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Protective Roles of Mast Cells Against Enterobacterial Infection Are Mediated by Toll-Like Receptor 4¹

Volaluck Supajatura^*,, Hiroko Ushio, Atsuhito Nakao, Ko Okumura^,, Chisei Ra^2, and Hideoki Ogawa^*,

Departments of ^* Dermatology and Immunology and Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Toll-like receptors (TLRs) are mammalian homologues of the Drosophila Toll receptors and are thought to have roles in innate recognition of bacteria. We demonstrated that TLR 2, 4, 6, and 8 but not TLR5 were expressed on mouse bone marrow-derived mast cells (BMMCs). Using BMMCs from the genetically TLR4-mutated strain C3H/HeJ, we demonstrated that functional TLR4 was required for a full responsiveness of BMMCs to produce inflammatory cytokines (IL-1, TNF-, IL-6, and IL-13) by LPS stimulation. TLR4-mediated stimulation of mast cells by LPS was followed by activation of NF-B but not by stress-activated protein kinase/c-Jun NH₂-terminal kinase signaling. In addition, in the cecal ligation and puncture-induced acute septic peritonitis model, we demonstrated that genetically mast cell-deficient W/W^v mice that were reconstituted with TLR4-mutated BMMCs had significantly higher mortality than W/W^v mice reconstituted with TLR4-intact BMMCs. Higher mortality of TLR4-mutated BMMC-reconstituted W/W^v mice was well correlated with defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Taken together, these observations provide definitive evidence that mast cells play important roles in exerting the innate immunity by releasing inflammatory cytokines and recruitment of neutrophils after recognition of enterobacteria through TLR4 on mast cells.

	Introduction

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Mast cells have been preserved through evolution even among the lowest orders of animals. In addition to production and secretion of a wide spectrum of mediators and cytokines, their functional capacity, including the ability to phagocytose and process Ags, has lead us to propose a potential role of mast cells in innate immune response against infectious organisms as well as in allergic diseases (1, 2, 3, 4). As shown in two different models of acute bacterial infection, mast cell-deficient W/W^v mice were less efficient in clearing the pathogenic bacteria in the cecal ligation and puncture (CLP)³-induced peritonitis and from the lungs of mice intranasally challenged with Klebsiella pneumoniae (5, 6). It has been shown that mast cell-derived TNF- and leukotriene-dependent recruitment of circulating leukocytes with bactericidal properties is crucial for a full response against acute infection (5, 6, 7). Although the molecular basis for the interaction between mast cells and various Gram-negative and Gram-positive bacteria is thought to be mediated by "pattern recognition receptors" that display binding specificity for structural pattern molecules common to many microorganisms (8), precise mechanisms for mast cell activation by these microorganisms are yet to be clarified.

Toll-like receptor (TLR) families are transmembrane proteins containing repeated leucine-rich motifs in their extracellular portions similar to other pattern recognition proteins of the innate immune system, and a cytoplasmic domain that is homologous to the signaling domain of the IL-1R which mediates activation of NF-B (9). Although 10 such receptors have been identified (10), functional data are available only for two TLRs, TLR2 and TLR4. TLR2 appears to participate in the innate recognition response to soluble peptidoglycan, lipoteichoic acid, or whole Gram-positive bacteria (11, 12, 13, 14, 15), whereas TLR4 is implicated in cellular responses to LPS, the major constituent of the Gram-negative bacteria outer membrane (16, 17). Immunocompetent cells express a variety of specific transcripts of TLRs (18). TLR1 is expressed in all leukocytes including monocytes, polymorphonuclear cells, T and B cells, and NK cells, whereas TLR2, 4, and 5 are expressed in myelomonocytic cells. Specific expression of TLR3 is observed only in dendritic cells (18). Thus, the difference in TLR expression may reflect their specialized functions in immune responses.

