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Published online before print July 20, 2005
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,1
,¶,,,¶
* Departments of Surgery,
Medicine, and
¶ Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville; and Departments of
Surgery I and
Microbiology, National Defense Medical College, Tokorozawa, Japan
2Correspondence: Department of Surgery, University of Florida College of Medicine, Room 6116, Shands Hospital, 1600 SW Archer Road, Gainesville, FL 32610-0286. E-mail: moldawer{at}surgery.ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–1) and plasmacytoid (plasmacytoid DC antigen+ B220+) DC subsets, whereas LPS only increased myeloid DCs in the draining lymph node. In addition, the footpad injection of flagellin or LPS induced significant CD4+ T cell activation in the draining popliteal lymph node, as judged by increased CD69 or CD25 expression. We illustrate, for the first time, that flagellin also increases natural killer (NK) cell number and activation status in the draining lymph node after footpad injection. Using coculture with enriched carboxy-fluorescein diacetate succinimidyl ester-labeled NK cells, flagellin-treated DCs induce significant NK cell proliferation and activation. In fact, direct treatment of NK cells with flagellin induces a greater increase in cell proliferation than treatment with LPS. In contrast, flagellin treatment of NK cells was not a strong inducer of interferon-
(IFN-) production, indicating that NK cell proliferation and IFN- production may be regulated differentially. These data suggest that flagellin is a capable maturation agent for murine myeloid-derived DCs, and flagellin-activated DCs and flagellin itself are potent inducers of NK cell proliferation.
Key Words: Toll-like receptor • migration • chemokine • chemokine receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCs are antigen-presenting cells, which take up and process antigens in tissues and peripheral blood. Immature DCs, which are particularly good at antigen capture and processing, migrate to draining secondary lymphoid tissues, where they present antigen to naive T cells and NK cells [10 ]. To initiate a productive T cell response, immature DCs must undergo a maturation or activation process, which results in the expression of high levels of major histocompatibility complex (MHC) and costimulatory molecules to present peptides to and activate T cells. DCs serve as sentinel cells in virtually every tissue, and very small numbers of activated DCs are efficient at generating adaptive immune responses against invading pathogens through direction of T helper (Th) cell differentiation and processing of pathogen molecules.
Recent studies have shown that DCs may also enhance the cytotoxic functions of NK cells [11 ], which are critical effector cells of the innate immune system, recognizing and killing target cells expressing virus-encoded proteins and tumor cells, which have lost their expression of MHC class I antigens [12 ]. Subpopulations of NK cells are also responsible for an early inflammatory cytokine response [13 , 14 ]. Activated DCs can prime resting NK cells, which in turn, induce DC maturation or death of immature DCs [11 , 15 ]. Indeed, this DC-NK cell cross-talk may play a pivotal role as the first line of host defense against bacterial and viral infection.
In the present report, we compared the phenotypic and functional DC maturation response to flagellin and LPS in vitro and in vivo. Prior studies have reached different conclusions regarding whether flagellin can induce maturation of murine myeloid-derived DCs in vitro [16 17 18 ], and there has been only one study examining direct interactions between flagellin and NK cells [7 ]. In addition to examining the capacity of flagellin to induce DC maturation in vivo and in vitro, we illustrate that flagellin and LPS recruit DCs to draining lymph nodes, where flagellin increases NK cell recruitment. Finally, flagellin and LPS can induce NK cell proliferation. These data suggest that pathogen-dependent DC-NK cell cross-talk occurs in lymph nodes, and the biological responses induced by flagellin and LPS may contribute to the physiological responses in bacterial infection and sepsis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
LPS from Escherichia coli strain 0111:B4 was purchased from Sigma Chemical Co. (St. Louis, MO).
