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(Journal of Leukocyte Biology. 2001;70:977-984.)
© 2001 by Society for Leukocyte Biology

Toll-like receptor 4-mediated activation of murine mast cells

J. D. McCurdy*, T.-J. Lin{dagger} and Jean S. Marshall*,{ddagger}

Departments of
* Microbiology and Immunology,
{dagger} Pediatrics, and
{ddagger} Pathology, Dalhousie University, Halifax, Nova Scotia, Canada

Correspondence: Jean S. Marshall, Ph.D., Dept. of Pathology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. E-mail Jean.Marshall{at}Dal.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toll-like receptors (TLRs) are a family of pattern recognition receptors that are critical for cellular responses to a variety of bacterial, viral, and fungal products. Mast cells are important to host survival in a number of models of bacterial infection and might act as sentinel cells in host defense. We therefore examined the expression of TLRs and associated molecules by murine bone marrow-derived mast cells (BMMCs). BMMCs and the murine mast cell line MC/9 expressed mRNA for TLR2, TLR4, and TLR6 but not TLR5 and for both adapter molecule MD-2 and signaling molecule MyD88 but lacked surface CD14. After activation with the TLR2- and TLR4-dependent stimuli Staphylococcus aureus-derived peptidoglycan and Escherichia coli-derived lipopolysaccharide (LPS), respectively, mast cells produced significant levels of interleukin-6 (IL-6) and tumor necrosis factor {alpha} (TNF-{alpha}). To determine whether mast cells require TLR4 for cellular responses to LPS, mast cells were derived from the bone marrow cells of C3H/HeJ and C57Bl/10ScNCr mice containing a point mutation and a null mutation, respectively, in TLR4. Using these models, we demonstrated that the BMMC IL-6 and TNF-{alpha} responses to LPS were completely dependent on functional TLR4 with no significant LPS response observed in its absence. These findings have important implications for the mechanism of mast cell responses to pathogens and their products and suggest that different TLR4-expressing cells might have different thresholds for activation with LPS.

Key Words: LPS • bacteria • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of lines of evidence suggest that mast cells play a protective role in host defense against bacteria. Mast cells are found in high numbers in vascularized tissues at the host-environment interface [1 , 2 ]. This strategic location facilitates their exposure to invading organisms. By virtue of their ability to synthesize large numbers of inflammatory mediators, mast cells can generate potent inflammatory reactions [3 4 5 ], and numerous accounts have shown that mast cells are responsive to a large number of bacteria and bacterial products [6 7 8 9 ]. Studies using mast cell-deficient mice have provided direct evidence for a protective role of mast cells in host defense against bacterial infections. Echtenacher et al. [10 ] demonstrated, in a model of acute septic peritonitis, that mast cell-deficient (KitW/KitW-v) mice display significantly increased mortality compared with wild-type mice. Correction of this mast cell deficiency restores survival to nearly the level observed in wild-type mice [10 ]. Similarly, enhanced mortality in KitW/KitW-v mice after intraperitoneal instillation of enterobacteria could be prevented by correction of the mast cell deficiency [11 ]. These studies clearly demonstrate that mast cells are required for host defense in some murine models of bacterial infection; however, the mechanisms utilized remain largely undefined.

Both gram-positive and gram-negative bacteria elicit a number of proinflammatory responses in mammalian cells. Many of these responses are induced by cell wall components, including peptidoglycan (PGN) and lipopolysaccharide (LPS) from gram-positive and gram-negative bacteria, respectively. Production of proinflammatory cytokines such as tumor necrosis factor (TNF) {alpha}, interleukin (IL)-6, and IL-1ß play an indirect role in eliminating bacterial infection. However, excessive cytokine production might also lead to endotoxic shock. Determining the molecular mechanism of LPS recognition by responsive cells has been a priority in the development of strategies to artificially modulate the LPS response.

In most cells of the myeloid lineage, including the macrophage, LPS is captured by a serum protein, LPS-binding protein (LBP) and subsequently is transferred to CD14, a glycosylphosphatidylinositol-anchored membrane protein [12 ]. Cells that lack membrane expression of CD14, including endothelial and epithelial cells, utilize soluble CD14 (sCD14) for efficient activation [13 ]. Despite the known function of CD14, this receptor lacks an intracellular signaling domain. Therefore, additional molecules with intracellular-signaling capacity are required. Members of the Toll-like receptor (TLR) superfamily have been demonstrated to serve this function.