Despite the assumption that mast cells and TLRs are both involved in innate immune response, no data are available regarding the expression patterns of TLRs on mast cells. Signaling pathways upstream of cytokine expression of mast cells in response to LPS also have not yet been well defined. Using TLR4-intact LPS-responsive C3H/HeN and TLR 4-mutated LPS-hyporesponsive C3H/HeJ mice (19), we therefore first examined the TLR expression on mast cells and functional roles of TLR 4 on mast cells in LPS-induced cytokine production, degranulation, and signal transduction pathways. In addition, in vivo TLR 4-mediated protective roles of mast cells were examined in an acute peritonitis model using genetically mast cell-deficient W/W^v mice that were reconstituted with bone marrow derived-mast cells (BMMCs) from C3H/HeN or C3H/HeJ mice.

	Materials and Methods

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Mice

WBB6F₁-W/W^v, WBB6F₁^+/+, C3H/HeN, C3H/HeJ, BALB/c, and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). It has been reported that C3H/HeJ mice are hyporesponsiveness to LPS because of the point mutation at codon 712 within the cytoplasmic domain of Tlr4 gene by substitution of highly conserved proline by histidine, which leads to the defect in the signal transduction through TLR4 (19). NC/Nga mice were originally obtained from Kowa Institute (Ibaraki, Japan) and maintained in the specific pathogen-free animal facility at Juntendo University. All animal experiments were performed under the approved manual of the Institutional Review Board of Juntendo University.

Generation of BMMCs

BMMCs were generated from the femoral bone marrow cells of mice as described previously (20). Cells were grown in the RPMI 1640 (Sigma, St. Louis. MO) supplemented with 10% heat-inactivated FCS (Biological Industries, Haemek, Israel), 100 U/ml penicillin, 100 µg/ml streptomycin, 10^-4 M 2-ME, 10 mM sodium pyruvate, 10 µM MEM nonessential amino acids solution, and 10% pokeweed mitogen-stimulated spleen-conditioned medium as a source of mast cell growth factors (21) by replacement of half of the medium weekly. After 4–5 wk of culture, >98% of the cells were identifiable as mast cells as determined by toluidine blue staining and FACS analysis of cell surface expression of c-kit and FcRI.

-Hexosaminidase release assay

A total of 5 x 10⁵ cells/ml BMMCs in Tyrode’s buffer (10 mM HEPES buffer (pH 7.4), 130 mM NaCl, 5 mM KCl, and 5.6 mM glucose) containing 10% FCS (as a source of soluble CD14), 1 mM CaCl₂, and 0.6 mM MgCl₂ was stimulated with the indicated concentration of LPS (Escherichia coli serotype 0111:B4; Sigma) for 1 h at 37°C. The BMMCs stimulated with IgE (1 µg/ml; BD PharMingen, San Diego, CA) plus anti-IgE (1 µg/ml; PharMingen) or with PMA (10 ng/ml) plus ionomycin (100 ng/ml) were used as positive controls. Cell supernatants and total cell lysates solubilized with 1% Nonidet P-40 were collected, and -hexosaminidase in the supernatants and cell lysates was quantified by spectrophotometric analysis of hydrolysis of p-nitrophenyl-N-acetyl--D-glucopyranoside (Sigma). The percentage of -hexosaminidase release was calculated using the following formula: percent release = (OD of the stimulated supernatant - OD of the unstimulated supernatant) x 100/(OD of the total cell lysate - OD of the unstimulated supernatant). Viability of the cells under each condition was >98% as assessed by the trypan blue dye exclusion test.

Evaluation of cytokine concentrations

BMMCs (1 x 10⁶ cell/ml) in complete cultured medium were stimulated at 37°C with the indicated concentration of LPS, 3 h for TNF-, and 6 h for IL-1, IL-6, and IL-13. From preliminary experiments, these time points were optimal for production of these cytokines from BMMCs upon LPS stimulation. The levels of each cytokine in the culture supernatants were determined by ELISA kits according to the manufacturer’s instructions (Genzyme Techne, Minneapolis, MN). Viability of the cells under each condition was >98% as assessed by a trypan blue dye exclusion test.

