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Originally published online as doi:10.1189/jlb.0203062 on September 2, 2003

Published online before print September 2, 2003
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(Journal of Leukocyte Biology. 2003;74:1064-1073.)
© 2003 by Society for Leukocyte Biology

CD40-mediated up-regulation of Toll-like receptor 4-MD2 complex on the surface of murine dendritic cells

Davor Frleta, Randolph J. Noelle and William F. Wade1

Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire

1 Correspondence: Department of Microbiology and Immunology, 630W Borwell Building, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756. E-mail: William.F.Wade{at}Dartmouth.Edu


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ABSTRACT
 
Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, which are non-self macromolecular components of pathogens that allow the innate-immune system to recognize infection. TLRs are expressed on macrophages and dendritic cells (DC). TLR stimulation or CD40 agonists can induce inflammatory cytokine secretion from macrophages and DC, and promote DC maturation. The regulation of TLR expression by inflammation has begun to be explored. Our studies have focused on the regulation of TLR4 surface expression on DC. TLR4, along with the adaptor molecule MD2, is involved in the recognition of lipopolysaccharide (LPS). CD40 stimulation via cross-linked anti-CD40 monoclonal antibody (mAb) up-regulates TLR4-MD2 surface expression on a DC cell line (DC2.4) and on ex vivo-cultured splenic DC. LPS treatment down-regulated surface TLR4-MD2 on DC2.4 cells, but if combined with anti-CD40 mAb, increased TLR4-MD2 expression was observed. The increased TLR4-MD2 surface expression by any treatment did not correlate with TLR4 mRNA levels. The functional consequence of increased TLR4-MD2 expression following LPS and anti-CD40 treatment was examined. Although CD40 prestimulation did slightly enhance interleukin-12p70 secretion after LPS restimulation, simultaneous anti-CD40 mAb and LPS treatment, which up-regulates TLR4-MD2 complex, does not restore DC responsiveness to subsequent LPS.

Key Words: lipopolysaccharide • CD40 • toll-like receptors


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INTRODUCTION
 
ToXll-like receptors (TLRs) are mammalian homologues of the Drosophila protein Toll. A common extracellular leucine repeat region and an intracytoplasmic Toll/interleukin (IL)-1 receptor homology (TIR) domain identify TLRs [1 ]. TLRs are involved in the recognition of pathogen-associated molecular patterns (PAMPs), macromolecular components of pathogens such as bacteria and viruses. PAMPs are absent or are rarely found in vertebrates, thus allowing the vertebrate-immune system to distinguish them as "non-self." TLR4 recognizes lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell membrane [2 ]. Gram-positive bacterial cell-wall components such as peptidoglycan (PGN) are recognized by TLR2 [3 ]. TLR3 recognizes double-stranded RNA [4 ], which is indicative of many viral infections, and TLR5 recognizes bacterial flagella [5 ]. TLR9 is critical for immune stimulation in response to bacterial DNA or CpG-DNA [6 ].

Some TLRs have been shown not to directly bind to the PAMPs to which they respond [7 ]. Rather, scavenger receptors, e.g., CD14, bind to PAMPs and interact with the TLRs as a signaling complex [8 ]. One of the best-understood TLRs is TLR4, whose knockout (KO) mice do not respond to LPS [9 ]. Mutations in the TIR domain of TLR4 ablate LPS responsiveness and are the reason for LPS nonresponsiveness exhibited by C3H/HeJ mice [2 ]. However, TLR4 does not directly bind to LPS. The plasma membrane-expressed receptor CD14 binds LPS, and LPS-bound CD14 forms a complex with TLR4 to initiate the TLR signaling pathway [8 ]. Another protein that has been shown to be important for this interaction is the extracellular adaptor protein MD2 [10 ], which interacts with TLR4 and is critical for TLR4-mediated LPS recognition and signaling, as MD2 KO mice do not respond to LPS [10 ]. Furthermore, TLR4 does not reach the cell surface in the absence of MD2, instead TLR4 remains blocked in the Golgi complex [10 ].

TLR stimulation has been shown to induce various innate immune responses and modulate adaptive immune response in humans and mice [11 ]. TLR stimulation of macrophages with LPS, PGN, or CpG-DNA induces inflammatory cytokine secretion and the production of reactive oxygen intermediates [12 13 14 ]. B cell proliferation can be induced with LPS [12 ], and CpG-DNA has been shown to enhance the lytic activity of natural killer (NK) cells [15 ]. TLR stimulation also promotes dendritic cell (DC) maturation [16 , 17 ], which is biphasic: Immature DC are adept at antigen uptake but not antigen presentation. Upon maturation with various stimuli, they lose their antigen-uptake ability but up-regulate major histocompatibility complex class I and II molecules, as well as costimulatory molecules that make them efficient at activating naive T cells [18 ]. DC are believed to act as the bridge between the innate and adaptive immune response, and DC maturation is critical for initiating an adaptive immune response [18 ]. LPS has been well described as a potent maturation stimulus for DC [17 18 19 20 ]. LPS induces high levels of IL-12 secretion from DC, thereby potentiating a T helper 1 immune response [20 ]. As LPS is important for the DC priming in response to a bacterial infection, we investigated how the TLR4-MD2 complex is regulated in response to CD40 stimulation, a critical component of the adaptive immune response. CD40 is expressed on a variety of cells, especially professional antigen-presenting cells (APCs) such as B cells, macrophages, and DC [21 ]. The ligand for CD40 (CD154) is primarily found on activated T cells, and CD40 is a costimulatory molecule that has an important role in modulating APC activity [22 ]. CD40 ligation on DC enhances T cell expansion through conditioning of DC maturational phenotype [22 ]. Whereas it is well known that LPS up-regulates CD40 on DC, it is not known how CD40 stimulation of DC affects TLR4-MD2 expression.