Among the rapidly expanding family of TLRs [14 15 16 17 18 19 ], TLR4 has been most closely linked with LPS signaling. Initially, a number of reports suggested that TLR2 was the signaling receptor for LPS in macrophages [20 , 21 ]. However, protein contamination in commercial preparations of LPS might be responsible for such signals through TLR2 [22 ]. Convincing evidence provided by Poltorak et al. [23 ], Hoshino et al. [24 ], and Qureshi et al. [25 ] demonstrated that mutations in the Lps gene, found in hyporesponsive murine strains including C3H/HeJ and C57Bl/10ScCr, mapped to TLR4. Sequence analysis of the TLR4 gene in C3H/HeJ and C57Bl/10ScCr revealed a single point mutation at amino acid 712 and a null mutation, respectively [23 ]. Later, Takeuchi and colleagues [24 ] used TLR2 and TLR4 knockout mice to confirm that TLR4 but not TLR2 is the major receptor for LPS in vivo.

Although LPS preparations from most species of gram-negative bacteria appear to activate cells via TLR4, recent studies [26 , 27 ] determined that LPS from Leptospira interrogans and Prophyromonas gingivalis, respectively, signal through TLR2 suggesting that LPS from different species of bacteria may be recognized by distinct TLR molecules. However, synthetic P. gingivalis-type as well as Escherichia coli lipid A did not activate peritoneal macrophages from C3H/HeJ mice in a further report [28 ]. Even though LPS responses mediated by TLR2 have been controversial, it is well accepted that products from gram-positive bacteria such as PGN signal via TLR2. In addition to the number of in vitro studies [29 ], studies using TLR2-deficient mice have confirmed that TLR2 is important for survival and cytokine responsiveness in vivo [30 ]. The role of mast cells in the in vivo response to LPS and other TLR ligands is not known.

TLRs and members of the IL-1 receptor family share a highly homologues intracellular domain which has been designated the Toll/IL-1R-like region (TIR). The TIR domain is important for recruitment of the intracellular adapter molecule myeloid differentiation factor 88 (MyD88). Association of these receptors with MyD88 occurs through a homotypic interaction that is dependent on a homologous TIR domain in the C terminus of MyD88 [31 , 32 ]. MyD88 is critical for IL-1 receptor signaling [33 ] and many TLR-mediated responses [34 , 35 ]. Studies conducted by Shimazu et al. [36 ] identified that human TLR4 alone is not capable of sensing the presence of LPS. An additional molecule, MD-2, forms a complex with the extracellular domain of TLR4 for effective LPS recognition. Although TLR2 signaling is not dependent on MD-2, responses to a number of bacterial products including PGN are significantly enhanced by the expression of MD-2 [37 ].

We have previously shown that LPS activates mast cells to produce IL-6 and TNF-{alpha} without degranulation [8 , 38 ]. However, efficient stimulation of such mast cells requires much higher doses of LPS than classical LPS-responsive cells such as monocytes and macrophages [39 ]. The ability of TLRs to mediate activation of mast cells has not been previously determined; we therefore examined both the expression of TLRs and associated molecules and the ability of LPS to utilize TLR4 to activate mast cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
E. coli LPS (serotype O55:B5), Pseudomonas aeruginosa LPS (serotype 10), and PGN purified from Staphylococcus aureus were from Sigma, St. Louis, MO. S. aureus PGN contained < 0.0025 ng/µL of endotoxin as assessed using the Limulus amoebocyte lysate test from Sigma. Purified rat anti-mouse CD14 monoclonal antibody [mAb; clone rmC5-3, immunoglobulin (Ig) G1{kappa}], purified rat IgG1{kappa} (R3-34) isotype control antibody, and goat anti-rat Ig-fluorescein isothiocyanate (FITC) polyclonal antibody were all purchased from PharMingen (San Diego, CA).

Mice
C57BL/6, C3H/HeJ, C3H/HeOuJ, and C57BL/10ScSn mouse strains (The Jackson Laboratory, Bar Harbor, MA) and C57BL/10ScNCr (National Cancer Institute, Bethesda, MD) were housed in sterilized, filter-hooded cages and provided food and water ad libitum. All experiments were approved by the animal research ethics boards of Dalhousie University.