RT-PCR analysis of TLR expressions on mast cells

Total RNA was extracted from BMMCs using STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturer’s instructions. First-strand cDNA was constructed from 3 µg of total RNA with oligo(dT)_12–18 as primers using Superscript II RNase H^- reverse transcriptase (Life Technologies, Rockville, MD). PCR was performed using primers for mouse TLR2 (GenBank accession no. AF185284: 5'-CTT CCT GGT TCC CTG CTC GTT CTC and 5'-CAA GAA CAA AGA AAA TGA GTC AAG), mouse TLR4 (accession no. AF110133) (22), mouse TLR5 (accession no. AF186107) (23), mouse TLR6 (accession no. NM011604) (24), mouse TLR8 (accession no. AF113795: 5'-TCT TGC CCT TGG CAG AGA AGT T and 5'-GGA GCT GAC ATT CCA GAC AGA ACA), and species nonspecific GAPDH (5'-AGT ATG ACT CCA CTC ACG GCA A and 5'-TCT CGC TCC TGG AAG ATG GT) (25).

Western blotting

A total of 5 x 10⁶ cell/ml BMMCs was stimulated with LPS (50 ng/ml) for the indicated time period. At the indicated time points, the reaction was stopped with cold Tyrode’s (-) buffer. The cells were lysed with 20 µl of lysis buffer (1% Triton X-100, 150 mM NaCl, 25 mM Tris-HCl (pH 7.5), 1 mM EDTA, containing 1 µM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 50 µg/ml aprotinin, and 2 mM sodium orthovanadate), and the lysates were subjected to 12% SDS-PAGE (Bis Tris; NOVEX, San Diego, CA). The resolved protein was then transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and the membrane was probed with specific polyclonal Abs to tyrosine-phosphorylated IB- and c-Jun NH₂-terminal kinase (JNK; New England Biolabs, Beverly, MA) according to the manufacturer’s instruction. To confirm equal loading, immunoblots were stripped with 40% methanol and stripping buffer (62.5 mM Tris-HCl (pH 6.8) and 2% SDS containing 100 mM 2-ME) and reprobed with polyclonal Abs to signal-related kinases. The concentrations of total protein in each lysate were measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The membrane was developed with an ECL detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). The density of resolved bands was evaluated by a Personal Densitometer Scan (version 1.30) and ImageQuant software (version 3.3; Molecular Dynamics, Sunnyvale, CA).

Mast cell reconstitution in W/W^v mice

Mast cell deficiency of W/W^v mice was selectively reconstituted by the injection of 2 x 10⁶ BMMCs (4 wk old) from C3H/HeN or C3H/HeJ mice into the peritoneal cavity as previously described (5, 20). Five weeks after injection of BMMCs, the mice were used for experiments. Reconstitution of mast cells was confirmed by toluidine blue or Alcian blue/safranin staining of the cytospun preparation of peritoneal cells. Functional relevance of reconstituted mast cells was confirmed by the -hexosaminidase release assay using purified peritoneal mast cells 5 wk after reconstitution through the stimulation by FcRI cross-linking or calcium ionophore.

Cecal ligation and puncture

CLP was performed as previously described with slight modification (5, 6, 26). In brief, the mice were anesthetized by i.p. injection of 50 mg/kg sodium pentobarbital (Abbott Laboratories, Abbott Park, IL) in 200 µl of sterile PBS. A 1-cm midline incision on the anterior abdominal wall was made. The cecum was exposed and filled with feces by squeezing stool back from the ascending colon. The cecum was 50% ligated below the ileocecal valve and then punctured using a 0.9-mm needle followed by gentle squeezing of the cecum. Mice were observed for mortality at least five times daily over a period of 10 days. Before CLP was performed, the mice were coded so that the CLP was done without notifying individual groups.

