Departments of
* Microbiology and Immunology,
Pediatrics, and
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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(TNF-). 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- 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
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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) , 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- 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.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
], purified rat IgG1 (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 ; PharMingen) or
purified rat IgG1 (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- 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- was measured using a similar protocol. All incubations were
carried out at room temperature. Plates were coated with
TNF--coating antibody and polyclonal goat anti-murine TNF-
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- ( 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- ELISA was 30 pg/mL unless otherwise indicated.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
that were more than eightfold
higher than unstimulated cells (mean values of 42±26 pg/mL and <5
pg/mL of TNF- for PGN-stimulated and -unstimulated cells,
respectively, representative of two independent experiments). To ensure
that the IL-6 and TNF- 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- (data not shown) production was observed compared with that
in unstimulated control cells.
 View larger version (36K): [in a new window] |
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.
|
 View larger version (33K): [in a new window] |
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.
|
 View larger version (31K): [in a new window] |
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.
|
 View larger version (87K): [in a new window] |
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.
|
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- 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- 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- compared with unstimulated cells.
 View larger version (42K): [in a new window] |
Figure 5. BMMCs cultured from wild-type mice but not TLR4-deficient mice produced
IL-6 and TNF- 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- (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- 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.
|
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- as a
readout (data not shown).
 View larger version (30K): [in a new window] |
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.
|
 View larger version (32K): [in a new window] |
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.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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.
Received April 18, 2001;
revised July 13, 2001;
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and IL-6 production but not degranulation from murine bone marrow-derived mast cells J. Leukoc. Biol. 69,253-262This article has been cited by other articles:
 |
|
 |
|
  S. R. Holdsworth and S. A. Summers Role of Mast Cells in Progressive Renal Diseases J. Am. Soc. Nephrol., December 1, 2008; 19(12): 2254 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Sutherland, J. S. Olsen, A. McKinstry, S. A. Villalta, and P. J. Wolters Mast Cell IL-6 Improves Survival from Klebsiella Pneumonia and Sepsis by Enhancing Neutrophil Killing J. Immunol., October 15, 2008; 181(8): 5598 - 5605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Sherman The Role of Mast Cells in Bacterial Enteritis Am. J. Pathol., August 1, 2007; 171(2): 399 - 401. [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kikawada, J. V. Bonventre, and J. P. Arm Group V secretory PLA2 regulates TLR2-dependent eicosanoid generation in mouse mast cells through amplification of ERK and cPLA2{alpha} activation Blood, July 15, 2007; 110(2): 561 - 567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Mullaly and P. Kubes The Role of TLR2 In Vivo following Challenge with Staphylococcus aureus and Prototypic Ligands J. Immunol., December 1, 2006; 177(11): 8154 - 8163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Jawdat, G. Rowden, and J. S. Marshall Mast Cells Have a Pivotal Role in TNF-Independent Lymph Node Hypertrophy and the Mobilization of Langerhans Cells in Response to Bacterial Peptidoglycan J. Immunol., August 1, 2006; 177(3): 1755 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kanehira, T. Kaifu, K. Maya, M. Kaji, A. Nakamura, M. Obinata, and T. Takai Novel Mast Cell Lines with Enhanced Proliferative and Degranulative Abilities Established from Temperature-Sensitive SV40 Large T Antigen Transgenic Mice J. Biochem., August 1, 2006; 140(2): 211 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. I. Nigo, M. Yamashita, K. Hirahara, R. Shinnakasu, M. Inami, M. Kimura, A. Hasegawa, Y. Kohno, and T. Nakayama Regulation of allergic airway inflammation through Toll-like receptor 4-mediated modification of mast cell function PNAS, February 14, 2006; 103(7): 2286 - 2291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Tavener and P. Kubes Cellular and molecular mechanisms underlying LPS-associated myocyte impairment Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H800 - H806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Qiao, M. V. Andrade, F. A. Lisboa, K. Morgan, and M. A. Beaven Fc{epsilon}R1 and toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells Blood, January 15, 2006; 107(2): 610 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. B. Goralski, D. Abdulla, C. J. Sinal, A. Arsenault, and K. W. Renton Toll-like receptor-4 regulation of hepatic Cyp3a11 metabolism in a mouse model of LPS-induced CNS inflammation Am J Physiol Gastrointest Liver Physiol, September 1, 2005; 289(3): G434 - G443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 J. M. Daley, T. Ivanenko-Johnston, J. S. Reichner, and J. E. Albina Transcriptional regulation of TNF-{alpha} production in neutropenia Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R409 - R412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Matsushima, N. Yamada, H. Matsue, and S. Shimada TLR3-, TLR7-, and TLR9-Mediated Production of Proinflammatory Cytokines and Chemokines from Murine Connective Tissue Type Skin-Derived Mast Cells but Not from Bone Marrow-Derived Mast Cells J. Immunol., July 1, 2004; 173(1): 531 - 541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Sundstrom, D. M. Little, F. Villinger, J. E. Ellis, and A. A. Ansari Signaling through Toll-Like Receptors Triggers HIV-1 Replication in Latently Infected Mast Cells J. Immunol., April 1, 2004; 172(7): 4391 - 4401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. T. Edelson, Z. Li, L. K. Pappan, and M. M. Zutter Mast cell-mediated inflammatory responses require the {alpha}2{beta}1 integrin Blood, March 15, 2004; 103(6): 2214 - 2220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Okumura, J.-i. Kashiwakura, H. Tomita, K. Matsumoto, T. Nakajima, H. Saito, and Y. Okayama Identification of specific gene expression profiles in human mast cells mediated by Toll-like receptor 4 and Fc{epsilon}RI Blood, October 1, 2003; 102(7): 2547 - 2554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Forsythe and A. D. Befus CCR3: A Key to Mast Cell Phenotypic and Functional Diversity? Am. J. Respir. Cell Mol. Biol., April 1, 2003; 28(4): 405 - 409. [Full Text] [PDF]
|
|
|
|
|
|
A. Di Nardo, A. Vitiello, and R. L. Gallo Cutting Edge: Mast Cell Antimicrobial Activity Is Mediated by Expression of Cathelicidin Antimicrobial Peptide J. Immunol., March 1, 2003; 170(5): 2274 - 2278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. McCurdy, T. J. Olynych, L. H. Maher, and J. S. Marshall Cutting Edge: Distinct Toll-Like Receptor 2 Activators Selectively Induce Different Classes of Mediator Production from Human Mast Cells J. Immunol., February 15, 2003; 170(4): 1625 - 1629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Genovese, G. Borgia, L. Bjorck, A. Petraroli, A. de Paulis, M. Piazza, and G. Marone Immunoglobulin Superantigen Protein L Induces IL-4 and IL-13 Secretion from Human Fc{varepsilon}RI+ Cells Through Interaction with the {kappa} Light Chains of IgE J. Immunol., February 15, 2003; 170(4): 1854 - 1861. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Mori, R. Suzuki, T. Furuno, D. M. McKay, M. Wada, R. Teshima, J. Bienenstock, and M. Nakanishi Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite Am J Physiol Cell Physiol, December 1, 2002; 283(6): C1738 - C1744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Masuda, Y. Yoshikai, K. Aiba, and T. Matsuguchi Th2 Cytokine Production from Mast Cells Is Directly Induced by Lipopolysaccharide and Distinctly Regulated by c-Jun N-Terminal Kinase and p38 Pathways J. Immunol., October 1, 2002; 169(7): 3801 - 3810. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