Mast cells
MC/9 cells (ATCC CRL 8306) were routinely grown in modified Dulbecco’s modified Eagle’s medium (Life Technologies, Burlington, ON, Canada) containing 36 mg/mL of L-aspartate, 0.1 mM nonessential amino acids, 5 x 10-5 M 2-mercaptoethanol (ME), 10% fetal calf serum (FCS), and 3 ng/mL of recombinant mouse (rm) IL-3 (Pepro Tech, Inc., Rocky Hill, NJ) in 10% CO2. Bone marrow-derived mast cells (BMMCs) were generated from the bone marrow of C57BL/6, C3H/HeJ, C3H/HeOuJ, C57BL/10ScSn, and C57Bl/10ScNCr mice according to the method of Tertian et al. [40 ]. Briefly, mice were sacrificed, and intact femurs and tibias were removed. Sterile endotoxin-free medium was repeatedly flushed through the bone shaft using a needle and syringe, and the bone marrow cells were passed through a sterile wire screen to remove any bone fragments. The cell suspension was centrifuged at 320 g for 20 min at 4°C and cultured at a concentration of 0.5–1 x 106 nucleated cells/mL in RPMI 1640 (Life Technologies) supplemented with 10% FCS (Sigma-Aldrich, Mississuaga, ON, Canada), 10% (v/v) concentrated WEHI-3 cell-conditioned medium as a source of IL-3, 1% penicillin/streptomycin (Life Technologies), 10-7 M prostaglandin E2 (PGE2), and 50 µM 2-ME (BMMC medium). The medium was replaced three times a week with fresh BMMC medium. BMMCs were monitored for purity after 4 weeks by Alcian blue (pH 0.3) staining of fixed cytocentrifuge preparations. Once the BMMC population reached a purity of >98% (5–8 weeks), they were used in subsequent experiments.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from MC/9 cells, BMMCs, and the murine macrophage-like cell line J774 using Trizol reagent (Life Technologies), according to the manufacturer’s instructions. To remove possible genomic-DNA contamination, RNA samples were treated with deoxyribonuclease I (DNase I) (Life Technologies). Briefly, 1 gmg of RNA was incubated with 1 µg of DNase I, 1 µL of DNase reaction buffer, and 7 µL of H2O for 15 min at room temperature, after which DNase I activity was inactivated by the addition of 2 mM EDTA and heating between 60 and 65°C for 20 min. Reverse transcription of the RNA samples was achieved using Moloney murine leukemia virus transcriptase (Life Technologies) and oligodeoxythymidylic acid primers as random templates. Primers used for PCR amplification of MD-2, MyD88, GAPDH, TLR2, TLR4, TLR5, and TLR6 were purchased from Research Genetics, Inc. (Huntsville, AL), and sequences were as follows: (1) mouse MD-2: sense, 5'–GAG AAG CAA CAG TGG TTC TG-3'; antisense, 5'-CTC CTT TCA GAG CTC TGC AA-3'; (2) MyD88: sense, 5'–CGA GTT TGT GCA GGA GAT GA-3'; antisense, 5'-GGA TAC TGG GAA AGT CC T TC-3'; (3) GAPDH: sense, 5'–ACT CAC GGC AAA TTC AAC GGC-3'; antisense, 5'-ATC ACA AAC ATG GGG GCA TCG-3'; (4) TLR2: sense, 5'–GCC TTG ACC TGT CTT TCA AC-3'; antisense, 5'-GGA CTG ATA ATT CCG GAG AC-3'; (5) mouse TLR4: sense, 5'-CAG CTT CAA TGG TGC CAT CA-3'; antisense, 5'-CTG CAA TCA AGA GTG CTG AG-3'; (6) TLR5: sense, 5'–AGT GCT CAG TGC CTG TAC TA-3'; antisense, 5'-GCA TGT GCTAGG TTC TAG GT-3'; and (7) TLR6: sense, 5'–GGC ATC TAG ACC TCT CAT TC-3'; antisense, 5'-ATG GAT AAC GGT GGT ATT GG-3'. The PCR products for murine MD-2, MyD88, GAPDH, TLR2, TLR4, TLR5, and TLR6 were 277, 287, 245, 407, 438, 305, and 230 bp, respectively. PCR was performed in a 50-µL reaction mixture composed of 1 µM (final concentration) of each forward and reverse oligonucleotide primer, 50 mM MgCl2, 5 mM of the four deoxynucleotide triphosphates, 5 µL of cDNA preparation, and 0.02 U/µL (final concentration) of Taq DNA polymerase. Thirty-four cycles were used (94°C for 1 min, 1 min at 56°C, and 2 min at 72°C) followed by 7 min at 72°C. Products were run out on a 1.8% agarose gel containing ethidium bromide. Gel images were captured and visualized using a Gel Doc image analysis system (Bio-Rad, Hercules, CA).