Differential cell counts and estimation of cytokine concentrations in peritoneal exudates

Peritoneal exudates were collected from CLP-induced mice at the indicated time points and total cell numbers were counted. Cytospun preparations were made from the exudates of each mouse and differential cell counts of infiltrating leukocytes were done by counting 500 leukocytes under oil immersion fields after staining with DiffQuik (Kokusaishi, Yaku, Japan). The percentage of mast cells in the exudates was determined by toluidine blue (pH 4.0) or Alcian blue/safranin staining. The concentrations of cytokines in peritoneal fluids were determined by ELISA kits according to the manufacturer’s instruction (Genzyme Techne).

Statistical analysis

Statistical analysis of most data was performed using the Student t test. Statistical analysis of survival data in CLP experiments was performed using the log rank test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TLR mRNA expressions in mouse mast cells

TLR mRNA expressions of mouse mast cells were initially assessed by RT-PCR. cDNA from mouse bone marrow-derived dendritic cells was used as a known source for mouse TLR expression to confirm the specificity of the primers and PCR. As shown in Fig. 1, transcripts of TLR2, TLR4, TLR6, and TLR8 were detected in both C3H/HeN and C3H/HeJ BMMCs; in contrast, no transcript of TLR5 was detected in these BMMCs. Similar results were obtained in BMMCs from BALB/c, C57BL/6, and NC/Nga mice (data not shown). RT-PCR analysis of GAPDH expression confirmed the equality of all RNA preparations used for these experiments.

View larger version (108K): [in this window] [in a new window]   FIGURE 1. TLR mRNA expressions in BMMCs. Gene expressions of TLR2 (385 bp), TLR4 (540 bp), TLR5 (540 bp), TLR6 (696 bp), and TLR8 (250 bp) in C3H/HeN and C3H/HeJ BMMCs were analyzed by PCR following RT. Bone marrow-derived mouse dendritic cells (DC) were used as positive controls. Equality of the RT reaction of isolated RNA was confirmed by amplification of the housekeeping gene GAPDH. The result shown is representative of three independent experiments that had similar results.

Significant degranulation and cytokine release were associated with mast cell activation by soluble microbacterial products. To investigate whether LPS-stimulated cytokine production and degranulation activities of mast cells were mediated via functional TLR4, we developed BMMCs from TLR4-intact C3H/HeN or TLR4-mutated C3H/HeJ mice, then assessed the cytokine production (TNF-, IL-1, IL-6, and IL-13) and -hexosaminidase release from these mast cells upon LPS stimulation. As shown in Fig. 2A, C3H/HeN BMMCs could respond to produce TNF-, IL-1, IL-6, and IL-13 by LPS stimulation in a dose-dependent manner, whereas C3H/HeJ BMMCs failed to produce any cytokines by stimulation with LPS (1–1000 ng/ml). The viability of BMMCs from both strains of mice after LPS stimulation was >95%, indicating cytokine release was not due to cytotoxic effects of LPS.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Cytokine production and -hexosaminidase release from BMMCs upon LPS stimulation. A, BMMCs from C3H/HeN () or C3H/HeJ () were stimulated with the indicated concentration of LPS, 3 h for TNF-, and 6 h for IL-1, IL-6, and IL-13. The levels of cytokines in the supernatants were estimated by ELISA kits. Data shown are mean ± SD of three independent experiments. B, Percent release of -hexosaminidase from BMMCs of C3H/HeN () or C3H/HeJ () was evaluated as described in Materials and Methods upon stimulation with the indicated concentration of LPS for 1 h. IgE and anti-IgE- or PMA-ionomycin-stimulated BMMCs were used as positive controls. The result shown is representative of three independent experiments that had similar results.

These results suggested that cytokine productions from BMMCs by LPS stimulation were highly dependent on the functional expression of TLR4 and mechanisms of cytokine and -hexosaminidase release from BMMCs were different.