The regulation of selected TLR expression has recently been studied in DC [23 24 25 26 27 ]. DC subsets express specific TLRs [23 ] that likely play a role in regulating the effector arm of immune responses. Inflammatory mediators can regulate TLR expression [24 25 26 27 ]. LPS up-regulates TLR2, -4, and -9 mRNA in murine DC [24 ] and up-regulates TLR9 mRNA in murine macrophages [25 ]. In contrast to its effect on TLR4 mRNA, LPS stimulation down-regulates TLR4-MD2 surface expression on murine peritoneal macrophages [26 ]. Inflammatory cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}) have also been shown to up-regulate TLR2 mRNA in murine macrophages [27 ]. TLR responsiveness is regulated at the signaling level as well as TLR expression. Initial LPS stimulation inhibits DC and monocytes from responding to secondary LPS stimulation. This is a phenomenon referred to as "endotoxin or LPS tolerance" [26 ].

We assessed how CD40-mediated maturation affects TLR4-MD2 expression on DC. We found that CD40 stimulation up-regulates TLR4-MD2 surface expression on the DC cell line, DC2.4, as well as splenic DC cultured ex vivo. LPS down-regulates TLR4-MD2 on the surface of DC2.4 cells, but simultaneous LPS and anti-CD40 monoclonal antibody (mAb) treatment up-regulate TLR4-MD2 on DC2.4 cells and ex vivo-cultured splenic DC. The surface levels of TLR4-MD2 were higher than with anti-CD40 mAb treatment alone, indicating that LPS enhances CD40-mediated up-regulation of surface TLR4-MD2. The CD40-mediated up-regulation of TLR4-MD2 induced higher IL-12p70 production on LPS stimulation. However, CD40 prestimulation was not able to fully overcome LPS tolerance, indicating that DC are still less responsive to LPS after primary exposure to LPS, even with CD40 stimulation.


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MATERIALS AND METHODS
 
Mice
Three- to 5-week-old female recombination-activating gene (RAG) KO and wild-type C57BL/6 mice provided by the National Cancer Institute (Bethesda, MD) were housed under pathogen-free conditions in the Animal Resource Center at the Dartmouth-Hitchcock Medical Center (Lebanon, NH). Mice were injected intraperitoneally with 10 µg fetal liver tyrosine kinase 3 ligand (Flt3L) every day for 8 days. Flt3L was made in Dr. Noelle’s laboratory and represents a recombinant protein: Flt3L attached to human immunoglobulin G (IgG) Fc region. Spleens were harvested from mice after 8 days of Flt3L treatment and were used to prepare splenocytes for culture as described previously [28 ].

Cells
DC2.4 cells (kindly provided by Dr. Kenneth L. Rock, University of Massachusetts, Worcester) were established from bone marrow cells transduced with murine granulocyte macrophage-colony stimulating factor (GM-CSF) followed by retroviral transfection with raf and myc oncogenes [29 ]. Cells were cultured in RPMI-1640 media supplemented with 10% fetal clone serum (Hyclone, Logan, UT), antibiotics, ß-mercaptoethanol, and L-glutamine. DC2.4 cells were grown at 37°C in a humid environment with 5% CO2. Splenocytes were prepared by mechanical disruption of spleens harvested from Flt3L-treated mice followed by ammonium chloride treatment to lyse red blood cells. Splenocytes were cultured under the same media and conditions as DC2.4 cells. Splenic DC were purified from splenocytes by magnetic sorting using anti-CD11c microbeads (Miltenyi, Auburn, CA), according to the manufacturer’s instructions as described previously [28 ].

Reagents
FGK45, a CD40 agonist (a gift from Antonius Rolink, University of Basel, Basel, Switzerland) is a rat IgG2a mAb. Rat IgG polyclonal Ab, antibody control for FGK45, was purchased from Sigma Chemical Co. (St. Louis, MO). FGK45 and rat IgG had levels of endotoxin that were below detection with a limulus amebocyte assay (BioWhittaker, Walkersville, MD). MTS510 (biotinylated) is a rat IgG2a mAb specific for murine TLR4-MD2 complex and was purchased from Cell Sciences (Norwood, MA). Anti-CD11c-fluorescein isothiocyanate (FITC; 0.5 mg/mL), anti-CD80-phycoerythrin (PE; 0.2 mg/mL), anti-CD54-PE (0.2 mg/mL), and biotinylated anti-transferrin receptor (TfR; 0.5 mg/mL) were purchased from BD PharMingen (San Diego, CA). Streptavidin-conjugated allophycocyanin (StrepAPC; 0.1 mg/mL) was purchased from Caltag (Burlingame, CA). LPS from Escherichia coli serotype 055:B5 was purchased from Sigma Chemical Co. Murine TNF-{alpha} and GM-CSF were purchased from Peprotech (Rocky Hill, NJ).