Flow-cytometric analysis
In 96-well U-bottom plates (Nunc, Burlington, ON, Canada), cells (5x105/well) were incubated with purified rat anti-mouse CD14 mAb (clone rmC5-3, IgG1 {kappa}; PharMingen) or purified rat IgG1{kappa} (R3-34) isotype control antibody (PharMingen) in immunofluorescence (IF) buffer [phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.2% sodium azide] for 1 h at 4°C. After washing, cells were further incubated for 1 h with goat anti-rat Ig-FITC polyclonal antibody (PharMingen). Cells were washed three times (with IF buffer) and resuspended in 400 mL of 1% paraformaldehyde (in PBS), and 10,000 cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The results obtained with specific antibodies were compared with those using isotype-matched-control antibodies in parallel.

Mast cell activation
Mast cell activation was conducted using an endotoxin-free system. Prior to BMMC activation, cells were washed several times in PGE2-free BMMC medium and incubated for 16–20 h. BMMCs cultured from the various strains (specified in Results) of mouse MC/9 cells were washed several times in experimental medium. The experimental medium for MC/9 cells consisted of culture medium with the addition of 100 µg/mL of soybean trypsin inhibitor (Sigma-Aldrich, reconstituted in saline) whereas the experimental medium for BMMCs consisted of RPMI 1640 (Life Technologies) supplemented with 10% FCS (Sigma-Aldrich), 1% penicillin/streptomycin (Life Technologies), 50 µM 2-ME (BMMC medium), 1.5 ng/mL of recombinant IL-3 (rIL-3), and 100 µg/mL of soybean trypsin inhibitor (Sigma). In some experiments, cells were activated in the absence of FCS. For these experiments, BMMCs were washed four times in serum-free medium (AIM V medium; Sigma) supplemented with 1% penicillin/streptomycin (Life Technologies), 50 µM 2-ME (BMMC medium), 1.5 ng/mL of rIL-3, and 100 µg/mL of soybean trypsin inhibitor (Sigma-Aldrich; reconstituted in saline). For control medium, 10% FCS was added to this medium. Cells were incubated at 106/mL for 6 and 24 h at 37°C with the following reagents: E. coli LPS (serotype O55:B5), P. aeruginosa LPS (serotype 10), and calcium ionophore (A23187). Samples were stored at -20°C until assayed.

Cytokine enzyme-linked immunosorbent assays (ELISAs)
Murine IL-6 and TNF-{alpha} were measured by "in-house" sandwich ELISAs. For IL-6, maxisorp ELISA plates (Nunc/Inter Med) were coated for 18–24 h at 4°C with 100 µL/well of 2-µg/mL anti-mouse IL-6 capture antibody (Endogen, Cambridge, MA) diluted in 0.1 M bicarbonate buffer (0.1 M NaHCO3, 0.5 M NaCl, dH2O). The wells were washed three times and incubated for 1 h with 200 µL/well of blocking solution (1% BSA in PBS). The blocking solution was decanted, and the wells were washed four times with PBS containing 0.05% Tween 20. Standards (Genzyme, Cambridge, MA) and samples were added to the plate at 50 µL/well and incubated at 4°C overnight. The wells were washed as described above, and secondary biotinylated anti-mouse IL-6 antibody (Endogen) at 0.5 µg/mL in assay solution (0.3% BSA, 0.05% Tween 20 in PBS) was added at 50 µL/well. After 2 h, the wells were washed, and 50 µL/well of streptavidin-alkaline phosphatase (Life Technologies) prepared in blocking solution were added to the plates for an hour. The wells were washed, and bound secondary antibody was detected with the Gibco ELISA amplification system (Life Technologies). The colored product was read at 492 nm. Sensitivity of the IL-6 ELISA was 300 pg/mL.