LPS-TLR4-mediated pathways activate NF-B signaling in mast cells

Signaling through the TLRs has been focused on NF-B activation that is required for the transcription of many immune responsive genes including cytokine genes. It has been reported that LPS can activate NF-B in human intestinal epithelial cells expressing TLRs (28) and activate NF-B and c-Jun/activating transcription factor 2/T cell-specific factor in human leukocytes (29). We determined whether NF-B and stress-activated protein kinase (SAPK)/JNK cascade were also triggered in mast cells upon LPS stimulation. The phosphorylation of IB- at Ser³², essential for release of active NF-B, is a marker of NF-B activation. As shown in Fig. 3, LPS (50 ng/ml) strongly up-regulated the phosphorylated IB- in C3H/HeN BMMCs but not in C3H/HeJ BMMCs. The activity was at maximum 15 min after stimulation and then gradually decreased within 60 min (Fig. 3A). However, the cell lysates of both BMMCs did not exhibit specific induction of SAPK/JNK activity at any indicated time, although positive control provided by the manufacturer showed significant phosphorylation of SAPK/JNK (Fig. 3B). Although we confirmed that an equal amount of protein was loaded in each lane, total amount of IkB- from C3H/HeJ BMMCs was always lower than that from C3H/HeN BMMCs.

View larger version (56K): [in this window] [in a new window]   FIGURE 3. LPS-induced activation of NF-B in mast cells. BMMCs were stimulated with 50 ng/ml LPS for 5, 15, 30, and 60 min and then lysed in the lysis buffer. Proteins were resolved by SDS-PAGE, electroblotted, and immunoblotted with Abs specific for phospho-IB- (A) and phospho-SAPK/JNK (B). The same blots were stripped and reblotted with Abs for the nonphosphorylated form of each respective signaling protein. LPS-stimulated Raw 264.7 was used as positive controls. Positive and negative controls provided by the kits were also used as control. The result shown is a representative of three independent experiments that had similar results.

The role of TLR4 in mast cell-dependent innate immunity was examined by subjecting genetically mast cell-deficient W/W^v mice to CLP, a mouse model of acute septic peritonitis. Deficiency of mast cells in W/W^v mice was reconstituted with BMMCs either from TLR4-intact C3H/HeN or TLR4-mutated C3H/HeJ mice. The CLP peritonitis was induced 5 wk after i.p. transfer of BMMCs. As shown in Fig. 4, in unreconstituted W/W^v mice, all of the animals died within 3 days. In contrast, the lethal effect of acute bacterial peritonitis was greatly diminished by reconstitution of W/W^v mice with BMMCs from the C3H/HeN strain (p < 0.05 from days 3 to 10). Interestingly, among the mast cell-reconstituted groups, the W/W^v mice reconstituted with BMMCs from the C3H/HeJ strain showed a significantly higher mortality rate than the W/W^v mice reconstituted with BMMCs of the C3H/HeN strain, 80% in the C3H/HeJ BMMC-reconstituted group vs 20% in the C3H/HeN BMMC-reconstituted group at day 7 (p < 0.05). Even though it appeared that W/W^v mice reconstituted with BMMCs from C3H/HeJ mice had a better survival rate after CLP than that of W/W^v mice without reconstitution of BMMCs, there were no statistically significant differences in the mortality rate between these two groups of mice during the experimental periods (from days 3 to 10). During the study period (2 mo), the recipient mice were all healthy and showed no sign of graft rejection. The cytospun preparations of peritoneal exudates from both groups 5 wk after the reconstitution of BMMCs had a similar number of peritoneal mast cells (1.23 ± 0.14 x 10⁵ cell/mouse in W/W^v-C3H/HeN vs 1.22 ± 0.18 x 10⁵ cell/mouse in W/W^v-C3H/HeJ). These numbers of mast cells were slightly less but not significantly different from those of W/W^v that received BMMCs from control WBB6F₁^+/+ mice (1.48 ± 0.12 x 10⁵ cells/mouse). The staining properties of peritoneal mast cells derived from W/W^v that received BMMCs from C3H/HeN, C3H/HeJ, and +/+ were similar; all cells exhibited positive staining with both Alcian blue and safranin, a prominent feature of connective tissue-type mast cells. Also, we confirmed that there were no functional differences in the peritoneal mast cells derived from W/W^v that received C3H/HeN BMMCs or C3H/HeJ BMMCs by measuring -hexosaminidase release upon FcRI cross-linking (W/W^v-C3H/HeN vs W/W^v-C3H/HeJ, 31 vs 33%) or calcium ionophore stimulation (W/W^v-C3H/HeN vs W/W^v-C3H/HeJ, 42 vs 34%). Thus, the difference in the mortality rate between W/W^v-C3H/HeN and W/W^v-C3H/HeJ was not due to the differences in development of transferred cells in the W/W^v environment.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Requirement of mast cells with intact TLR 4 for the protection of W/Wv mice against the potentially lethal effects of CLP. Five weeks after reconstitution of W/Wv mice with or without BMMCs, peritonitis was induced by CLP. CLP-induced mortality rates of different mouse groups (10 mice/group) are shown. The result is obtained from two independent experiments.