In vitro CD40 stimulation of cells
DC2.4 or splenocytes were cultured in a six-well plate with 2 mL media and were first treated with 10 µg/mL anti-CD40 mAb (FGK45) or 10 µg/mL rat IgG control Ab or were left untreated. Cells were kept at 4°C for 45 min to allow Ab binding and then treated with 10 µg/mL donkey anti-rat IgG Ab and/or 100 ng/mL LPS. Some samples were also treated with 10 ng/mL TNF-{alpha}. Cells were then cultured at 37°C in 5% CO2 for 6 h. After 6 h, cells harvested were prepared for fluorescence-activated cell sorting (FACS) or real-time reverse transcriptase-polymerase chain reaction (RT-PCR). For kinetic experiments, cells were cultured as described for 3, 6, 12, or 24 h before harvesting for FACS analysis. For dose titration experiments, anti-CD40 mAb was used at 0.1, 1, or 10 µg/mL, and LPS was used at 0.1, 1, or 100 ng/mL.

Flow cytometry
DC2.4 cells and splenocytes were treated with 2.4G2 mAb to block any nonspecific Ab binding through Fc receptors [19 ]. Cells were then stained with biotinylated anti-TLR4-MD2 (MTS510 mAb) followed by secondary staining with StrepAPC and anti-CD80-PE or anti-CD54-PE. Splenocytes were further stained with anti-CD11c-FITC to distinguish DC. All antibodies were diluted 1:50 for staining. Stained cells were analyzed using a Becton Dickinson FACScalibur flow cytometer and Cellquest software (BD Immunocytometry Systems, San Jose, CA). Viable cells were discriminated by forward- and side-scatter. The mean fluorescence intensity (MFI) of TLR4-MD2, CD80, and CD54 was determined. MFI of the various marker per given treatment was normalized as a percentage of the MFI using the same marker on untreated cells.

IL-12 enzyme-linked immunosorbent assay (ELISA)
Ex vivo DC were pretreated with rat IgG Ab or anti-CD40 mAb, LPS, or both or were left untreated as described above for 24 h, and then media with stimuli were removed, and the cells were washed 2x with fresh media. Cells were restimulated with 100 ng/mL LPS or were left untreated for an additional 24 h, and then supernatants were harvested. IL-12p70 levels in supernatants were assessed by ELISA using the IL-12 OptEIA kit from BD PharMingen, according to the manufacturer’s instructions. Briefly, enzyme immunoassay/radioimmunoassay plates (Costar, Corning, NY) were coated with anti-IL-12p70 capture antibody and then blocked with phosphate-buffered saline + 10% fetal clone serum. Individual supernatant were then added followed by secondary reagents consisting of biotin-conjugated anti-IL-12p70 mAb and horseradish peroxidase-conjugated avidin, with thorough washing in between individual steps. The color reaction was developed using tetramethyl benzidine (KPL, Gaithersburg, MD), and IL-2p70 levels were determined in supernatant samples by comparison to an IL-12p70 standard curve.

Real-time RT-PCR
Whole RNA was extracted from ~2 x 106 DC2.4 cells with Trizol reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer. Isolated RNA samples were digested with DNase I (Invitrogen) to remove contaminating genomic DNA. RNA (3 µg) from each treatment sample was reverse-transcribed using Superscript RNase H- RT (Invitrogen). cDNA from various samples was analyzed for TLR4 levels by real-time PCR using a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) after 40 cycles in an iCycler Thermal Cycler (Bio-Rad, Hercules, CA). Relative levels of TLR4 cDNA were determined by analyzing SYBR Green Fluorescence and were normalized to ß-actin levels. All primers were provided by Integrated DNA Technologies (Coralville, IA) and are as follows: TLR4 forward primer: 5'-GGAAGTTTCTCTGGACTAACAAGTTTAGA-3', TLR4 reverse primer: 5'-AAATTGTGAGCCACATTGAGTTTC-3'; ß-actin forward primer: 5'-AGAGGGAAATCGTGCGTGAC-3', ß-actin reverse primer: 5'-CAATAGTGATGACCTGGCCGT-3' [30 ].

Statistics
Statistical analysis was performed using PRISM 3.02. (Graphpad, San Diego, CA). Means and SEM were calculated for each experimental condition. Unpaired Student’s t-test was performed to test for significance in Figures 1 and 2 ; an ANOVA test was used to determine statistical significance in Figures 3 4 5 6 . A P value of <0.05 was considered significant. Standard error bars are shown in all figures, and data with no standard error bars indicate a SEM that is too small to be seen.