Expression and purification of flagellin
The fliC gene was inserted as a 1.2-kb polymerase chain reaction product into the Nde-BamHI sites of the plasmid pET15bVP. The resulting plasmid, pET15bVPC, was introduced into E. coli BL21 (pLysS; Novagen, Madison, WI), which contains the T7 polymerase gene on the chromosome under the control of the lacUV5 promoter. Bacterial cultures were grown to an absorbance at 550 nm 0.4–0.5, and the T7 promoter was induced by the addition of a 1.0-mM final concentration of isopropyl-ß-D-thiogalactopyranoside. The cultures were grown for 3 h more and harvested. These pellets were resuspended in 1x binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The cell lysate was prepared by disrupting the cells in a French pressure cell at 16,000 lb/in2 and spinning at 20,000 g for 20 min, and inclusion bodies having insoluble His-FliC were collected and resuspended in 1x binding buffer with 6 M urea and incubated on ice for 1 h. Insoluble material was removed by centrifugation at 39,000 g for 20 min. Supernatants were filtered and loaded onto a column containing 2.5 ml chelating Sepharose Fast Flow resin (Pharmacia Biotech, Inc., Piscataway, NJ). The His-FliC protein was eluted with 1x elution buffer (1 M imidazole, 0.4 M NaCl, 20 mM Tris-HCl, pH 7.9) containing 6 M urea. The protein was dialyzed against 1 L vol phosphate-buffered saline (PBS) with step-wise decreases in urea concentration. To remove any possible contaminating LPS, the preparation was passed through a polymyxin B column, resulting in a flagellin preparation, which contained less than 1 pg endotoxin/ìg protein using Pyrochrome limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA). Purified proteins were quantified by Bio-Rad protein assay (Bio-Rad, Hercules, CA). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining (Laemmli gels).
Other investigators have used recombinant flagellin in similar in vivo studies [19 ]. To verify that the recombinant flagellin preparation was acting specifically through Toll-like receptor 5 (TLR5) or TLR11 ligation and not through some unknown contaminant or improper folding, the preparation was tested for its ability to release interleuklin (IL)-8 from A549 cells. Included as a control for interaction with TLR5 was another recombinant flagellin preparation, wherein the flagellin TLR5 binding site was mutated at a single amino acid residue replacing leucine 88 with alanine using site-directed mutagenesis (manuscript in revision,Infect. Immun.). This mutant flagellin was expressed in E. coli and purified on a nickel column in a manner identical to the wild-type flagellin preparation. This mutant flagellin resulted in an 80-fold reduction in the release of IL-8 compared with the wild-type flagellin.
Generation of bone marrow-derived DCs
DCs derived from murine bone marrow were generated as described previously [20
]. Briefly, bone marrow cells harvested from tibiae and femurs of mice were depleted of red blood cells (RBC) with lysis buffer (150 mM NH4Cl, 10 mM NaHCO3, and 0.4% EDTA). Cells (2.0x106) were cultured in Petri dishes (100 mm diameter, Falcon, Heidelberg, Germany) in 10 ml culture media (RPMI 1640, Cellgro, Herndon, VA) with 10% heat-inactivated fetal bovine serum, 0.000375% 2-mercaptoethanol (Sigma Chemical Co.), and 1% penicillin-streptomycin-neomycin (Gibco, Grand Island, NY), supplemented with 2000 U granulocyte macrophage-colony stimulating factor (GM-CSF; Peprotec, Frankfurt, Germany). On Day 3, 10 ml culture medium containing 2000 U GM-CSF was added to the dishes. On Day 6, 10 ml media were removed and centrifuged, and the pelleted cells were resuspended in 10 ml fresh culture medium containing 2000 U GM-CSF. On Day 8, nonadherent and loosely adherent cells were harvested, washed twice with PBS, and used for subsequent experiments.
Negative isolation of NK cells
NK cells were isolated from spleens of naïve C57BL/6 mice. Single-cell suspensions of splenocytes were made, RBC were lysed, and NK cells were negatively selected using a labeling kit from Miltenyi Biotec (Auburn, CA, magnetic cell sorter) in accordance with the manufacturer’s instructions. Purity of NK cells was confirmed by flow cytometric staining with DX5 and NK1.1 antibody as described below and was greater than 60%.
Cell stimulation
DCs (1x106 cells) were treated with LPS (1 µg/ml), flagellin (1 µg/ml or 10 µg/ml), or media control in 1 ml culture medium at 37°C with 5% CO2 in 24-well plates (Costar, Corning, NY) for 24 h. In some studies, comparing the activity of a wild-type versus a mutated flagellin with diminished binding to the TLR5 receptor, 10 ng/ml of the preparations was used. Subsequently, cells and media were harvested, centrifuged at 1200 rpm for 5 min, and cells were washed three times with culture medium. The supernatants were collected and stored at –80°C for cytokine analysis, and the pelleted cells were used for subsequent experiments or stained for flow cytometric analysis as described below.
