin pre-treatment partially inhibited the induced increase in dectin-1 expression on the surface of C57BL/6 BMMC (c). Comparing mean fluorescence from three independent experiments further confirmed that laminarin pre-treatment significantly inhibited dectin-1 expression (d). Resident peritoneal macrophages which had been cultured for overnight were used as a positive control for surface dectin-1 receptor assessment (e). Shaded histograms represented IgG2b isotype control and unshaded histograms represented surface staining with mab2A11. P: plasma, HP: heated plasma. Representative of three independent experiments.
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control to assess surface expression of dectin-1 receptors (Fig. 2e).
Generation of intracellular ROS in zymosanactivated BMMCs Intracellular ROS generation in BMMCs was measured by loading reduced fluorescent indicator, DCFH-DA, which can be rapidly oxidized by H2O2 (Bass et al., 1983). DCFH-DA-loaded C57BL/6 BMMCs were stimulated with zymosan (100 mg/ml) and intracellular ROS production was measured at one hour intervals for 3 h. Intracellular ROS was initially generated at 1 h and gradually increased in extent. At 3 h, intracellular ROS production terminated (Fig. 3a). Fifty nanogram per milliliter PMA treatment for 30 min was used as a positive control to confirm the reliability of ROS assay system (Fig. 3b). TLR2/ BMMCs activated by zymosan for 1 and 2 h, respectively, (Fig. 4a) also produced intracellular ROS. Comparing ROS levels between C57BL/6 and TLR2/ BMMCs at the same incubation time (Fig. 4b), C57BL/6 BMMCs produced significantly more ROS than that of TLR2/ after 2 h zymosan stimulation (po0.05). However, there was no significant difference in ROS generation between these two cell types activated by zymosan for 1 h.
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Dectin-1-mediated intracellular ROS generation Laminarin, a well-known dectin-1 inhibitor (Brown et al., 2002; Gantner et al., 2003), was used to investigate the potential role of dectin-1 receptors in mediating intracellular ROS generation in this study. Pretreatment of C57BL/6 BMMCs with 50 mg/ml laminarin in PBS (pH 7.4) for 30 min at 4 1C inhibited intracellular ROS generation following 2 h zymosan activation (Fig. 5a). Laminarin pretreatment significantly inhibited ROS generation (Fig. 5b, po0.05). An alternate analysis (Fig. 5c), which compares the difference between median RFU (percentage of negative control) of zymosan-activated cells with and without laminarin pretreatment, also shows significant differences between these two treatment groups (po0.05).
Discussion Dectin-1 is a c-type lectin, which can interact with zymosan, the major yeast cell wall component, as well as other fungal pathogens, such as Candida albicans, Coccidiodies posadasii, Pneumocystis carinii and Aspergillus fumigatus (Hohl et al., 2005; Rogers et al., 2005; Steele et al., 2003; Underhill et al., 2005; Viriyakosol et al., 2005). Dectin-1 receptors have been found to be expressed on monocyte/macrophage
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Fig. 3. Zymosan-activated intracellular ROS generation in murine mast cells. DCFH-DA-loaded C57BL/6 BMMCs were stimulated with zymosan (100 mg/ml) and intracellular ROS production was measured at hourly intervals for 3 h. Shaded area represents control cells, open area represents activated cells (a). Intracellular ROS generation by PMA (50 ng/ml) activation for 30 min was used as a positive control (b). Representative of 4–5 independent experiment.
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Fig. 4. Comparison of zymosan-induced intracellular ROS generation in the presence and absence of TLR2. DCFH-DA-loaded TLR2/ BMMCs were stimulated with zymosan (100 mg/ml) in PBS and intracellular ROS generation was measured for 1 and 2 h, respectively, (a). Shaded histograms represented ROS generation from TLR2/ BMMCs without any treatment. Intracellular ROS generation by PMA (50 ng/ml) activation for half an hour was used as a positive control. Intracellular ROS generation was compared between C5BL6/7 and TLR2/ BMMCs at 1 and 2 h, respectively, (b). Representative of 4–5 independent experiment. Denotes po0.05.
lineage, neutrophils, NK cells, fibroblasts and dendritic cells (Ariizumi et al., 2000; Kougias et al., 2001; Taylor et al., 2002; Williams, 1997). In our previous study, human mast cells were found to express functional dectin-1 receptors on the cell surface, which respond to zymosan, and participate in leukotriene generation (Olynych et al., 2006). In this study, we demonstrated that dectin-1 receptors were expressed on murine mast cell surface at a very low level and their mRNA expression could be increased by zymosan activation. The addition of mouse plasma or heated plasma alone did not induce dectin-1 protein expression as analyzed by FACs. Plasma treatment along with zymosan did, however, stimulate murine BMMC to express two dectin-1 mRNA isoforms, an effect that was lost by heating plasma at 56 1C for 30 min to inactivate the
complement cascade. These results suggest that the mast cell mRNA expression of dectin-1 regulated by zymosan activation of complement. However, surface protein expression data from FACS analysis consistently indicated (in four separate experiments) that cell surface expression of dectin-1 could also be enhanced by zymosan treatment alone at 3 h and was rapidly down regulated at the later (6 h) time point. Laminarin, a specific inhibitor for b-glucan receptors does not block the activity of the mannose receptor or the CR3 receptor (Brown et al., 2002). Therefore, it has been used in several studies to confirm the role of dectin1 receptors in response to zymosan or PGN activation (Brown et al., 2003; Gantner et al., 2003; Olynych et al., 2006). Adding laminarin at the same time as zymosan to mast cell suspensions can reduce zymosan-induced
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Fig. 5. Laminarin-inhibited zymosan-induced intracellular ROS generation in C57BL/6 BMMCs. Pre-treatment of C57BL/6 BMMCs with laminarin (50 mg/ml) for 30 min at 4 1C inhibited intracellular ROS generation following zymosan activation for 2 h. Representative of 4 independent experiment. Denotes po0.05.