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  		Figure 1. CD40 stimulation up-regulates TLR4-MD2 surface expression on DC2.4 cells, which were treated with anti-CD40 mAb, rat IgG isotype-control Ab, TNF-{alpha}, or LPS for 6 h as described in Materials and Methods and then prepared for FACS analysis. (A) FACS profiles of TLR4-MD2 levels on untreated, LPS, or anti-CD40 mAb-treated cells. The isotype control for nonspecific staining is shown (dotted line). Cells treated with rat IgG control or LPS/anti-CD40 mAb are not shown for sake of clarity; cells treated with rat IgG Ab have TLR4-MD2 levels identical to that on untreated cells, and LPS/anti-CD40 mAb-treated cell staining for TLR4-MD2 obscures the histogram of cells treated with anti-CD40 mAb alone. (B) Graphical analysis of TLR4-MD2 levels of cells under indicated treatment conditions as A. Results are shown as the percentage of the TLR4-MD2 MFI for variously treated cells to the TLR4-MD2 MFI of untreated cells. Data represent the mean of three independent experiments with SEM shown. *, A P value of <0.05 is considered statistically significant from untreated levels. (C) Expression of TfR on DC2.4 cells. (D) TLR4-MD2 levels were assessed by FACS analysis of DC2.4 cells treated with anti-TfR mAb followed by cross-linking with donkey anti-rat IgG Ab or treated with donkey anti-rat IgG Ab only.



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  		Figure 2. TLR4-MD2 expression in response to dose titration of LPS or anti-CD40 mAb treatment of DC2.4 cells. DC2.4 were treated with (A) 0.1, 1, or 10 µg/mL anti-CD40 mAb for 6 h, as described in Materials and Methods, (B) 0.1, 1, or 100 ng/mL LPS for 6 h, as described in Materials and Methods, (C) 100 ng/mL LPS with indicated doses of anti-CD40 mAb, as described in Materials and Methods, or (D) 10 µg/mL anti-CD40 mAb with indicated doses of LPS as described in Materials and Methods. TLR4-MD2 surface levels were quantified by FACS analysis. Results are shown as the percentage of the TLR4-MD2 MFI for variously treated cells to the TLR4-MD2 MFI of untreated cells. Data represent the mean of at least three independent experiments with SEM shown. *, A P value of <0.05 is considered statistically significant from untreated levels.



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  		Figure 3. Kinetics of surface TLR4-MD2 up-regulation on DC2.4 cells following anti-CD40 mAb treatment. (A) DC2.4 cells were treated with anti-CD40 mAb, rat IgG isotype control, or LPS for 3, 6, 12, and 24 h as described in Materials and Methods and were then prepared for FACS analysis. Results are shown as the percentage of the TLR4-MD2 MFI for variously treated cells to the TLR4-MD2 MFI of untreated cells. (B) DC2.4 cells were first treated with 100 ng/mL LPS or left untreated for 6 h at 37°C in 5% CO2. After 6 h, cells were treated further with anti-CD40 mAb or rat IgG isotype-control Ab, as described in Materials and Methods, or were left untreated and cultured for an additional 6 h. TLR4-MD2 levels were quantified by FACS analysis. Results are shown as the percentage of the TLR4-MD2 MFI for variously treated cells to the TLR4-MD2 MFI of untreated cells. Data represent the mean of at least two independent experiments with SEM shown. *, A P value of <0.05 is considered statistically significant from TLR4-MD2 levels on untreated or rat IgG-treated cells. #, TLR4-MD2 levels on anti-CD40 mAb-treated cells that had received LPS prestimulation are statistically significant (a P value of <0.05) from TLR4-MD2 levels on anti-CD40 mAb-treated cells that had not received LPS prestimulation.



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  		Figure 4. Effect of CD40 stimulation on TLR4 mRNA in DC2.4 cells, which were treated with anti-CD40 mAb, rat IgG isotype control, or LPS for 6 h as described in Materials and Methods, and total RNA was isolated and analyzed for TLR4 mRNA by real-time RT-PCR. TLR4 mRNA levels were first normalized to ß-actin mRNA levels (housekeeping gene control). The ß-actin-normalized TLR4 mRNA levels for all treatment samples were divided by the ß-actin-normalized TLR4 mRNA levels in untreated cells. A value of 1 indicates baseline levels. Data represent the mean of at least two independent experiments with SEM shown. *, A P value of <0.05 is considered statistically significant from baseline levels.



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  		Figure 5. CD40 stimulation up-regulates TLR4-MD2 on the surface of ex vivo-cultured splenic DC. Spleens were harvested from Flt3L-treated RAG KO mice and mechanically disrupted to prepare splenocytes, which were treated in ex vivo culture with anti-CD40 mAb, rat IgG isotype-control Ab, or LPS for 6 h, as described in Materials and Methods, and then TLR4-MD2 levels were assessed by FACS analysis. (A) FACS profiles of TLR4-MD2 levels on untreated cells (filled histogram). The isotype control for nonspecific staining is shown (dotted line). (B) FACS profiles of TLR4-MD2 levels on cells treated with LPS and cross-linked anti-CD40 mAb (open histogram) or cross-linked anti-CD40 mAb only (filled histogram). Histograms of cells treated with rat IgG Ab and LPS treatments are not shown for clarity; they did not differ from that of untreated cell staining. (C) Results are shown as the percentage of the TLR4-MD2 MFI for variously treated cells to the TLR4-MD2 MFI of untreated cells. Data represent the mean of two independent experiments with SEM shown. *, A P value of <0.05 is considered statistically significant from untreated levels.