TNF-{alpha} was measured using a similar protocol. All incubations were carried out at room temperature. Plates were coated with TNF-{alpha}-coating antibody and polyclonal goat anti-murine TNF-{alpha} antibody (R&D Systems, Minneapolis, MN) as mentioned above. Plates were blocked for 2 h followed by the administration of samples and standards (source). Biotinylated secondary antibody, anti-murine TNF-{alpha} ( MM35000-B; Endogen) was added next for 2 h. Subsequent steps were the same as for the IL-6 ELISA protocol. The sensitivity of this TNF-{alpha} ELISA was 30 pg/mL unless otherwise indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytokine production by murine mast cells was induced by high doses of LPS and by S. aureus-derived PGN
BMMCs were cultured from C57BL/6 mice (>98% purity) and used as a murine mast cell model. In light of the possible contribution of non-mast cell contaminants, the IL-3-dependent murine mast cell line MC/9 was also used. Cells were stimulated with E. coli-derived LPS at concentrations ranging from 0.05 to 5 µg/mL. After 24 h, cell-free supernatants were harvested and analyzed for IL-6 production by ELISA. Activation of BMMCs and MC/9 cells with 0.5 and 5 µg/mL but not 0.05 µg/mL of E. coli LPS resulted in significantly higher levels of IL-6 compared with unstimulated cells (Fig. 1 A and B). Levels of IL-6 were greatest after stimulation with 5 µg/mL of LPS. Using similar activation conditions, C57Bl/6 BMMCs were stimulated with the known TLR2-activator, PGN purified from S. aureus. After 24 h, PGN treatment induced significantly higher levels of IL-6 compared with unstimulated cells (Fig. 1C) . Similarly, C57BL/6 BMMCs stimulated with 100 µg/mL of PGN for 6 h produced levels of TNF-{alpha} that were more than eightfold higher than unstimulated cells (mean values of 42±26 pg/mL and <5 pg/mL of TNF-{alpha} for PGN-stimulated and -unstimulated cells, respectively, representative of two independent experiments). To ensure that the IL-6 and TNF-{alpha} responses of BMMCs to PGN stimulation were not the result of LPS contamination, BMMCs cultured from TLR4-deficient C57Bl/10ScNCr mice were activated with PGN. Significant IL-6 (Fig. 1D) and TNF-{alpha} (data not shown) production was observed compared with that in unstimulated control cells.



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  		Figure 1. Both the TLR4 activator LPS and TLR2 activator S. aureus PGN induce IL-6 production by murine mast cells. (A) C57Bl/6 BMMCs and (B) the murine mast cell line MC/9 were stimulated with a range of doses of E. coli LPS in the presence of FCS and 100 µg/mL of soybean trypsin inhibitor. Under similar conditions, (C) C57Bl/6 and (D) C57BL/10ScNcr BMMCs were stimulated with S. aureus PGN (1–100 µg/mL). Unstimulated cells were analyzed for constitutive IL-6 production. Cell-free supernatants from each condition were harvested after 24 h and analyzed for IL-6 by ELISA. C57Bl/6 BMMC data are representative of three independent experiments from separate BMMC cultures. ND, IL-6 levels that were below the level of detection of the ELISA system used. ***, P < 0.001 compared with medium control.

 
CD14 expression by murine mast cells
It is well documented that CD14 is a primary LPS-binding molecule on myeloid cells such as macrophages, monocytes, and neutrophils. However, previous studies of human mast cells have suggested that it might not be expressed on this cell type [41 42 43 ]. Therefore, the murine mast cell expression of CD14 was analyzed. To determine whether these cells express CD14 protein on the cell surface, BMMCs (>98% pure) were incubated with anti-CD14 antibody for 1 h followed by an FITC-labeled secondary antibody. Fluorescence of cells stained with anti-CD14 antibody was compared with that of cells stained with an irrelevant isotype control antibody. Using the mouse macrophage-like cell line J774 as a positive control, we observed an increase in fluorescence with anti-CD14 antibody-stained J774 cells compared with that of isotype control-stained cells (Fig. 2 A ). The fluorescence distribution of BMMCs stained with anti-CD14 antibody was identical to that of cells stained with the isotype control antibody (Fig. 2A) , demonstrating a lack of CD14 protein expression on the surface of mast cells.



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  		Figure 2. Flow-cytometric analysis of murine mast cells for surface CD14 expression. (A) the murine macrophage-like cell line J774 and (B) C57Bl/6 BMMCs (>98% purity) were stained with purified rat anti-mouse CD14 mAb (black line histogram) or purified rat IgG1 isotype control antibody (shaded, histogram). Cells were then stained with an FITC-labeled secondary antibody and analyzed for fluorescence. The data presented were representative of two and three independent experiments for BMMC and J774 cells, respectively.

 
Mast cell LPS responsiveness required the presence of serum
It has been previously reported that surface CD14-negative cells such as endothelial cells [13 , 44 ] and epithelial cells [45 ] required soluble CD14, an abundant serum protein, to respond to LPS. Because mast cells do not express detectable levels of surface CD14, we analyzed the requirement of serum factors for mast cell responses to LPS. C57Bl/6 BMMCs were stimulated with LPS or the positive control calcium ionophore A23187 in the presence or absence of serum. In the presence of serum, BMMCs produced significant levels of IL-6 when challenged with 0.5 and 5 µg/mL of E. coli LPS (Fig. 3 ). In contrast, IL-6 remained below detectable levels (<0.3 ng/mL, n=3) when C57Bl/6 BMMCs were stimulated with 0.5 and 5 µg/mL of LPS in the absence of serum (Fig. 3) .



