Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation

Robert J. Rossi, Guruprasaadh Muralimohan, Joseph R. Maxwell and Anthony T. Vella

Division of Immunology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA

Corresponding author: A. T. Vella; E-mail: vella{at}uchc.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report we examined overlap between superantigen (SAg) and Toll-like receptor 4 (TLR4) stimulation of the innate immune system. Before in vivo stimulation we found that mouse splenic DCs expressed unexpectedly low levels of surface TLR4 compared to macrophages. In response to LPS, TLR4 gene expression in fractionated spleen cells was downregulated. By comparison, surface TLR4 staining with the Sa15-21 mAb showed little downregulation, and the anti-TLR4 MTS510 mAb showed decreased staining, suggesting that LPS was bound to TLR4 at the time points examined. Interestingly, SAg stimulation induced decreased TLR4 staining as measured by the MTS510 mAb, even though the TLR4 gene was not downregulated. Nevertheless, LPS potently induced DCs to produce TNF and IL-12, but SAg did not, even though they efficiently activated DCs. Notwithstanding, in vivo stimulation with staphylococcal enterotoxin SAg conditioned the innate immune system to hyper-respond to various pathogen-associated molecular patterns (PAMPs). Specifically, pre-priming with SAg enhanced LPS-mediated DC synthesis of TNF and IL-12. Thus, SAgs may exert their pathogenesis on the host by conditioning DCs, in a T cell activation dependent manner to potentiate responses to PAMPs.

Keywords: superantigens, T cells and shock


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TLRs are evolutionarily conserved proteins that can sense PAMPs and mediate intracellular signaling (1). One of the most heavily studied is TLR4, a key molecule for bacterial LPS (or endotoxin) responsiveness (2,3). TLR4 activation promotes proinflammatory cytokine secretion and upregulation of costimulatory molecules on antigen presenting cells. Many of the signaling molecules that mediate TLR activation are common between the various TLRs, including TRAF6, MyD88 and IRAK (2). The details of how these signaling molecules interplay with each other is likely to be a complex pattern of cross talking and feedback loops, but ultimately they regulate NF{kappa}B function (4).

The sensing of PAMPS by dendritic cells and other innate APCs is thought to be central in activating innate immunity (5). Additionally, PAMPS can act as adjuvants in promoting productive T cell immunity to specific Ag and have been used for many decades to study in vivo lymphocyte activation. It has been shown that gene expression of various TLRs can rise and fall depending on the type of stimulus and the subpopulation of immune cells analyzed (612). Nevertheless, many of these studies did not examine surface expression of TLRs. Therefore, it is difficult to rely on mRNA levels as a measurement of surface TLR since gene expression is not necessarily congruent with surface protein expression.

To address this issue we examined TLR4 expression on multiple populations of innate immune splenocytes. Using direct ex vivo analysis we found that mature splenic CD11c+ or CD11b+ MHC class IIhi dendritic cells expressed relatively low levels of surface TLR4. In contrast, murine splenic macrophages characterized by high granularity were profoundly positive for surface TLR4. We explored how these populations were influenced in the presence or absence of TLR4 stimulation using an in vivo system free of culturing procedures. Our in vivo data demonstrate rapid downregulation of TLR4 gene expression in response to LPS. Surface TLR4 on the other hand, was detected on macrophages as measured by the anti-TLR4 mAb Sa15-21 (13). The anti-TLR4 MTS510 mAb did show decreased TLR4 surface staining but this is likely an indication of LPS being bound to TLR4 thereby hampering MTS510 binding (13).

Irrespective of TLR4 expression, primary activation with LPS facilitated endotoxin tolerance by inhibiting DC production of TNF and IL-12 in response to a second injection of LPS. In contrast, SAg potentiated LPS responses by inducing DCs to secrete greater levels of TNF and IL-12. Thus, T cell independent innate responses may squelch secondary innate responses, but T cell dependent innate responses via SAgs condition DCs for enhanced effector function.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice, reagents and in vivo treatments
B10.A, C57BL/6, BALB/c and BALB/c Nude mice were purchased from Charles River–National Cancer Institute (Frederick, MD). C57BL/6 SCID were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in the Central Animal Facility at the University of Connecticut Health Center in accordance with federal guidelines.