dectin-1 expression substantially, which confirms the role of zymosan in enhancing the dectin-1 expression on murine mast cell surface. ROS generation in mast cells may play a critical role in innate immune reactions (Forman and Torres, 2001; Henricks and Nijkamp, 2001; Malaviya et al., 1999; Malaviya et al., 1996). However, its cellular pathway is still unclear. Mast cells can be stimulated by a range of mechanisms such as IgE/Ag, ionophore, etc to generate ROS (Brooks et al., 1999; Niu et al., 1996; Tsinkalovsky
and Laerum, 1994; Wolfreys and Oliveira, 1997). ROS generated by some mechanisms, such as IgE/Ag stimulation, was accompanied by mast cell degranulation, while no degranulation was detected by other mechanisms (Brooks et al., 1999; Tsinkalovsky and Laerum, 1994). ROS production in both IgE-sensitized mast cells after antigen challenge and PMA-stimulated mast cells is probably attributed to NAD(P)H oxidase (Suzuki et al., 2003; Swindle et al., 2002). Comparing ROS generation in macrophages with that in mast cells
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suggests much lower level of intracellular ROS generation by mast cells (Swindle et al., 2002). There has been some controversy over ROS generated by PMA-stimulated mast cells. One study (Swindle et al., 2002) suggested a small amount of ROS generation by PMA stimulation in rat peritoneal mast cells. Significant intracellular ROS was generated in human mast cells via PMA stimulation and its production reached a peak at 30 min (Kim and Ro, 2005), which is in full agreement with our data using similar stimulation in BMMC. DCFH-DA oxidation into DCF reflects the level of intracellular H2O2 generation with high sensitivity and was also used in our study to detect intracellular ROS generation upon zymosan stimulation. The mast cells we used were all non-adherent, of extremely high purity and not contaminated by detectable numbers of macrophages. Our data revealed significant intracellular ROS generation in zymosanstimulated BMMCs at 2 h. Furthermore, comparing intracellular ROS production from zymosan-activated C56BL/7 BMMCs for 2 h with and without pretreatment of laminarin, shows that laminarin pretreatment significantly inhibited ROS generation (po0.05). These results demonstrate that dectin-1 receptors on BMMCs surface-mediated ROS generation. Zymosan-derived intracellular ROS generation in this study was not accompanied by degranulation (data not shown). Zymosan also does not induce significant degranulation of human mast cells (McCurdy et al., 2003). In a previous study, low level of dectin-1 receptors on macrophage surface were also demonstrated to mediate zymosan-derived ROS generation without being accompanied by degranulation (Gantner et al., 2003). However, faster kinetics of ROS production might be exhibited in macrophages than that in mast cells. Pattern-recognition receptors, such as TLRs and lectin-like receptors are critical in the early detection of pathogens and initiating innate immune responses against them. There have been several studies attempting to clarify the relationship between TLR2 and dectin1 (lectin type) receptors in immune responses. Both receptors can recognize microbial components such as zymosan and stimulate inflammatory responses in either a co-operative or independent manner. Dectin-1 receptor expression on macrophages and dendritic cells is able to upgrade TLR-mediated cytokine release in response to zymosan. Also dectin-1 receptors act synergistically with TLR2 receptors in phagocytosis as well as ROS generation. However, dectin-1 expressed by macrophage lineage was shown to mediate phagocytosis of zymosan and subsequent ROS generation in a TLR-independent manner. In this study, both C57BL/6 and TLR2/ BMMCs were able to produce intracellular ROS in response to zymosan activation. After 2 h zymosan stimulation, ROS level of C57BL/6 BMMCs was significantly higher than that of TLR2/ BMMCs
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(po0.05). Therefore, from these data analysis it may be concluded that TLR2 receptors co-operate with dectin-1 receptors in zymosan-derived intracellular ROS generation from murine mast cells. In summary, we have demonstrated that low levels of surface expression of dectin-1 receptors on murine mast cells can be up-regulated by zymosan activation. Such dectin-1 receptors mediate zymosan-derived intracellular ROS generation by a mechanism that is also partially dependent on TLR2 function. The analysis of surface expression of dectin-1 receptors and ROS generation in response to zymosan has provided novel insights into the recognition of b-glucan by murine mast cells and into the cellular responses mediated by dectin-1.
Acknowledgments The authors would like to thank Ms. Yi-Song Wei, Dr. Wojciech Dawicki, Dr. Dunia Jawdat, Dr. Ian Haidl and Dr. Nong Xu for their advice and assistance on aspects of these studies. This work was supported by the Canadian Institutes of Health Research.