Next, we determined whether defective neutrophil recruitment in the W/W^v mice contributes to impaired resistance of animals to microbial agents as previously reported (5, 6). The peritoneal exudates were examined for leukocyte infiltration after CLP induction. The majority of infiltrating cells in the peritoneal cavity were neutrophils, 3 and 6 h after CLP induction (Fig. 5A). Interestingly, W/W^v mice that did not receive BMMCs or that received C3H/HeJ BMMCs showed significantly less neutrophil influx than W/W^v mice reconstituted with C3H/HeN BMMCs (p < 0.01). Although, W/W^v mice that received C3H/HeJ BMMCs showed significantly more leukocytes (neutrophils) influx than unreconstituted W/W^v, 6 h after CLP (p < 0.01), the value was still significantly lower than that of W/W^v mice reconstituted with C3H/HeN BMMCs (p < 0.01). Also, the levels of cytokines (TNF-, IL-1, IL-6, and IL-13) in peritoneal fluids, especially 6 h after CLP, were significantly higher in W/W^v mice reconstituted with C3H/HeN BMMCs than in W/W^v mice with C3H/HeJ BMMCs or those unreconstituted (p < 0.01, Fig. 5B). These results again suggest that functional TLR4 on mast cells is required for in vivo production of proinflammatory cytokines and for recruitment of neutrophils into the peritoneal cavity after CLP.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Neutrophil recruitment and inflammatory cytokine productions following CLP treatment is dependent on the functional TLR4 on mast cells. A, Total leukocytes in the peritoneal exudates were counted at the indicated time and the number of neutrophils was estimated by differential cell counts of the cytospun preparations of peritoneal exudates. Data shown are the mean ± SD of five mice. B, Peritoneal exudates were collected at the indicated time after CLP induction. Levels of cytokines in the peritoneal fluids were determined using ELISA kits. Data shown are the mean ± SD of five mice. *, p < 0.05; **, p < 0.01, significantly different from the mean value of the corresponding control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We analyzed the expression of five TLRs in mouse mast cells. Transcripts of TLR2, 4, 6, and 8 were present in mast cells but no transcript of TLR5 was detectable. Although several reports have suggested that the expressions of TLR4 and TLR2 on macrophages and T cells could be regulated after interaction with bacterial products such as LPS, lipoarabinomannan, or proinflammatory cytokines (18, 30), we could not detect any modulation of the expression of TLR2, 4, 5, 6, and 8 on BMMCs by LPS, lipoteichoic acid (Gram-positive bacterial product), or FcRI cross-linking (data not shown). Since it has been reported that different immunocompetent cells express specific transcripts of TLRs (18), it would be interesting to see whether mast cells in different tissues (e.g., mucosal vs connective tissue) have different expression patterns of TLRs.