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  		Figure 6. LPS responsiveness of ex vivo DC after CD40 stimulation. Splenic DC (96–98% pure) were purified from spleens of Flt3L-treated RAG KO mice. Cells were first treated with rat IgG Ab or anti-CD40 mAb, LPS, or both for 24 h, as described in Materials and Methods, and were then restimulated with 100 ng/mL LPS or were left untreated for an additional 24 h. ELISA assessed IL-12p70 levels, which were normalized to IL-12p70 levels from rat IgG-pretreated cells. Cells that were not restimulated with LPS did not produce any detectable IL-12p70 (data not shown). Data represent the mean of at least three independent experiments with SEM shown. *, A P value of <0.05 is considered statistically significant from rat IgG-treated cells that were restimulated with LPS. #, LPS/rat IgG and LPS/anti-CD40 mAb-pretreated cells are statistically different.


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RESULTS
 
CD40 stimulation up-regulates TLR4-MD2 on the surface of DC2.4 cells
To investigate receptor cross-talk, we treated DC2.4 cells for 6 h with LPS and/or rat anti-mouse CD40 mAb (FGK45), followed by donkey anti-rat IgG Ab. Cross-linking of the FGK45 mAb was necessary, as anti-CD40 mAb treatment alone failed to modulate TLR4-MD2 levels (data not shown). Representative FACS profiles of TLR4-MD2 surface expression after the various treatments are shown in Figure 1A . Treatment with a rat IgG control Ab followed by donkey anti-rat IgG for 6 h did not alter baseline expression of TLR4-MD2 (Fig. 1B) . Cross-linking CD40 increased the TLR4-MD2 surface expression six- to 15-fold higher than baseline (Fig. 1B) . Although CD40 expression is homogeneous (data not shown), CD40-mediated up-regulation of TLR4-MD2 is heterogeneous with some DC2.4 expressing more TLR4-MD2 than other anti-CD40 mAb-treated cells (Fig. 1A) .

LPS was reported to down-regulate TLR4-MD2 on monocytic cells [26 ]. LPS treatment of DC2.4 cells for 6 h also down-regulated TLR4-MD2 surface expression to 35–40% of untreated levels (Fig. 1B) . The LPS-mediated down-regulation of TLR4-MD2 is homogenous compared with the increased heterogeneous expression induced by CD40 stimulation (Fig. 1A) . Combined LPS and anti-CD40 mAb treatment resulted in up-regulation of TLR4-MD2 surface expression, implying that the CD40-mediated up-regulatory effect is dominant over the LPS effect (Fig. 1B) . Treatment of DC2.4 cells with TNF-{alpha} did not up-regulate surface TLR4-MD2, nor did it overcome the LPS down-regulatory effect on TLR4-MD2 (Fig. 1B) . TNF-{alpha} did increase CD80 and CD54 (data not shown).

To demonstrate that the specificity of the TLR4-MD2 surface expression was a result of CD40 stimulation and not an artifact of cross-linking an Ab bound to any cell-surface molecule, DC2.4 cells were treated for 6 h with rat anti-mouse TfR mAb followed by donkey anti-rat IgG. The anti-TfR mAb control differs from the rat IgG control in Figure 1B , as anti-TfR mAb binds to a plasma membrane protein (Fig. 1C) . Cross-linking TfR did not modulate TLR4-MD2 levels nor did treatment with donkey anti-rat IgG (Fig. 1D) .

LPS enhances CD40-mediated up-regulation of TLR4-MD2
To further explore how LPS and anti-CD40 mAb treatment affect TLR4-MD2 surface expression on DC2.4 cells, we titered the doses of LPS and anti-CD40 mAb but held constant (10 µg/mL) the cross-linking Ab. Anti-CD40 mAb treatment failed to induce TLR4-MD2 up-regulation at doses of 0.1 or 1 µg/mL (Fig. 2A ), although a three- to fourfold up-regulation was noted with 5 µg/mL (data not shown). As reported, 10 µg/mL is the dose for optimal CD40 stimulation [28 , 31 ]. The LPS down-regulation of TLR4-MD2 was titerable. DC2.4 cells treated with 1 ng/mL or 100 ng/mL LPS express TLR4-MD2 at 60% and 40%, respectively, of the levels of untreated cells (Fig. 2B) .

LPS (100 ng/mL) treatment in the context of increasing anti-CD40 mAb did not down-regulate TLR4-MD2 surface expression (Fig. 2C) . LPS plus 0.1 or 1 µg/mL anti-CD40 mAb did not reduce TLR4-MD2 expression as did LPS alone (Fig. 2C , compare with Fig. 2B ). Conversely, LPS treatment enhanced CD40-mediated up-regulation of TLR4-MD2 surface expression with the optimal dose of anti-CD40 mAb (Fig. 2D) . Increased concentrations of LPS increased the amount of TLR4-MD2 up-regulation in response to 10 µg/mL anti-CD40 mAb (Fig. 2D) . LPS treatment increases CD40 surface expression on DC2.4 cells (data not shown), which may play a role in the enhanced anti-CD40 mAb stimulation. However, LPS does not up-regulate CD40 surface expression at a dose of 0.1 or 1 ng/mL (data not shown), and at these doses, the enhancement of CD40-mediated TLR4-MD2 up-regulation is not statistically significant.