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Di Carlo, F.J., Fiore, J.V., 1958. On the composition of zymosan. Science 127, 756–757. Forman, H.J., Torres, M., 2001. Redox signaling in macrophages. Mol. Aspects Med. 22, 189–216. Gantner, B.N., Simmons, R.M., Canavera, S.J., Akira, S., Underhill, D.M., 2003. Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J. Exp. Med. 197, 1107–1117. Heinsbroek, S.E., Taylor, P.R., Rosas, M., Willment, J.A., Williams, D.L., Gordon, S., Brown, G.D., 2006. Expression of functionally different dectin-1 isoforms by murine macrophages. J. Immunol. 176, 5513–5518. Henricks, P.A., Nijkamp, F.P., 2001. Reactive oxygen species as mediators in asthma. Pulm. Pharmacol. Ther. 14, 409–420. Hohl, T.M., Van Epps, H.L., Rivera, A., Morgan, L.A., Chen, P.L., Feldmesser, M., Pamer, E.G., 2005. Aspergillus fumigatus triggers inflammatory responses by stage-specific beta-glucan display. PLoS Pathog. 1, e30. Kim, J.Y., Ro, J.Y., 2005. Signal pathway of cytokines produced by reactive oxygen species generated from phorbol myristate acetate-stimulated HMC-1 cells. Scand. J. Immunol. 62, 25–35. Kolaczkowska, E., Seljelid, R., Plytycz, B., 2001. Role of mast cells in zymosan-induced peritoneal inflammation in Balb/c and mast cell-deficient WBB6F1 mice. J. Leukoc. Biol. 69, 33–42. Kolaczkowska, E., Lelito, M., Kozakiewicz, E., van Rooijen, N., Plytycz, B., Arnold, B., 2007. Resident peritoneal leukocytes are important sources of MMP-9 during zymosan peritonitis: superior contribution of macrophages over mast cells. Immunol. Lett. 113, 99–106. Kougias, P., Wei, D., Rice, P.J., Ensley, H.E., Kalbfleisch, J., Williams, D.L., Browder, I.W., 2001. Normal human fibroblasts express pattern recognition receptors for fungal (1-3)-beta-D-glucans. Infect. Immun. 69, 3933–3938. Malaviya, R., Ikeda, T., Ross, E., Abraham, S.N., 1996. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 381, 77–80. Malaviya, R., Gao, Z., Thankavel, K., van der Merwe, P.A., Abraham, S.N., 1999. The mast cell tumor necrosis factor alpha response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48. Proc. Natl. Acad. Sci. USA 96, 8110–8115. Marshall, J.S., 2004. Mast-cell responses to pathogens. Nat. Rev. Immunol. 4, 787–799. McCurdy, J.D., Olynych, T.J., Maher, L.H., Marshall, J.S., 2003. Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J. Immunol. 170, 1625–1629. Mullaly, S.C., Kubes, P., 2007. Mast cell-expressed complement receptor, not TLR2, is the main detector of zymosan in peritonitis. Eur. J. Immunol. 37, 224–234. Niu, X.F., Ibbotson, G., Kubes, P., 1996. A balance between nitric oxide and oxidants regulates mast cell-dependent neutrophil–endothelial cell interactions. Circ. Res. 79, 992–999.
Olynych, T.J., Jakeman, D.L., Marshall, J.S., 2006. Fungal zymosan induces leukotriene production by human mast cells through a dectin-1-dependent mechanism. J. Allergy Clin. Immunol. 118, 837–843. Peden, D.B., Dailey, L., DeGraff, W., Mitchell, J.B., Lee, J.G., Kaliner, M.A., Hohman, R.J., 1994. Hydrogen peroxide effects on rat mast cell function. Am. J. Physiol. 267, L85–93. Rogers, N.C., Slack, E.C., Edwards, A.D., Nolte, M.A., Schulz, O., Schweighoffer, E., Williams, D.L., Gordon, S., Tybulewicz, V.L., Brown, G.D., Reis e Sousa, C., 2005. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C-type lectins. Immunity 22, 507–517. Steele, C., Marrero, L., Swain, S., Harmsen, A.G., Zheng, M., Brown, G.D., Gordon, S., Shellito, J.E., Kolls, J.K., 2003. Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor. J. Exp. Med. 198, 1677–1688. Suzuki, Y., Yoshimaru, T., Matsui, T., Inoue, T., Niide, O., Nunomura, S., Ra, C., 2003. Fc epsilon RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals. J. Immunol. 171, 6119–6127. Swindle, E.J., Hunt, J.A., Coleman, J.W., 2002. A comparison of reactive oxygen species generation by rat peritoneal macrophages and mast cells using the highly sensitive real-time chemiluminescent probe pholasin: inhibition of antigen-induced mast cell degranulation by macrophage-derived hydrogen peroxide. J. Immunol. 169, 5866–5873. Swindle, E.J., Metcalfe, D.D., Coleman, J.W., 2004. Rodent and human mast cells produce functionally significant intracellular reactive oxygen species but not nitric oxide. J. Biol. Chem. 279, 48751–48759. Taylor, P.R., Brown, G.D., Reid, D.M., Willment, J.A., Martinez-Pomares, L., Gordon, S., Wong, S.Y., 2002. The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J. Immunol. 169, 3876–3882. Tsinkalovsky, O.R., Laerum, O.D., 1994. Flow cytometric measurement of the production of reactive oxygen intermediate in activated rat mast cells. Apmis. 102, 474–480. Underhill, D.M., Rossnagle, E., Lowell, C.A., Simmons, R.M., 2005. Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood 106, 2543–2550. Viriyakosol, S., Fierer, J., Brown, G.D., Kirkland, T.N., 2005. Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1. Infect. Immun. 73, 1553–1560. Williams, D.L., 1997. Overview of (1-3)-beta-D-glucan immunobiology. Mediators Inflammation 6, 247–250. Wolfreys, K., Oliveira, D.B., 1997. Alterations in intracellular reactive oxygen species generation and redox potential modulate mast cell function. Eur. J. Immunol. 27, 297–306.