One of the biological responses of mast cells to stimuli is release of preformed mediators in their granules and production of cytokines that are not necessarily stored within the cells as preformed substances (31, 32). We could detect the production of TNF-, IL-1, IL-6, and IL-13 from TLR4-intact BMMCs but not from TLR4-mutated BMMCs through stimulation with LPS. We also observed similar impairment of proinflammatory cytokine productions in peritoneal fluids of W/W^v mice reconstituted with C3H/HeJ BMMCs after CLP. Although the levels of cytokines were much less than those in W/W^v reconstituted with C3H/HeN BMMCs, a slight but significant increase of some cytokines (IL-1 at 3 and 6 h, IL-6 at 6 h) was observed in peritoneal fluids of W/W^v reconstituted with C3H/HeJ BMMCs. This suggested that not all cytokine production in the peritoneal cavity after CLP was dependent on the activation of mast cells via functional TLR4. TNF- and IL-1 are the potent monocyte/macrophage activator (33), and TNF- production from mast cells is thought to be critical for some of the acute inflammatory events, including the local influx of neutrophils (34). IL-6 also plays a role in local inflammatory reactions by amplifying leukocyte (monocyte, PMN, lymphocyte) recruitments (35). In contrast, since IL-13 has been reported to show anti-inflammatory properties by modulating the production of macrophage/monocyte-derived TNF-, IL-1, and IL-8, it might have some roles for sweeping exaggerated inflammatory responses (36, 37). Compared with other cytokines (TNF-, IL-1, IL-6), which were produced more in vivo than in vitro after CLP (Fig. 5B), production of IL-13 seemed to be much less in vivo than in vitro. Although we do not know the precise reason for this, it may be due to the different ability to produce IL-13 upon stimulation with microorganisms between BMMCs and peritoneal mast cells or due to different stimulants, LPS for BMMCs vs whole microorganisms for peritoneal mast cells.

Our data demonstrated that mast cells responded to LPS for these cytokine productions in a dose-dependent manner, but failed to degranulate in response to LPS at concentrations that induced cytokine production. This result was consistent with a previous study using rat peritoneal mast cells in which LPS could induce substantial IL-6 production without releasing a significant amount of histamine (38). It is therefore suggested that mast cells are capable of releasing cytokines into the extracellular environment, independent of the classical degranulation pathway, in response to infection.

LPS elicits several immediate proinflammatory responses in peripheral blood leukocytes through described pathways involving serine-threonine kinases and NF-B transcription factor (28). However, the functional responses of mouse mast cells to stimulation with LPS via TLR4 were unknown. Our results demonstrated that LPS-stimulated TLR4 on mast cells led to phosphorylation of IB- but not of SAPK/JNK. These results were in agreement with previous studies that LPS stimulation of some peripheral blood leukocytes or other cell lines did not always result in the activation of p42/p44 mitogen-activated protein kinase, JNK or P38 (28, 39). LPS-induced signal transduction in a different manner might be due to the distinct cell origin, the state of differentiation, or idiosyncratic alterations of the signal transduction pathway. It is possible that, in mast cells, TLR4 signal for NF-B activation is not via the mitogen-activated protein kinase cascade and that the signaling cascade of mast cells upon LPS stimulation may be transversed by MyD88, IL-1 R-associated kinase, TNFR-associated factor 6 and NF-B, like signal elicited by IL-1 (28, 29, 39).

Cell surface CD14 (LPS receptor), by making the LPS-CD14 complex, has been implicated in sensing the LPS through TLRs on cells such as monocytes, macrophages, and PMN (40). But we could not detect CD14 expression on the surface of any strains of BMMCs (C3H/HeN, C3H/HeJ, BALB/c, C57BL/6, and NC/Nga) by FACS analysis (data not shown). It is possible that the LPS-binding protein and soluble CD14, both of which have been shown to be present in serum (41, 42), could transfer LPS signaling to TLR on mast cells, as demonstrated in other CD14^- cells which require the presence of soluble CD14 for the response of LPS (41, 42, 43).