Footpad injection of the flagellin and LPS and lymph node single-cell suspensions
Following induction of anesthesia with ketamine HCl, mice were injected into the left hind footpad with 1 µg or 10 µg flagellin or LPS in 50 µl PBS, using an insulin syringe and 29-gauge needle (BD Biosciences, Franklin Lakes, NJ). The doses used were comparable with those recently reported by Honko and Mizel [19
]. Twenty-four hours later, mice were killed, and bilateral popliteal lymph nodes were harvested. In some cases, peripheral blood was collected from subclavian vessels for serum cytokine assay. The lymph nodes were resuspended in 4 ml Hanks’ balanced salt solution (HBSS) without phenol red containing Ca+3, Mg+2 (Cellgro) and 100 U/ml collagenase D solution (Boehringer-Mannheim, Norwich, CT). Lymph nodes were placed into 60 x 15 mm tissue-culture dishes (Falcon, Becton Dickinson, San Jose, CA) on ice. The lymph nodes were dissected with two 30-gauge needles. The suspension was placed into 30 ml HBSS without phenol red, Ca+3, or Mg+2 (Cellgro) on ice. The tissue fragments remaining in the dish were resuspended in 4 ml HBSS with 400 U/ml collagenase D and transferred to a 15-ml conical tube. These tubes were placed in a 37°C water bath for 30 min, and the solutions were pipetted vigorously, creating a single-cell suspension. The cell suspension was filtered through a 70-µm mesh filter (Falcon) into a 50-ml conical tube containing the previously acquired cells. The cells were suspended with 50 ml HBSS and centrifuged at 1500 rpm for 10 min at 4°C. The supernatant was aspirated, and the cells were resuspended in 50 ml HBSS and centrifuged. The cells were resuspended in 5 ml HBSS and counted on a hemacytometer. Cells (1x106) were dispensed into 5 ml polystyrene round-bottom tubes and stained for flow cytometry as described below.
Flow cytometry
Cells were washed twice with 1% bovine serum albumin (BSA) flow buffer, 1 mM EDTA (Fisher Scientific, Atlanta, GA), and 0.1% sodium azide (Sigma Chemical Co.) in HBSS without phenol red, Ca+3, and Mg+2 (Cellgro) and were resuspended in 4% BSA flow buffer and blocked with Fc antibodies (PharMingen, San Diego, CA) for 30 min. Stimulated DCs or cells from popliteal lymph nodes were stained using fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse MHC class II antibody (PharMingen), R-phycoerythrin (PE)- or allophycocyanin (APC)-conjugated hamster anti-mouse CD11c antibody (PharMingen), 7-amino-actinomycin D (7-AAD; PharMingen), and PE- or biotin-conjugated rat anti-mouse CD86 and biotin-conjugated anti-mouse CD8 antibody (PharMingen). For monitoring plasmacytoid DCs, FITC-conjugated anti-CD86, MHC class II, or CD11c was combined with PE-conjugated rat anti-mouse plasmacytoid DC antigen-1 (PDCA-1) antibody (Miltenyi Biotec) and APC-conjugated rat anti-mouse B220 antibody (PharMingen). When examining T cells, FITC- or PE-conjugated hamster anti-mouse CD69 antibody (PharMingen), FITC- or PE-conjugated rat anti-mouse CD25 antibody (PharMingen) and biotin, or FITC-conjugated rat anti-mouse CD4 antibody (PharMingen) were used. For monitoring NK cells, PE-conjugated rat anti-mouse DX5 antibody (PharMingen), APC-conjugated anti-mouse NK1.1 antibody (PharMingen), and FITC-conjugated hamster anti-mouse CD3
(PharMingen), CD25, or CD69 were used. Cells were incubated for 15 min at room temperature, washed with 1% BSA flow buffer, and then centrifuged at 1200 rpm for 5 min. Staining groups, which used a biotin-conjugated antibody, cells were then placed in 100 µl 1:1000 APC-conjugated streptavidin (Molecular Probes, Eugene, OR) in 1% BSA flow buffer. During analysis, debris and 7-AAD+ cells were excluded through gating. DCs were identified using anti-CD11c and anti-MHC class II antibody, and DC maturation was determined based on the relative levels of MHC class II and CD86 expression. Samples were analyzed on a FACScan® (Becton Dickinson) and a specialized software package (CELLQuestTM, Becton Dickinson). Isotype controls (PharMingen) were used for all analyses and included FITC, PE, or APC-conjugated anti-rat immunoglobulin G1 (IgG1), anti-rat IgG2a, anti-rat IgG2b, anti-rat IgM, and anti-hamster IgG1.