Immunobiology 214 (2009) 321–330 www.elsevier.de/imbio
Zymosan treatment of mouse mast cells enhances dectin-1 expression and induces dectin-1-dependent reactive oxygen species (ROS) generation Zhan Yangb, Jean S. Marshalla, a
Department of Microbiology & Immunology, Dalhousie University, 5850 College Street, Halifax, N.S., Canada B3H 1X5 Atlantic Baptist University, N.B., Canada
b
Received 7 March 2008; received in revised form 29 July 2008; accepted 10 September 2008
Abstract Dectin-1 is a major b-glucan receptor expressed in the innate immune cells such as macrophages, neutrophils and dendritic cells. It can mediate pro-inflammatory mediator release and other cellular responses such as phagocytosis and respiratory burst in response to pathogens. Mast cells are sentinel cells of the immune system found in greater numbers at sites of pathogen exposure such as the skin and airways than in other body sites. Dectin-1 on human mast cells has been shown to mediate the production of leukotrienes in response to yeast zymosan. In this study, using RT-PCR and FACS analysis, we examined both mRNA and protein expression of dectin-1 on bone marrow-derived cultured mast cells (BMMC) from either C57BL/6 or TLR2 deficient mice. Low levels of surface dectin-1 were detected on the mast cell surface, which could be up-regulated by zymosan activation. Neither mouse plasma nor decomplemented plasma (56 1C, 30 min) induced altered dectin-1 protein expression although zymosan-activated C57BL/6 BMMCs expressed two dectin-1 mRNA isoforms. Addition of laminarin, a well-established dectin-1 inhibitor, significantly inhibited surface expression of dectin-1 (po0.05), which further confirmed that dectin-1 surface expression was up-regulated by non-opsonized zymosan activation. Further studies showed that zymosan stimulated both C57BL/6 and TLR2(/) deficient BMMCs to generate intracellular oxidative burst. Pretreatment of BMMCs with laminarin inhibited ROS generation significantly (po0.05) after 2 h zymosan activation. Therefore, intracellular ROS generation in murine mast cells in response to zymosan is dependent on dectin-1 receptors. r 2008 Elsevier GmbH. All rights reserved. Keywords: Dectin-1; Innate immunity; Reactive oxygen species; Mast cells; Zymosan
Introduction Dectin-1, an important b-glucan surface receptor, is expressed on a broad range of innate immune cells such as monocytes/macrophages, neutrophils, NK cells, fibroblasts and dendritic cells (Ariizumi et al., 2000; Corresponding author. Tel.: +1 902 494 5118; fax: +1 902 494 5125. E-mail address: [email protected] (J.S. Marshall).
0171-2985/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2008.09.002
Kougias et al., 2001; Taylor et al., 2002; Williams, 1997). b-glucan is a major fungal cell wall component, and dectin-1 has been found to play an important role in the innate immunity against pathogenic fungi such as Coccidiodies posadasii, Pneumocystis carinii and Aspergillus fumigatus (Hohl et al., 2005; Steele et al., 2003; Viriyakosol et al., 2005). Recently, two different, functionally distinct dectin-1 isoforms were observed in murine macrophages. Either isoform can stimulate TNF production, but to different degrees (Heinsbroek et al.,
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2006). Dectin-1 is a type II transmembrane protein receptor with a C-type lectin-domain and a cytoplasmic tail with ITAM motif (Adachi et al., 2004) phosphorylation of which can stimulate pro-inflammatory mediators release and other cellular responses such as phagocytosis and respiratory burst in collaboration with TLR-2 (Brown et al., 2002; Gantner et al., 2003). However, recent studies showed that early inflammatory responses of mast cells to pathogens can often be attributed to complement receptors, not TLR-2 (Mullaly and Kubes, 2007). Several studies also showed that dectin-1 could interact with zymosan as well as yeast from Candida albicans, which stimulated phagocytosis and ROS generation in a TLR-independent manner (Rogers et al., 2005; Underhill et al., 2005). Little information is available concerning dectin-1 expression on murine mast cells and its role in regulating cellular responses to b-glucan. Zymosan derived from the cell wall of Saccharomyces cerevisiae is rich in both b-glucan and mannan (Di Carlo and Fiore, 1958). It has been widely used as a model fungal particle to study immune responses conducted by different innate and adaptive immunity cells. Zymosan not only interacts with leukocytes, such as macrophages via non-opsonic dectin-1 receptors to stimulate proinflammatory mediator or cytokine release (Brown et al., 2002; Heinsbroek et al., 2006), but also can interact with complement or TLR-2 receptors in mediating inflammatory responses, such as phagocytosis (Gantner et al., 2003; McCurdy et al., 2003; Mullaly and