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  		Figure 3. Murine mast cell-responsiveness to LPS and requirement of FCS. C57Bl/6 (>98% purity) BMMCs were stimulated with E. coli LPS (0.5 and 5 µg/mL) in the presence (black bars) or absence (shaded bar) of FCS. Unstimulated cells were analyzed for constitutive IL-6 production, and 0.5 µM A23187 (A23) was used as the positive control. Cell-free supernatants from each condition were harvested after 24 h and analyzed for IL-6 by ELISA. C57Bl/6 BMMC data were representative of three independent experiments from separate BMMC cultures. ND, IL-6 levels below the level of detection of the ELISA system used. ***, P < 0.001 compared with medium control.

 
Expression of TLRs by murine mast cells
TLR mRNA expression by murine mast cells was examined by RT-PCR. Two proteins implicated in the LPS response, TLR2 and TLR4, along with two less-well-characterized TLRs, TLR5 and TLR6, were chosen for analysis. The murine mast cell line MC/9 and BMMCs cultured from C57BL/6 mice (>98% pure) were used as mast cell models whereas the mouse macrophage-like cell line J774 was used as a positive control source of TLR mRNA. cDNA was amplified using primers specific for TLR2, TLR4, TLR5, and TLR6. PCR products of the appropriate size were obtained using J774 cDNA (Fig. 4 ). Appropriate PCR products from BMMCs and MC/9 cell cDNA were also observed using primers for TLR2, TLR4, and TLR6 but not TLR5 (Fig. 4) . All PCR products were absent when the reverse transcription step was omitted (Fig. 4) .



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  		Figure 4. RT-PCR analysis of murine mast cells for TLR2, TLR4, TLR5, and TLR6 expression. Total RNA extracted from C57Bl/6 BMMCs and the mast cell line MC/9 was treated with DNase followed by reverse transcription and PCR amplification using specific primers for 34 cycles. RNA isolated from the macrophage-like cell line J774 was used as a positive source of murine TLR RNA. RNA without reverse transcription was used as a negative control. The data shown are representative of three independent experiments.

 
TLR4 function in murine mast cells
We next sought to determine whether the induction of TNF-{alpha} and IL-6 by LPS in mast cells required a functional TLR4 for efficient activation. To address this, we compared the LPS responsiveness of BMMCs derived from TLR4-deficient mice to control BMMCs from a similar genetic background. The TLR-deficient murine strains used were C3H/HeJ and C57Bl/10ScNCr, characterized by a point mutation and a null mutation, respectively, in the TLR4 gene. BMMCs were cultured from these mice and their respective wild-type, congenic littermates C3H/HeOuJ and C57Bl/10ScSn, respectively. BMMCs (>97% pure) were stimulated with a range of doses of E. coli LPS for periods of 6 and 24 h after which cell-free supernatants were harvested and analyzed for cytokines. Time points of 6 and 24 h were chosen to measure TNF-{alpha} and IL-6, respectively, based on earlier time course studies in control(C57Bl/6) mast cells (data not shown). BMMCs cultured from TLR4-deficient mice were nonresponsive to stimulation by E. coli LPS (Fig. 5 ). Despite high levels of cytokine production after stimulation with the positive control, calcium ionopohore (A23187), levels of TNF-{alpha} and IL-6 production after LPS stimulation were consistently below the limit of detection of each ELISA, 30 pg/mL and 300 pg/mL, respectively. In contrast, control mice responded rigorously to the highest concentrations of E. coli LPS and produced markedly increased levels of IL-6 and TNF-{alpha} compared with unstimulated cells.



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  		Figure 5. BMMCs cultured from wild-type mice but not TLR4-deficient mice produced IL-6 and TNF-{alpha} in response to E. coli LPS stimulation. BMMCs (106 cells/mL) cultured from C3H/HeJ (A) and C3H/HeOuJ (C) mice or C57Bl/10ScNCr (B) and C57Bl/10ScSn (D) mice were stimulated for 24 h with a range of doses of E. coli LPS. Cell-free supernatants were harvested and analyzed for IL-6 (A, B) and TNF-{alpha} (C, D) by ELISA after incubation periods of 24 and 6 h, respectively. ND, samples that were below 300 µg/mL and 30 µg/mL, the limits of detection of the IL-6 and TNF-{alpha} ELISA assays, respectively. ***, P < 0.001 compared with medium control. The data presented are representative of two independent experiments for the C3H/HeJ model and three independent experiments for the C57Bl/10 model.