NK cell proliferation
Enriched NK cells were labeled with carboxy-fluorescein diacetate succinimidyl ester (CFSE; Renovar, Madison, WI) using the manufacturer’s instructions. Briefly, NK cells were resuspended in PBS, and CFSE was then added to a final concentration of 1 µM. The suspension was mixed immediately following CFSE addition and incubated for 10 min in a 37°C water bath. The labeled cells were washed twice with PBS and resuspended in culture medium. NK cells (2x105)/100 µl were then seeded in 96-well round-bottom plates (Costar). Then, 2 x 105/100 µl flagellin, LPS, and untreated DC or 1 µg/ml flagellin, 1 µg/ml LPS, or medium alone were added and incubated for 48 h. Cells were washed twice with 1% flow buffer and stained with 7-AAD and APC-conjugated NK1.1. The division profiles of NK cells were analyzed based on the CFSE content, reflected by halving of CFSE fluorescence intensity in progeny cells on 7-AAD-negative and NK1.1-positive cells. The supernatants were collected and stored at –80°C for cytokine analysis.
Cytokine measurement
Murine IL-10, IL-12 p40, IL-12 p70, tumor necrosis factor (TNF-), and interferon- (IFN-) in the supernatant were measured by specific enzyme-linked immunosorbent assay (ELISA) using commercially available reagents (PharMingen).
Statistical analysis
Data were analyzed using the statistical software program StatView 5.0 statistical software package (Abacus Concepts, Berkeley, CA) and are reported as the mean ± SEM. For multivariant comparison among groups, a one-way ANOVA was performed with post-hoc analysis using Fisher least significant difference method. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by DCs, and flagellin was generally more stimulatory regarding cytokine production than the same dose (1 µg) of LPS (Fig. 1B
and 1D)
. Although not shown, flagellin- and LPS-stimulated DCs demonstrated reduced endocytosis with FITC-conjugated dextran and increased chemotaxis toward macrophage-inflammatory protein-3ß, the chemokine for mature but not immature DCs (data not shown). Thus, flagellin and LPS induced the activation of murine bone marrow-derived DCs in a comparable manner. To confirm that the induction of DC maturation was secondary to specific interaction to a TLR-binding site, the mutant flagellin, which was attenuated in its ability to induce IL-8 expression from A549 cells, was used as a control. At concentrations as low as 10 ng/ml, wild-type flagellin, but not the mutant, induced significant expression of IL-12p40, the most sensitive activation marker of DC maturation (Fig. 2
).
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, IL12 p70, and IL10 were below the level of detection in control, flagellin, and LPS-injected mice, whereas p40 concentrations were detectable but unchanged. Only the footpad injection of 10 µg LPS had a modest but significant increase in serum IL12 p40 levels compared with vehicle injections (data not shown), suggesting that this dose of LPS had local and systemic consequences. We subsequently examined the cellularity in the bilateral popliteal lymph nodes 24 h after the footpad injection of flagellin or LPS to measure the local ipsilateral response to footpad injection versus the systemic response in the contralateral lymph node. As shown in Figure 3A , flagellin and LPS dramatically increased the size of the ipsilateral popliteal lymph nodes, as compared with the contralateral lymph node or following vehicle injection. No difference in lymph node size among the three groups was observed in the contralateral lymph nodes. Consistent with the lymph node size, flagellin and LPS dose-dependently increased the total cellularity in the ipsilateral lymph nodes (Fig. 3B) . It is interesting that the 10 µg LPS injection increased the total cell numbers in the contralateral lymph nodes. Taken with the serum IL-12 p40 results, this suggests that the footpad injection of 10 µg LPS may have induced a systemic response.