Kubes, 2007). Both mast cells and other leukocyte populations have been shown to have important roles in the response to zymosan in models of peritonitis (Kolaczkowska et al., 2001, 2007). Recently, we described that fungal zymosan can stimulate human mast cells to produce LTC4 in a dectin-1-dependent manner (Olynych et al., 2006). Mast cells are distributed widely throughout the body tissues, which are commonly found at submucosal surfaces of skin, airway and the intestine. Mast cells function as sentinel cells in host defense, including enhancement of both innate and specific defenses, against pathogens. Mast cells are well known to release a large amount of pro-inflammatory and inflammatory mediators, which contribute to both immediate allergic reactions and inflammation (Marshall, 2004). They are also capable of phagocytosis of invaded pathogens, which plays an important role in innate immunity (Abraham and Malaviya, 1997). Following phagocytosis, the production of reactive oxygen species (ROS) is critical for pathogen killing (Forman and Torres, 2001; Henricks and Nijkamp, 2001; Malaviya et al., 1999, 1996). There are many substances such as IgE/Ag, calcium ionophore A23187, gold compound, D-penicillamine, compound 48/80, nerve growth factor and others (Brooks et al., 1999; Niu et al., 1996; Wolfreys
and Oliveira, 1997) that can stimulate mast cells to generate intracellullar ROS. ROS generation has also been demonstrated to be frequently accompanied by degranulation (Swindle et al., 2002, 2004). Inhibition of ROS generation leads to decreased release of mediators such as histamine and LTC4 in RBL-2H3 mast cells (Suzuki et al., 2003). Several studies showed ROS such as potassium superoxide or hydrogen peroxide could induce mast cells to degranulate (Akagi et al., 1994; Peden et al., 1994). In macrophage cell lines, dectin-1 mediates zymosan-induced ROS generation (Gantner et al., 2003). It is not yet known whether zymosan can induce intracellular ROS generation in mast cells after phagocytosis and the role of dectin-1 in ROS generation has not been determined in mast cells. In this study, we examined murine mast cell expression of dectin-1 and intracellular ROS generation in response to zymosan as a model of fungal-induced ROS generation. Even though the level of dectin-1 expression on murine mast cells was far lower than that on the cell surface of macrophages, it was demonstrated that zymosan-induced intracellular ROS generation was dectin-1 dependent in mast cells. Furthermore, dectin1 expression can be up-regulated by zymosan activation alone. To our knowledge, this is the first study to show the relationship between dectin-1 and ROS production in mast cells.
Methods Reagents Yeast zymosan from S. cerevisiae and laminarin were purchased from Sigma (St. Louis, MO). Dichlorodihydrofuorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Eugene, OR). Zymosan suspensions were made in endotoxin free saline and vortexed immediately before use to ensure particles were equally distributed in suspension. Laminarin was also dissolved in endotoxin free saline and sonicated immediately prior to use. The primary Abs used in this study were 2A11, monoclonal rat anti-mouse dectin-1 receptors conjugated with biotin (ABD Serotec, Raleigh, NC). The secondary Abs were Streptavidin-PE obtained from BD pharmingen (San Diego, CA). IgG2b-biotin (eBioscience, San Diego, CA) was used as an isotype control.
Mice C57BL/6 (Jackson Labs, Bar Harbor, Maine) mice and TLR2-deficient mice on a C57BL/6 background (a gift from Dr. S. Akira, Osaka, Japan) were used in this study. All mice were 6–10 weeks of age and housed
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under specific pathogen-free conditions with food and water provided ad libitum. All experiments were approved by the animal research ethics board of Dalhousie University.
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modifications: (1) 50 -CAAGTGCTCTGCCTACCTAG; (2) CACCA TCTTTATATTCTCACATAC. These primers amplified A and B isoforms of mouse dectin-1. PCR products were detected by electrophoresis on a 1.5% agarose gel and ethidium bromide staining.
Cell culture Bone marrow-derived cultured mast cells (BMMC) were obtained from 4- to 8-week-old C57BL/6 and TLR2-deficient mice according to a previously published method (Tertian et al., 1981). BMMC cells were incubated in RPMI-1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin and streptomycin (Life Technologies, Grand Island, NY), 5 105 M 2-mercaptoethanol (Sigma) and 10% WEHI3-B supernatant as a source of IL-3. Cells were grown at 37 1C, 5% CO2 for 5–8 weeks, at which time the purity of mast cells was determined morphologically and by the presence of metachromatic granules following toluidine blue staining (pH 1.0). Only 495% pure mast cells preparation were used.