Mast cell-deficient WBB6F₁-W/W^v mice and the model of "mast cells knock-in" mice could be used to analyze the expression of biological responses in tissues which do or do not contain mast cells (44). Although in the previous report they showed a successful reconstitution of embryonic stem cell-derived mast cells from 129Sv mice in the peritoneal cavity of W/W^v mice which only differ in minor histocompatibility Ag from 129Sv mice (45), it was a surprise that we could successfully reconstituted mast cell-deficient W/W^v mice with BMMCs from genetically unmatched C3H/HeN and C3H/HeJ mice as we mentioned in the results in Fig. 4. These results show that BMMCs of the C3H/He origin could survive and differentiate into functional CTMCs after injection into the peritoneal cavity of W/W^v mice, and it seemed that the TLR 4 mutation did not affect the differentiation of mast cells in the W/W^v environment.

The role of TLR4 on mast cells in bacterial infection, in a model of CLP-induced acute septic peritonitis was clearly demonstrated in this study. In accordance with the previous studies (5, 6, 7), the inability of W/W^v mice reconstituted with C3H/HeJ BMMCs to effectively clear bacteria in the peritoneum was directly correlated with impaired neutrophil recruitments. Along with the results of cytokine productions in vitro and in vivo, the above-mentioned results suggested that the TLR 4 mutation was the cause of defective LPS signaling in mast cells of C3H/HeJ and prone to develop severe infection and death.

Although C3H/HeJ BMMC-reconstituted W/W^v mice showed significantly greater mortality than C3H/HeN BMMC-reconstituted W/W^v mice, the mortality of the former mice seemed to be lower than that of unreconstituted W/W^v mice (but not significantly), suggesting that TLRs other than TLR4 expressed on mast cells (e.g., TLR6 and 8) whose functions have not yet been well characterized, or other molecules such as C3, might play protective roles in these responses, since it has been reported that the lack of C3 reduced the activation of mast cells by means of TNF- production, neutrophil recruitment, and bacterial clearance (46).

In conclusion, we demonstrated that activation of mast cells by LPS of Gram-negative bacteria via cell surface TLR4 resulted in production of inflammatory cytokines, leading to rapid infiltration of neutrophils into the peritoneal cavity, then expanded the inflammatory responses, and finally led to sufficient bacterial eradication. Our results again support the proposed roles of mast cells in the innate immune system and have revealed that the TLR4-mediated pathway can be activated by enterobacteria not only in monocytes/macrophages but also in mast cells.

	Acknowledgments

 
We are deeply grateful to Drs. Y. Akira and K. Hoshino at the University of Osaka (Osaka, Japan) for their kind support and helpful discussions. We also thank Drs. C. Nishiyama, K. Maeda, and H. Yagita for helpful discussions and Drs. Y. Furumoto, M. Masaki, T. Uchida, and K. Hira and T. Tokura for technical assistance.

	Footnotes

 
¹ This work was supported in parts by grants from the Ministry of Education, Science, and Culture, Japan, and from the Uehara Foundation. V.S. was supported by grants from the Japan International Cooperation Agency.

² Address correspondence and reprint requests to Dr. Chisei Ra, Atopy (Allergy) Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: cra{at}med.juntendo.ac.jp

³ Abbrevations used in this paper: CLP, cecal ligation and puncture; TLR, Toll-like receptor; BMMC, bone marrow-derived mast cell; CTMC, connective tissue-type mast cell; JNK, c-Jun NH₂-terminal kinase; SAPK, stress-activated protein kinase.

Received for publication January 31, 2001. Accepted for publication June 15, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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