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+ lymphoid DC, CD8– myeloid DCs, or PDCA-1+, B220+ plasmacytoid DCs. Flagellin and LPS footpad injection predominantly increased the CD8–DC in the draining lymph nodes 24 h after footpad injection (Fig. 5A
) and decreased CD8+MHC class IIlow cells in the lymph nodes (Fig. 5B)
. It is interesting that LPS did not significantly increase the numbers of plasmacytoid DCs (Fig. 6A
) in the ipsilateral lymph node, whereas flagellin did. Neither LPS nor flagellin, however, increased the percentage of or activation/maturation state of the plasmacytoid DCs (PDCA-1+, B220+; Fig. 6B
) nor altered the degree of CD11c expression.
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+ cell or NKT cells in the lymph nodes and found virtually identical results to those of total NK cells in each treatment group (data not shown).
Flagellin, LPS, and activated DCs differentially regulate NK cell proliferation, activation, and IFN- production in vitro
Because of the differential in vivo response in the popliteal lymph nodes, we studied the direct effects of flagellin and LPS on NK cells and on DC-NK cell interactions. First, NK cell proliferation was examined after 48 h of treatment with untreated or flagellin- or LPS-stimulated DCs using CFSE labeling of NK cells. Incubation of NK cells with flagellin or flagellin-treated DCs induced a dramatic increase in NK cell proliferation, whereas treatment with LPS or LPS-treated DCs only modestly increased NK cell proliferation when compared with unstimulated DCs or media alone (Fig. 9A
and 9B)
. Treatment with the flagellin-treated DCs was also a more potent activator of NK cells than LPS-treated DCs as measured by enhanced CD69 expression (Fig. 9C)
. However, direct treatment of NK cells with LPS increased the expression of CD69 more than treatment with flagellin. IFN- production was only modestly increased by treatment with flagellin-stimulated DCs or flagellin itself. Treatment of NK cells with LPS did induce IFN- production, whereas treatment with LPS-stimulated DCs only marginally increased IFN- production, although not significantly.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and IL-10. It is unlikely that contaminating endotoxin could contribute to the flagellin responses, as activation was still seen in DCs obtained from C3H/HeJ mice (endotoxin-resistant) treated with flagellin (Fig. 1
and data not shown). The recombinant flagellin used in these studies contained less than 1 pg residual LPS activity per µg protein. In addition, a mutant flagellin, which is defective in its ability to release IL-8 from A549 cells, induced less IL-12 secretion when incubated with bone marrow-derived DCs. Rather, the findings suggest that flagellin is a direct maturation agent for myeloid-derived, murine DCs. In this regard, the findings are consistent with those of Agrawal et al. [17
] and Didierlaurent et al. [18
], who observed maturation of murine myeloid-derived and human blood-derived DCs, respectively, by flagellin. The results differ, however, from the findings of Means, Luster, and co-workers [16
], who failed to observe increased maturation of bone marrow-derived DCs treated with flagellin. In their report, the myeloid DCs were generated from CD34+ progenitors using a protocol including GM-CSF and IL-4, which differed from the GM-CSF protocol we used to generate the immature DC population. GM-CSF/IL-4-derived, immature DCs also differ from GM-CSF-derived DCs in their capacity to activate NK cells [22
]. The present results also differ from the findings of Diderlaurent et al. [18
], who suggested that flagellin was not a potent inducer of IL-12 (the p40 or the p70 heterodimer) and appeared to direct the DCs toward a DC2 and Th2-promoting phenotype. In our hands, flagellin was as potent or more potent than LPS as an inducer of IL-12, particularly the p70 heterodimer (Fig. 1)
. In fact, as a maturation signal for myeloid-derived DCs, flagellin was equivalent to LPS in almost all regards. We also compared the in vivo inflammatory properties of flagellin and LPS using a local footpad injection and evaluated the cellularity and phenotype in the draining and contralateral popliteal lymph nodes. Comparison with the contralateral popliteal lymph node provided a means to distinguish between systemic and compartmentalized responses to the footpad injection of these TLR agonists. We have used this footpad injection in the past to examine the regulation of DC maturation in response to adenovirus and IL-10 [21 ]. Similarly, Terme et al. [22 ] injected immature and activated murine bone marrow-derived DCs into the footpads of mice and documented a differential NK cell activation in the draining popliteal lymph node. The model excels as an in vivo tool to examine the cellular responses of the innate immune response to a specific TLR agonist and to examine the interactions among antigen-presenting cells and effector cells of the innate and acquired immune responses. We observed that in vivo administration of LPS or flagellin increased the number and maturation status of DCs, as well as the activation of CD4+T cells in the draining popliteal lymph node, consistent with an in vivo maturation of DCs associated with their emigration to the draining lymph node.