Cell activation BMMC were washed and resuspended at a final concentration of 1 106 cells/ml in RPMI containing 1% bovine serum albumin (Sigma), 3 ng/ml IL-3 (PeproTech Inc., Rocky Hill, NJ) and 100 mg/ml of soybean trypsin inhibitor (Sigma) for activation. The cells were incubated with media alone or 100 mg/ml zymosan for 3 and 6 h, respectively, with the addition of mouse plasma or heated (56 1C, 30 min) mouse plasma (2% v/v) at 37 1C, 5% CO2. Cells were harvested by centrifugation for RT-PCR assays. The dose of zymosan was chosen based on preliminary dose response experiments (data not shown), which indicated that this dose gave a strong response but the viability of treated cells remained over 90%, as well as on the results of previous studies examining human mast cell responses to zymosan suspensions (Olynych et al., 2006)
RT-PCR Using RNA isolation kit (Qiagen, Mississauga, ON), RNA was isolated from each cell sample (2 106) with different stimulation as previously described and diluted to the same concentration based on the optical density. RNA integrity was assessed by examining ribosomal RNA bands, following gel electrophoresis. cDNA was then obtained using RT-PCR kit (Invitrogen, Carlsbad, CA) with Oligo(dT) primers according to manufacturer’s recommended methodology. Primers used for PCR amplification of reverse-transcribed RNA samples for mouse dectin-1 were manufactured according to the previous studies (Heinsbroek et al., 2006) with some
FACS analysis C57BL/6 BMMCs were activated by zymosan as described above. In the inhibition experiment, 50 mg/ml laminarin was added at the same time as zymosan to treat cells for 3 h at 37 1C, 5% CO2. 2.5 105 cells, following each different treatment, were added to wells of a 96-well tissue culture plate (Life Technologies). Cells were washed twice with phosphate buffered saline (PBS)–bovine serum albumin (BSA) (0.1%) buffer and incubated in a Fc receptor blocking buffer (human IgG in PBS–BSA) at 4 1C for 15 min. Cells were then incubated with primary Ab, mAb211-biotin (Brown et al., 2002), for 30 min at 4 1C, in separate tubes biotinylated IgG2b, with the same concentration, was used as an isotype control. After washing three times with PBS–BSA, cells were incubated in PBS–BSA buffer with secondary Ab, Streptavidin-PE at 4 1C for 30 min. Cells were fixed in 1% formaldehyde after washing three times, as described before. Finally cells were analyzed on a FACS Calibur flow cytometer using CellQuest software.
Measurement of intracellular ROS production by flow cytometry Mast cells (5 105) suspended in PBS (Life Technologies) were incubated with 5 mM DCFH-DA for 15 min at 37 1C. Then cells were washed twice or three times and resuspended in PBS at 4 1C, which were followed with activation by 100 mg/ml zymosan at 37 1C and 5% CO2 for different time periods. In inhibition experiments, cells were pre-incubated with 50 mg/ml laminarin on ice for 30 min before zymosan stimulation. Cells stimulated with 50 ng/ml PMA for 30 min at 37 1C and 5% CO2 were used as a positive control to check for authentic intracellular ROS generation. Intracellular ROS generation was analyzed on a FACS Calibur flow cytometer using CellQuest software.
Statistics Differences in fluorescence level between anti-dectin-1 and control antibody stained cells and fluorescent staining of cells subjected to different treatments were evaluated using the method of Komogorov–Smivov. Only p values of o0.05 were considered significant.
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Results
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Expression of dectin-1 mRNA in murine mast cells Using RT-PCR analysis, we examined the expression of dectin-1 mRNA in C57BL/6 and TLR2(/) BMMC mast cells. Without the addition of mouse plasma or heated plasma, BMMCs, either at rest or following activation with zymosan for 3 and 6 h, respectively, consistently showed no evidence of dectin-1 isoform expression. Following addition of mouse plasma and zymosan activation for 3 h, expression of two dectin-1 isoforms was consistently detected in C57BL/6 BMMC. Weaker expression of Dectin-1 mRNA was consistently observed in BMMC cells with the addition of heated plasma after zymosan stimulation within the same time period (Fig. 1a). TLR2/ BMMC treated with zymosan and plasma combined for 3 h showed lower expression of dectin-1 mRNA (Fig. 1b) compared with TLR2 expressing mast cells in each of the three separate experiments (Fig. 1a). After 6 h of zymosan stimulation in the presence of plasma, mast cells from both sources showed decreased dectin-1 mRNA expression compared with the earlier 3 h time point. RNA from splenic cells was used as a positive control (Fig. 1c). Results representative of 3–4 experiments/group.
Expression of dectin-1 receptors on cell surface We examined the surface expression of dectin-1 receptors on C57BL/6 BMMCs using a dectin-1 specific antibody (mAb211). After 3 h incubation in media alone, a very low expression of dectin-1 receptor was detected on the cell surface. Cells stimulated by zymosan for 3 h in the absence of mouse plasma consistently exhibited significantly higher surface expression of dectin-1 receptors by comparing the mean fluorescence with that of untreated negative control cells (po0.05). However, the addition of mouse plasma or heated plasma alone had no further inducing effect on dectin 1 expression (Fig. 2a). With or without the presence of plasma or heated plasma, C57BL/6 BMMCs, following zymosan activation for 6 h, did not show evidence of increased dectin-1 surface expression compared with
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untreated cells and treatment with heated plasma alone appeared to further reduce the low levels of dectin-1 expression observed in control cells (Fig. 2b). Pretreatment of 3 h, zymosan-activated C57BL/6 BMMCs with 50 mg/ml laminarin reduced the observed increase in dectin-1 surface expression (Fig. 2c), while the same laminarin treatment had no effect on dectin-1 expression
Fig. 2. Expression of dectin-1 receptors on C57BL/6 BMMCs. C57BL/6 BMMC cells expressed very low level of dectin-1 receptors on membrane surface with or without the addition of plasma or heated plasma. Activation by zymosan for 3 h consistently increased dectin-1 receptor expression, which was not enhanced by the addition of mouse plasma (a). Activation for 6 h by zymosan did not lead to a sustained increase in expression (b). Laminarin pre-treatment partially inhibited the induced increase in dectin-1 expression on the surface of C57BL/6 BMMC (c). Comparing mean fluorescence from three independent experiments further confirmed that laminarin pre-treatment significantly inhibited dectin-1 expression (d). Resident peritoneal macrophages which had been cultured for overnight were used as a positive control for surface dectin-1 receptor assessment (e). Shaded histograms represented IgG2b isotype control and unshaded histograms represented surface staining with mab2A11. P: plasma, HP: heated plasma. Representative of three independent experiments.