Kinetics of CD40-mediated up-regulation of TLR4-MD2 on the surface of DC2.4 cells
We examined over a 24-h time course the expression of TLR4-MD2 on DC2.4 cells treated with optimal doses of LPS and cross-linked anti-CD40 mAb or combinations thereof. Cells treated with rat IgG control Ab did not differ in TLR4-MD2 levels from untreated controls. CD40-stimulated TLR4-MD2 expression was maximal at 6 h of treatment, and TLR4-MD2 levels were six- to tenfold higher than levels on rat IgG-treated or untreated cells (Fig. 3A ). After 6 h of anti-CD40 mAb stimulation, TLR4-MD2 levels declined to levels still threefold higher than those on rat IgG treated or on untreated cells. CD40-mediated up-regulation of TLR4-MD2 was maintained for at least 24 h (Fig. 3A) . At 3 h post-LPS treatment and for 24 h after, DC2.4 cells down-regulated TLR4-MD2 surface expression to levels ~45% of that of untreated cells (Fig. 3A) .

LPS treatment along with cross-linked anti-CD40 mAb treatment up-regulated TLR4-MD2 expression sooner, which was sustained at higher levels than that seen with cross-linked anti-CD40 mAb treatment alone (Fig. 3A) . At 3 h, LPS/anti-CD40 mAb treatment had induced an ~threefold up-regulation in TLR4-MD2 surface expression, in contrast to less than a twofold increase with cross-linked anti-CD40 mAb alone (Fig. 3A) . Cells exposed to LPS and cross-linked anti-CD40 mAb (12 h) showed an ~tenfold increase in TLR4-MD2 levels relative to rat IgG-treated or untreated cells. In contrast, TLR4-MD2 levels were ~threefold lower on cells treated with cross-linked anti-CD40 mAb only (Fig. 3A) . At 24 h, both treatment groups (LPS/anti-CD40 mAb, anti-CD40 mAb only) had elevated (~threefold) TLR4-MD2 levels compared with baseline (Fig. 3A) .

We showed that CD40 stimulation of DC2.4 cells up-regulates TLR4-MD2 surface expression, and LPS treatment decreases it. We therefore addressed if CD40 stimulation could up-regulate TLR4-MD2 after LPS-mediated down-regulation of the complex. DC2.4 cells pretreated with LPS for 6 h were washed and then treated with cross-linked anti-CD40 mAb for an additional 6 h. CD40 stimulation increased TLR4-MD2 surface expression after 6 h of LPS treatment, which decreases TLR4-MD2 levels to ~45% of levels observed on untreated cells (Fig. 3B) . CD40 stimulation after LPS pretreatment up-regulated TLR4-MD2 surface expression ~fivefold higher than on untreated cells, whereas CD40 stimulation without LPS pretreatment induced ~tenfold higher TLR4-MD2 surface expression (Fig. 3B ; compare two solid bars).

CD40 stimulation does not affect TLR4 mRNA in DC2.4 cells
Treatment of DC2.4 cells with cross-linked anti-CD40 mAb significantly increases TLR4-MD2 surface expression at 6 h. To determine if the enhancement was a result of increased TLR4 mRNA at 6 h, we measured TLR4 mRNA using real-time RT-PCR. Consistent with its effect on TLR4-MD2 surface expression, rat IgG control Ab also did not affect TLR4 mRNA (Fig. 4 ). Cross-linked anti-CD40 mAb did not increase TLR4 mRNA (Fig. 4) . There was also no effect on TLR4 mRNA as a result of cross-linked anti-CD40 mAb treatment of DC2.4 cells for 3 h (data not shown). Six hours post-CD40 stimulation, DC2.4 cells did increase mRNA expression of the chemokine macrophage inflammatory protein-1{alpha} and IFN-inducible protein 10 by ~2.5- and ~sevenfold, respectively (data not shown). LPS treatment of DC2.4 cells for 6 h decreased TLR4 mRNA (Fig. 4) . DC2.4 cells that received LPS stimulation along with cross-linked anti-CD40 mAb treatment also had less TLR4 mRNA at 6 h (Fig. 4) , although this treatment caused the highest up-regulation of surface TLR4-MD2 at 6 h (Fig. 2) .

CD40 stimulation up-regulates TLR4-MD2 on the surface of ex vivo-cultured splenic DC
We determined if LPS and/or CD40 stimulation could regulate TLR4-MD2 surface expression on splenic DC, similar to that of DC2.4 cells. Splenocytes were isolated from Flt3L-treated, RAG KO mice and cultured in the presence of cross-linked anti-CD40 mAb, LPS, or both. RAG KO DC derived from Flt3L-treated mice have increased DC numbers, and the confounding effects of CD40 receptor competition for stimulation as a result of mature B are obviated. Experiments using Flt3L-treated C57BL/6 mice revealed the same type of modulation of the TLR4-MD2 surface expression seen using the RAG KO mice (data not shown).