DC subsets are characterized by the expression of different cell-surface markers [10
]. In lymph nodes, CD8 expression on DCs appears to reflect a state of activation, maturation, and mobilization rather than ontogeny. One hypothesis has been that CD8+ and CD8– DCs exhibit distinct biological functions, such as the ability of CD8– DCs to induce tolerance [23
, 24
], as well as exhibiting distinct localization; i.e., CD8+ DCs mainly reside in the T cell zone and CD8– DCs, in marginal zones [25
]. In the present study, LPS and flagellin induced a preferential increase in the number of mature DCs and CD8– DCs after footpad injection. It is surprising that flagellin and to a much lesser extent, LPS stimulated an increased number of plasmacyotid DCs in the draining lymph node (only the flagellin-treated group attained statistical significance). However, the increased numbers of plasmacytoid DCs were not associated with any increased activation state of these cells, consistent with a lack of direct effect of LPS or flagellin on plasmacytoid DC activation [26
].
NK cells are the principal effector cell population of the innate immune response and through their production of cytokines and the lysis of transformed cells, are crucial for controlling infection and immune surveillance. Recent studies have shown that NK cells express many of the TLR receptors, making them capable of interacting directly with products of microbial infection [7 , 27 ]. Chalifour et al. [7 ] has recently shown that splenic NK cells express TLR5 and can respond directly to flagellin. However, NK cells need not interact with microbial antigens directly but can also be activated through direct interactions with DCs. NK and DCs can communicate bidirectionally through paracrine and juxtacrine signaling, resulting in their cellular activation, maturation, and even death. Cell-to-cell contact, as may occur in the draining lymph node, is required for optimal costimulatory effects [11 ]. As NK cells are frequently found in the T cell region of resting lymph nodes, placing them in close proximity to activated DCs, the lymph node is likely crucial for the DC-mediated NK cell response within the lymph node microenvironment.
It is important that the footpad injection of flagellin dramatically increased NK cell number and activation status in the draining lymph node. Unfortunately, the footpad injection cannot address the specific question of whether the response to flagellin was mediated by direct interactions with the protein itself (via TLR5 receptor expression) or through cell-to-cell contact with flagellin-activated DCs. Coculture experiments with NK cell-enriched populations from the spleen suggest that flagellin itself as well as DCs activated with flagellin can induce NK cell proliferation, although these data are limited by the fact that the NK cell preparation was not pure. The former data are consistent with the findings of Chalifour et al. [7
], who observed increased NK and NKT cell activation and IFN- production. It is surprising that at the doses studied, LPS was a less-potent stimulant of NK cell proliferation, although IFN- production was induced to a greater extent than with flagellin. Chalifour et al. [7
] also observed a reduced IFN- production by NK cells in response to flagellin when compared with the TLR2 agonist Klebsiella membrane protein A.
In conclusion, flagellin has been shown to produce a number of pathological responses in vivo, and its recognition by the innate immune system through TLR5 signaling contributes to the pathogenesis of bacterial infection and septic shock. The studies reported here now provide mechanistic insights into how flagellin may induce these physiologic perturbations during Gram-negative sepsis. We demonstrate that flagellin is a potent in vivo activator of DCs and NK cells and enhances interactions between these cell types (increased NK proliferation and activation status) in coculture. We also show that local exposure to flagellin increases the number of activated DCs and T cells in the draining lymph nodes after local insult and also increases the number of NK cells. Increased NK and NKT cell activity may contribute directly to the pathogenesis of septic shock [28 ]. The increased NK cell number and activation may be secondary to direct interactions between flagellin and NK cells or indirectly, through flagellin-induced activation of DCs. These results suggest that although DC and NK cell responses through TLR4 and TLR5 are similar in vivo and in vitro, there are some subtle differences that may cause differential responses.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received January 25, 2005; revised June 2, 2005; accepted June 16, 2005.
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T. Uchida, P. O. Scumpia, D. M. Murasko, S. Seki, S. Woulfe, M. J. Clare-Salzler, and L. L. Moldawer Variable Requirement of Dendritic Cells for Recruitment of NK and T Cells to Different TLR Agonists J. Immunol., March 15, 2007; 178(6): 3886 - 3892. [Abstract] [Full Text] [PDF]
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