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control to assess surface expression of dectin-1 receptors (Fig. 2e).
Generation of intracellular ROS in zymosanactivated BMMCs Intracellular ROS generation in BMMCs was measured by loading reduced fluorescent indicator, DCFH-DA, which can be rapidly oxidized by H2O2 (Bass et al., 1983). DCFH-DA-loaded C57BL/6 BMMCs were stimulated with zymosan (100 mg/ml) and intracellular ROS production was measured at one hour intervals for 3 h. Intracellular ROS was initially generated at 1 h and gradually increased in extent. At 3 h, intracellular ROS production terminated (Fig. 3a). Fifty nanogram per milliliter PMA treatment for 30 min was used as a positive control to confirm the reliability of ROS assay system (Fig. 3b). TLR2/ BMMCs activated by zymosan for 1 and 2 h, respectively, (Fig. 4a) also produced intracellular ROS. Comparing ROS levels between C57BL/6 and TLR2/ BMMCs at the same incubation time (Fig. 4b), C57BL/6 BMMCs produced significantly more ROS than that of TLR2/ after 2 h zymosan stimulation (po0.05). However, there was no significant difference in ROS generation between these two cell types activated by zymosan for 1 h.
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Dectin-1-mediated intracellular ROS generation Laminarin, a well-known dectin-1 inhibitor (Brown et al., 2002; Gantner et al., 2003), was used to investigate the potential role of dectin-1 receptors in mediating intracellular ROS generation in this study. Pretreatment of C57BL/6 BMMCs with 50 mg/ml laminarin in PBS (pH 7.4) for 30 min at 4 1C inhibited intracellular ROS generation following 2 h zymosan activation (Fig. 5a). Laminarin pretreatment significantly inhibited ROS generation (Fig. 5b, po0.05). An alternate analysis (Fig. 5c), which compares the difference between median RFU (percentage of negative control) of zymosan-activated cells with and without laminarin pretreatment, also shows significant differences between these two treatment groups (po0.05).
Discussion Dectin-1 is a c-type lectin, which can interact with zymosan, the major yeast cell wall component, as well as other fungal pathogens, such as Candida albicans, Coccidiodies posadasii, Pneumocystis carinii and Aspergillus fumigatus (Hohl et al., 2005; Rogers et al., 2005; Steele et al., 2003; Underhill et al., 2005; Viriyakosol et al., 2005). Dectin-1 receptors have been found to be expressed on monocyte/macrophage
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Fig. 3. Zymosan-activated intracellular ROS generation in murine mast cells. DCFH-DA-loaded C57BL/6 BMMCs were stimulated with zymosan (100 mg/ml) and intracellular ROS production was measured at hourly intervals for 3 h. Shaded area represents control cells, open area represents activated cells (a). Intracellular ROS generation by PMA (50 ng/ml) activation for 30 min was used as a positive control (b). Representative of 4–5 independent experiment.
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Fig. 4. Comparison of zymosan-induced intracellular ROS generation in the presence and absence of TLR2. DCFH-DA-loaded TLR2/ BMMCs were stimulated with zymosan (100 mg/ml) in PBS and intracellular ROS generation was measured for 1 and 2 h, respectively, (a). Shaded histograms represented ROS generation from TLR2/ BMMCs without any treatment. Intracellular ROS generation by PMA (50 ng/ml) activation for half an hour was used as a positive control. Intracellular ROS generation was compared between C5BL6/7 and TLR2/ BMMCs at 1 and 2 h, respectively, (b). Representative of 4–5 independent experiment. Denotes po0.05.
lineage, neutrophils, NK cells, fibroblasts and dendritic cells (Ariizumi et al., 2000; Kougias et al., 2001; Taylor et al., 2002; Williams, 1997). In our previous study, human mast cells were found to express functional dectin-1 receptors on the cell surface, which respond to zymosan, and participate in leukotriene generation (Olynych et al., 2006). In this study, we demonstrated that dectin-1 receptors were expressed on murine mast cell surface at a very low level and their mRNA expression could be increased by zymosan activation. The addition of mouse plasma or heated plasma alone did not induce dectin-1 protein expression as analyzed by FACs. Plasma treatment along with zymosan did, however, stimulate murine BMMC to express two dectin-1 mRNA isoforms, an effect that was lost by heating plasma at 56 1C for 30 min to inactivate the
complement cascade. These results suggest that the mast cell mRNA expression of dectin-1 regulated by zymosan activation of complement. However, surface protein expression data from FACS analysis consistently indicated (in four separate experiments) that cell surface expression of dectin-1 could also be enhanced by zymosan treatment alone at 3 h and was rapidly down regulated at the later (6 h) time point. Laminarin, a specific inhibitor for b-glucan receptors does not block the activity of the mannose receptor or the CR3 receptor (Brown et al., 2002). Therefore, it has been used in several studies to confirm the role of dectin1 receptors in response to zymosan or PGN activation (Brown et al., 2003; Gantner et al., 2003; Olynych et al., 2006). Adding laminarin at the same time as zymosan to mast cell suspensions can reduce zymosan-induced
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Fig. 5. Laminarin-inhibited zymosan-induced intracellular ROS generation in C57BL/6 BMMCs. Pre-treatment of C57BL/6 BMMCs with laminarin (50 mg/ml) for 30 min at 4 1C inhibited intracellular ROS generation following zymosan activation for 2 h. Representative of 4 independent experiment. Denotes po0.05.