 
We further analyzed the requirement of TLR4 for responsiveness of BMMCs to LPS from an additional source, P. aeruginosa. Similar to our previous findings, BMMCs cultured from C3H/HeJ and C57Bl/10ScNCr mice were unresponsive to stimulation by P. aeruginosa. LPS. Levels of IL-6 (Fig. 6 ) production and TNF-{alpha} remained below the limit of detection of the ELISA despite high levels induced after A23187 stimulation. In contrast P. aeruginosa. LPS markedly up-regulated IL-6 (Fig. 6) for control BMMCs. Similar results were obtained with TNF-{alpha} as a readout (data not shown).



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  		Figure 6. BMMCs cultured from wild-type mice but not TLR4-deficient mice produce IL-6 in response to P. aeruginosa LPS stimulation. BMMCs (106 cells/mL) cultured from C3H/HeJ and C3H/HeOuJ control mice (A) or C57Bl/10ScNCr and C57Bl/10ScJn control mice (B) were stimulated for 24 h with 0.05, 0.5, or 5 µg/mL of P. aeruginosa LPS in the presence of 100 µg/mL of soybean trypsin inhibitor. Cell-free supernatants were harvested after 24 h and analyzed for IL-6 by ELISA. ND, samples below 300 pg/mL, the limit of detection of the IL-6 ELISA. ***, P < 0.001 compared with medium control. The data presented are representative of three independent experiments.

 
MD-2 and MyD88 mRNA expression by murine mast cells
The mast cell expression of two additional proteins implicated in the LPS response, MD-2 [46 , 47 ] and MyD88 [34 ], was characterized using the murine mast cell line MC/9 and C57Bl/6 BMMCs as mast cell models and the mouse macrophage-like cell line J774 as a positive control. After a positive PCR product was assessed for GAPDH, cDNA was amplified using primers specific for MD-2 and MyD88. Prominent bands were observed for MD-2 and MyD88 (Fig. 7 ) using cDNA from all cell types. All PCR products were absent when the reverse transcription step was omitted (Fig. 7) .



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  		Figure 7. RT-PCR analysis of murine mast cell RNA for MD-2 and MyD-88. Total RNA extracted from C57Bl/6 BMMCs and MC/9 cells was treated with DNase followed by reverse transcription and PCR amplification using specific primers for 34 cycles. RNA isolated from J774 cells was used as a positive source of murine MD-2 and MyD88 RNA. RNA without reverse transcription was used as a negative control. Note the expression of both MD-2 and MyD88 by both BMMCs and MC/9 cells. The data shown are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mast cells have a critical role in host defense against bacterial infections [10 , 11 ]. However, the essential mediators derived from mast cells and the mechanism by which these cells respond to infection remain largely undefined. Mast cells might respond through receptor-mediated mechanisms involving direct contact with the bacteria or mechanisms induced as a consequence of bacterial internalization (48–50). In addition, mast cells might be activated through indirect mechanisms involving mediators such as cytokines, produced by other cells, complement products [51 ], or lipid mediators [52 ].

This study demonstrates that mast cells express mRNA for several TLRs including TLR2, TLR4, and TLR6 but not TLR5. These findings suggest that mast cells have the potential to respond directly to a variety of invading pathogens through TLR ligation. The expression of TLR2, TLR4, and TLR6 are particularly important because previous studies have shown that these receptors mediate responsiveness to a number of pathogen products. TLR4 has been implicated in cellular responses to LPS [53 , 54 ], heat shock protein 60 [55 ], and the F protein from respiratory syncytial virus [56 ]. TLR2 is required for responses to products from gram-positive bacteria [29 , 57 ], Mycobacterium tuberculosis [58 ], spirochetes [59 ], and yeasts [60 ]. Furthermore, TLR6 has been shown to enhance the responsiveness of cells to a number of these TLR2 activators [61 , 62 ]. It has previously been shown that particular cell types may preferentially express certain TLRs. For example, TLR3 has been reported to be selectively expressed by dendritic cells but not monocytes/macrophages [63 ]. The lack of TLR5 expression by mast cells is intriguing and may provide a further marker to distinguish mast cells from other myeloid cells. TLR5 has recently been shown to mediate cellular responses to flagellin, a principal component of bacterial flagella [64 ]. The innate immune response to bacterial flagellin is mediated by TLR5. In light of this finding, it is predicted that BMMCs would not respond to purified flagellin. Moreover, flagellin activation of mast cells might not play a major role in Listeria monocytogenes. The vigorous cytokine responses that can be induced when murine mast cells are treated with either LPS (TLR4 activator) or PGN (TLR2 activator) suggest that multiple TLRs might be functional on mast cells.