After 6 h of treatment, TLR4-MD2 surface expression was assessed on DC (distinguished by gating on high CD11c-expressing cells) by FACS analysis. Untreated DC had undetectable levels of TLR4-MD2 (not significantly different from isotype-control staining; Fig. 5A ). When splenocytes were treated with cross-linked anti-CD40 mAb, DC up-regulated TLR4-MD2 surface expression (Fig. 5B and 5C) , as did DC2.4 cells. The up-regulation of TLR4-MD2 on ex vivo-treated DC was higher than on DC2.4 cells (~200-fold vs. six- to tenfold). The same treatment also induces higher absolute MFI levels of TLR4-MD2 on ex vivo-cultured DC (Fig. 5B ; compare with Fig. 1A ).

LPS did not appear to down-regulate TLR4-MD2 on ex vivo-cultured DC (Fig. 5C) , but this is because TLR4-MD2 levels on ex vivo-cultured DC are below the detectable limit of FACS, and so any down-regulation by LPS would not be discernable. However, as with DC2.4 cells, LPS enhanced the CD40-mediated up-regulation of TLR4-MD2 surface expression on ex vivo-cultured DC (Fig. 5B and 5C) . The fold up-regulation as well as the absolute MFI levels of TLR4-MD2 surface expression were higher on DC treated in culture with LPS and cross-linked anti-CD40 mAb in comparison with cells treated with cross-linked anti-CD40 mAb only (Fig. 5B and 5C) .

LPS responsiveness of ex vivo DC after CD40 stimulation
LPS stimulation and CD40 ligation induce the production of bioactive IL-12p70 from DC [32 ]. Prestimulation of splenic DC with LPS inhibits DC IL-12 production on a secondary stimulation with LPS [33 , 34 ]. We assessed functional TLR4 signaling after anti-CD40 mAb, LPS, or LPS/anti-CD40 mAb treatment that differentially modulated TLR4-MD2 expression. We tested IL-12p70 (bioactive IL-12) production in splenic DC isolated by magnetic sorting from Flt3L-treated RAG KO splenocytes. Splenic DC were 96–98% pure as determined by CD11c+ FACS analysis (data not shown). Splenic DC were treated with LPS, cross-linked anti-CD40 mAb, or both for 24 h and were then restimulated with LPS. Levels of IL-12p70 were normalized to levels secreted from cells pretreated with rat IgG control Ab. CD40 prestimulation significantly (P<0.01) enhanced LPS-mediated IL-12p70 secretion over rat IgG Ab-pretreated levels (Fig. 6 ). Prestimulation with LPS/rat IgG or LPS/anti-CD40 mAb inhibited IL-12p70 secretion upon a secondary treatment with LPS; however, IL-12p70 levels were marginally higher with LPS/anti-CD40 mAb (Fig. 6) . LPS tolerance is not complete with LPS and anti-CD40 mAb treatment; rather, the level of response is severely compromised. Rat IgG and anti-CD40 mAb-treated splenic DC produced lower IL-12p70 upon secondary stimulation with LPS compared with DC left untreated for the first 24 h (data not shown). We cannot rule out that undetectable LPS levels in the antibody aliquots may be mediating partial LPS tolerance in combination with Fc receptor effects. It is clear that anti-CD40 mAb treatment, if combined with LPS, enhances TLR4-MD2 expression, but this does not overcome the negative effect of LPS on subsequent TLR4-MD2-induced IL-12 production.


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DISCUSSION
 
The TLR4-MD2 complex detects LPS from Gram-negative bacteria [35 ]. MD2 expression is required for the surface expression of TLR4 [10 ]. Mice defective in TLR4 or MD2 do not respond to LPS [9 , 10 ]. TLR4-MD2 engagement on DC can shape how the adaptive immune system responds to infection, allowing more comprehensive effector functions [36 ]. Because of the link between innate and adaptive immunity, it is important to understand how TLR4-MD2 expression and function are regulated. We report that CD40 stimulation increases TLR4-MD2 surface expression on ex vivo splenic DC and on a DC cell line. Complex expression on DC2.4 cells peaks at 6 h before declining to levels slightly above baseline. Similar to other monocytic cells, LPS down-regulated TLR4-MD2 surface expression on DC2.4 [26 ]. Combined LPS and CD40 agonist significantly up-regulated surface TLR4-MD2 on DC2.4 cells and ex vivo splenic DC.

CD40 signaling might regulate TLR4-MD2 expression and function via a unique assemblage of TNF receptor-associated factor (TRAF) molecules. TLR and CD40 signaling pathways use the signaling intermediate protein TRAF6, which mediates nuclear factor (NF)-{kappa}B activation [32 , 37 ]. TNF receptors (TNFR1 and -2) use TRAF2 as a signaling intermediate, as does CD40 [37 ], but TNF treatment does not have the same effect on TLR4-MD2 expression as does CD40. CD40 and TNFR1 and -2 differ in that CD40 signaling uses TRAF6, and TNFR1 and -2 signaling does not. TLR4 signaling induces TRAF6 activation but not TRAF2 [37 ]. This argues for a CD40-specific signaling pathway that involves TRAF2 and TRAF6 as the means to mediate TLR4-MD2 expression.