dectin-1 expression substantially, which confirms the role of zymosan in enhancing the dectin-1 expression on murine mast cell surface. ROS generation in mast cells may play a critical role in innate immune reactions (Forman and Torres, 2001; Henricks and Nijkamp, 2001; Malaviya et al., 1999; Malaviya et al., 1996). However, its cellular pathway is still unclear. Mast cells can be stimulated by a range of mechanisms such as IgE/Ag, ionophore, etc to generate ROS (Brooks et al., 1999; Niu et al., 1996; Tsinkalovsky
and Laerum, 1994; Wolfreys and Oliveira, 1997). ROS generated by some mechanisms, such as IgE/Ag stimulation, was accompanied by mast cell degranulation, while no degranulation was detected by other mechanisms (Brooks et al., 1999; Tsinkalovsky and Laerum, 1994). ROS production in both IgE-sensitized mast cells after antigen challenge and PMA-stimulated mast cells is probably attributed to NAD(P)H oxidase (Suzuki et al., 2003; Swindle et al., 2002). Comparing ROS generation in macrophages with that in mast cells
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suggests much lower level of intracellular ROS generation by mast cells (Swindle et al., 2002). There has been some controversy over ROS generated by PMA-stimulated mast cells. One study (Swindle et al., 2002) suggested a small amount of ROS generation by PMA stimulation in rat peritoneal mast cells. Significant intracellular ROS was generated in human mast cells via PMA stimulation and its production reached a peak at 30 min (Kim and Ro, 2005), which is in full agreement with our data using similar stimulation in BMMC. DCFH-DA oxidation into DCF reflects the level of intracellular H2O2 generation with high sensitivity and was also used in our study to detect intracellular ROS generation upon zymosan stimulation. The mast cells we used were all non-adherent, of extremely high purity and not contaminated by detectable numbers of macrophages. Our data revealed significant intracellular ROS generation in zymosanstimulated BMMCs at 2 h. Furthermore, comparing intracellular ROS production from zymosan-activated C56BL/7 BMMCs for 2 h with and without pretreatment of laminarin, shows that laminarin pretreatment significantly inhibited ROS generation (po0.05). These results demonstrate that dectin-1 receptors on BMMCs surface-mediated ROS generation. Zymosan-derived intracellular ROS generation in this study was not accompanied by degranulation (data not shown). Zymosan also does not induce significant degranulation of human mast cells (McCurdy et al., 2003). In a previous study, low level of dectin-1 receptors on macrophage surface were also demonstrated to mediate zymosan-derived ROS generation without being accompanied by degranulation (Gantner et al., 2003). However, faster kinetics of ROS production might be exhibited in macrophages than that in mast cells. Pattern-recognition receptors, such as TLRs and lectin-like receptors are critical in the early detection of pathogens and initiating innate immune responses against them. There have been several studies attempting to clarify the relationship between TLR2 and dectin1 (lectin type) receptors in immune responses. Both receptors can recognize microbial components such as zymosan and stimulate inflammatory responses in either a co-operative or independent manner. Dectin-1 receptor expression on macrophages and dendritic cells is able to upgrade TLR-mediated cytokine release in response to zymosan. Also dectin-1 receptors act synergistically with TLR2 receptors in phagocytosis as well as ROS generation. However, dectin-1 expressed by macrophage lineage was shown to mediate phagocytosis of zymosan and subsequent ROS generation in a TLR-independent manner. In this study, both C57BL/6 and TLR2/ BMMCs were able to produce intracellular ROS in response to zymosan activation. After 2 h zymosan stimulation, ROS level of C57BL/6 BMMCs was significantly higher than that of TLR2/ BMMCs
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(po0.05). Therefore, from these data analysis it may be concluded that TLR2 receptors co-operate with dectin-1 receptors in zymosan-derived intracellular ROS generation from murine mast cells. In summary, we have demonstrated that low levels of surface expression of dectin-1 receptors on murine mast cells can be up-regulated by zymosan activation. Such dectin-1 receptors mediate zymosan-derived intracellular ROS generation by a mechanism that is also partially dependent on TLR2 function. The analysis of surface expression of dectin-1 receptors and ROS generation in response to zymosan has provided novel insights into the recognition of b-glucan by murine mast cells and into the cellular responses mediated by dectin-1.
Acknowledgments The authors would like to thank Ms. Yi-Song Wei, Dr. Wojciech Dawicki, Dr. Dunia Jawdat, Dr. Ian Haidl and Dr. Nong Xu for their advice and assistance on aspects of these studies. This work was supported by the Canadian Institutes of Health Research.
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