The current study demonstrated that TLR4 was essential for LPS responsiveness in murine mast cells. BMMCs from TLR4-mutated C3H/HeJ and C57Bl/10ScNCr mice were unresponsive to LPS stimulation whereas BMMCs from the control C3H/HeOuJ and C57Bl/10ScSn mice exhibited vigorous responses when stimulated with high doses of E. coli- or P. aeruginosa-derived LPS. Commercial LPS preparations might be contaminated with bioactive protein that activates cells via a TLR4-independent pathway. Mast cells did not respond to this contamination, however, because neither the C3H/HeJ or C57Bl/10ScNCr mice responded to LPS. P. aeruginosa was used as a second independent LPS source and produced similar results to those observed with E. coli-derived LPS. Expression of a functional TLR4 by murine mast cells was significant because it provided the mast cells with a further means of directly identifying and responding to bacterial challenge. Mast cells are resident in large numbers at most major tissues that interface directly with the external environment, including the skin, airways, and gastrointestinal tract. This brings the mast cell into contact with bacteria in the first line of host defense. The localization of mast cells close to nerves and blood vessels would add to the potential of these cells for initiating effective immune responses against bacterial infection [2 ].

High doses of LPS were required for activation of the murine mast cells compared with the macrophages. BMMCs required 0.5–5 µg/mL of LPS for effective cytokine induction. These findings are consistent with previous experiments using either BMMCs [39 ] or rat peritoneal mast cells [8 ]. In light of these observations, the mast cell expression and function of a number of molecules that have been previously implicated in LPS responsiveness were examined. As previously reported for other mast cell types [41 , 65 ], BMMCs did not express cell surface CD14, although the soluble form of this receptor and LBP would be available from the FBS in which most of the experiments were performed. When mast cells were activated with LPS, in the absence of serum, no significant IL-6 responses were observed (Fig. 3) , suggesting that CD14, LBP, or other serum factors were essential for the mast cell LPS responses. mRNA for both the adapter molecule MD-2 and the signaling molecule MyD88, which are critical for LPS responses in other cell types [32 , 34 ], were expressed by BMMCs. A number of factors might account for the mast cells’ reduced responses to these TLR ligands including decreased receptor expression, reduced receptor homo- or heterodimer formation, or a deficiency in the intracellular signaling mechanisms. However, mRNA expression of both the MD-2 and MyD88 molecules known to be critical for LPS signaling was observed in BMMCs. It is interesting that we have previously shown that mast cell activation with the TLR9 ligand CpG oligonucleotides also requires higher doses than those required to activate other cell types [66 ]. However, the doses of PGN required for mast cell activation were very similar to those reported for macrophage [29 ]. It is notable that human intestinal epithelial cells, which also lack surface CD14 expression, produced cytokines in response to much lower doses of LPS than the amounts required for BMMC activation [67 ].

These findings have important implications for our understanding of the responses of mast cells to bacterial products. The mast cells’ expression of a range of TLRs as well as MD-2 and MyD88 suggests that mast cells might respond to a wide variety of TLR-mediated signals; some such potential responses have already been reported [68 ]. The requirement for a high dose of LPS suggests that even if LPS signals are induced primarily through a shared TLR4-dependent mechanism, alternate cell types might have different thresholds for activation. The BMMCs have previously been considered as a model of the mast cells that reside at mucosal surfaces such as the intestine. In these sites, a higher threshold of activation for mast cells might help to distinguish tissue invasion and infection from the background levels of bacterial products associated with the microenvironment.

Overall, these studies demonstrated both cytokine response to a known TLR2 ligand and TLR4-mediated activation of murine mast cells in response to LPS. They also demonstrated the mRNA expression of a number of TLR and necessary accessory molecules. If mast cells truly serve as "sentinel" cells in host defense [2 ], pattern recognition receptors such as the TLR must have many important functions on mast cells, which we are only beginning to elucidate.


   ACKNOWLEDGEMENTS
 
This work was supported by the Canadian Institutes for Health. The authors thank Yi-Song Wei for her technical expertise and Rosa Bailey for her secretarial assistance during the preparation of this manuscript. Part of this work has previously been presented in abstract form [FASEB J. (2001) 15, A1019 (Abstr.)]

Received April 18, 2001; revised July 13, 2001;
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Z. Orinska, E. Bulanova, V. Budagian, M. Metz, M. Maurer, and S. Bulfone-Paus
TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment
Blood, August 1, 2005; 106(3): 978 - 987.
[Abstract] [Full Text] [PDF]