Our data suggest that the increased TLR4-MD2 expression is not regulated by TLR4 mRNA. The decrease in the TLR4 mRNA levels we report is supported by the results of An et al. [24 ], who showed that after 6 h, LPS treatment decreased TLR4 mRNA in murine bone marrow-derived DC. Even if CD40 stimulation is increasing MD2 mRNA transcripts at steady state, the negative effect of LPS treatment on TLR4 mRNA is dominant. Lower TLR4 mRNA would probably limit TLR4-MD2 assembly and thus transport/expression of the complex at the membrane. Collectively, these results suggest that CD40-mediated regulation of TLR4-MD2 expression is post-transcriptional. Similar to its effect on class II expression on immature DC, CD40 stimulation may regulate transport of preformed intracellular stores of TLR4-MD2. TLR4 has been localized to the Golgi complex [38 ]. The MTS510 mAb used to assess TLR4-MD2 plasma membrane expression did not detect intracellular TLR4-MD2 by fluorescent confocal microscopy (D. Frleta and W. F. Wade, unpublished observations). This may be because the association of TLR4 and MD2 occurs at the plasma membrane rather than intracellularly [10 ]. However, TLR4 and MD2 association is believed to occur in the endoplasmic reticulum and requires the chaperone protein gp96 [10 ]. MTS510 mAb might not recognize intracellular TLR4-MD2 as a result of the lack of a specific conformation-specific epitope, steric hindrance by gp96, or low abundance of complex.

The current paradigm of events during a Gram-negative bacterial infection includes DC binding LPS in the peripheral tissues followed by DC migration to secondary lymphoid organs, where CD40 stimulation is received if DC have also endocytosed antigen and thus are able to interact with antigen-specific T cells [39 ]. It is possible that DC can receive a CD40 signal in the periphery from other cell types associated with the innate immune response. CD40L is expressed on NKT cells, which can activate DC via a CD40-dependent manner [40 ]. DC in the lymph node could also receive additional LPS signals following CD40 stimulation if the infection in the periphery drains to the lymph node and traffics LPS to the areas of cognate interaction.

Our data suggest that CD40 agonist can induce high TLR4-MD2 levels, despite down-regulation of the receptor by an initial LPS pretreatment. Is there a functional consequence of CD40/LPS treatment-enhanced TLR4-MD2 expression? Recent data from Gold et al. [41 ] have shown CD40 contributes to acute sepsis. They report that CD40-/- mice have less pathology than wild-type mice during sepsis. The CD40-mediated up-regulation of surface TLR4-MD2 may offer a possible mechanism for the role of CD40 in sepsis, particularly if the up-regulation is similar in other monocytic cells, such as macrophages that produce inflammatory cytokines during sepsis, as well as in DC. The CD40 effect in the context of LPS treatment is an unexpected result, given the fact that macrophages or DC treated with LPS down-regulated TLR4-MD2 and also are refractory to a secondary LPS treatment [42 , 43 ]. Receptor desensitization is independent of LPS-mediated down-regulation of TLR4-MD2 [44 , 45 ]. In general, TLR stimulation increases IL-1 receptor-associated kinase-M (IRAK-M), an adaptor protein that inhibits further stimulation with any TLR agonist by interfering with cytoplasmic domain associations required for TLR signaling [46 ].

We report that CD40 stimulation cannot fully prevent LPS-induced desensitization yet preserves partial IL-12p70 secretion when given with LPS pretreatment. In support of the IL-12p70 results, we observed, using DC2.4 cells, loss of LPS-mediated NF-{kappa}B activation upon LPS/anti-CD40 mAb pretreatment, as measured by p-I{kappa}B levels. The ability to activate proximal signaling elements (a measure of function) was affected in the same hierarchical manner as the treatments that affected IL-12 secretion in ex vivo DC (D. Frleta and W. F. Wade, personal observation).

CD40-mediated maturation of DC is being considered for several therapeutic strategies [47 , 48 ]. The developing model is that a TLR and a CD40 agonist are required for potent activation of DC [49 , 50 ]. It is believed that the synergy between TLR and CD40 stimulation optimally shapes DC maturation and subsequent activation of antigen-specific T cells. Our data indicate that a CD40 agonist up-regulates TLR4-MD2 surface expression although cannot completely overcome LPS tolerance. The potential role of partial functionality but much-reduced LPS responsiveness is not known. If CD40 activation of DC cannot reverse LPS tolerance, why does CD40 stimulation up-regulate TLR4-MD2 surface expression on DC? There are several possible explanations: (i) to allow limited LPS signaling and subsequent cytokine release; (ii) to regulate LPS toxicity by limiting signaling but enhancing ligand scavenging; (iii) to increase intracellular binding sites, which compete away IRAK-M from other TLRs and therefore relieve IRAK-M-mediated inhibition of other TLR; and (iv) it is the response to DC maturation and a result of changes in membrane biology that are not linked to downstream functions. Future studies will define the in vivo functional consequences of the CD40/LPS regulation of TLR4-MD2.


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ACKNOWLEDGEMENTS
 
This work is supported by a NIH grant (P30CA23108) to the Norris Cotton Cancer Center and a NIH Immunology Training Grant (T32A107363) to D. F. The authors thank Evan F. Lind, Kathy Bennett, and Sergio A. Quezada for providing splenocytes from Flt3L-treated RAG KO mice. We acknowledge the staff of the Herbert C. Englert Cell Analysis Laboratory at Dartmouth Medical School for help with flow cytometry.

Received February 7, 2003; revised July 8, 2003; accepted July 30, 2003.


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