PE-conjugated anti-CD11c (HL3), anti-TNF (MP6-XT22), anti-IL-12p70 (C15.6) and control rat IgG1 (R3-34), FITC-conjugated anti-MHC Class II I-Ek (14-4-4S), anti-MHC Class II I-Ab (AF6-120.1), APC-conjugated anti-CD11c (HL3), biotin-labeled anti-CD86 (GL1), anti-CD80 (16-10A1), purified anti-rat IgG2a isotype control and biotin mouse anti-rat IgG2a (RG7/1.30) were purchased from BD Biosciences (Mountain View, CA). PE-conjugated anti-CD11b (M1/70), biotin-labeled anti-TLR4 (MTS510) and control rat IgG2a, anti-ICAM-1 (or CD54) (YN1/1.7.4) and FITC-conjugated anti-MHC Class II I-Ab (M5/114.15.2) were purchased from eBioscience (San Diego, CA). The TLR4 mAb Sa15-21 (13) was a kind gift from Dr Kensuke Miyake (University of Tokyo, Japan).

SEA and SEB (staphylococcal enterotoxin A and B) were purchased from Toxin Technology, Inc. (Sarasota, FL) and used in accordance with the genetic background of the mice (14). LPS was purchased from Sigma–Aldrich (St Louis, MO). All SEA treated mice received 0.3 µg unless noted otherwise. SCID and NUDE mice from Fig. 4(A and B) received 100 µg SEB and all LPS treated mice received 200 µg unless noted otherwise. In Fig. 7, mice received either 0.3 µg SEA or 200 µg LPS at 0 h and an additional dose of SEA or LPS at 24 h. CTLA4-Ig (used at 0.5 mg) was a kind gift from Dr Robert Mittler (Emory University, USA), and a human IgG was used as a control. The above reagents were in balanced salt solution (BSS) and administered to mice as intraperitoneal injections.



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Fig. 4. SEB-induced DC activation is T cell dependent. (A) Density-gradient fractionated cells from uninjected (upper panel) or mice injected with SEB 12 h earlier (lower panel) were stained and analyzed by flow cytometry. CD11c+ cells were gated and analyzed for MHC class II and ICAM-1 (CD54) expression with the number indicating the percent of high expressing MHC class II, ICAM-1 double positive cells. These data are from one experiment similar to a repeat experiment. (B) Wild-type (upper panel) or Nude mice (lower panel) were injected with nothing (dotted line) or SEB 12 h earlier (solid line), and gradient fractionated CD11c+ cells were analyzed for MHC class II, CD54 and CD86. Data are from one representative experiment of four performed.

 


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Fig. 7. Conditioning DCs with SEA enhances LPS-induced production of TNF and IL-12p70. Primary stimulation with SEA or LPS was followed 24 h later by a SEA or LPS challenge. One-hour post secondary challenge direct ex vivo intracellular cytokine staining on CD11c+ DCs as shown in Fig. 4A was performed (TNF, left panel: IL-12p70, right panel). Each bar represents the mean percentages ± SEM from a total of four separate experiments with one mouse per treatment.

 
Cell fractionation and flow cytometric analysis
Density gradient fractionation of innate APCs were followed as given in Swiggard et al. except that DC adherence was not completed since we were interested in analyzing macrophages as well as DCs (15). Briefly, spleens from mice were crushed mechanically with forceps and treated with 1 ml of a 3.3 mg/ml solution of Collagenase D (Roche Diagnostics Corporation, Indianapolis, IN) in MEM 2% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY), 10 mM HEPES (Gibco). Splenic tissue was incubated for 30 min at 37°C, 5% CO2. After treatment with 0.1 ml 0.1 M EDTA for 5 min at room temperature, spleen tissue was disassociated through cell strainers and rinsed with Ca/Mg free BSS. Red blood cells were lysed and cells were resuspended in 2 ml PBS 35% BSA, added to Ultra-Clear centrifuge tubes (Beckman, Palo Alto, CA) with an additional 2 ml Ca/Mg Free BSS layered on top. After centrifugation at 9500 g for 15 min at 4°C, cells from the interface were collected and washed with BSS. Cells were resuspended in either Complete Tumor Media (CTM: MEM with FBS, amino acids, salts and antibiotics) or FACS staining buffer (BSS, 3% FCS, 0.1% NaAz) and counted on a Z1 particle counter (Beckman Coulter, Miami, FL).

Surface staining of cells for flow cytometry was completed as before (16,17). Briefly, cells were resuspended in FACS staining buffer, nonspecific binding was blocked using a combination of 2.4G2 hybridoma supernatant, 5% heat inactivated normal mouse serum and 10 µg/ml human IgG. For staining with the Sa15-21 mAb a different block containing 40% heat inactivated normal mouse serum, 50% mouse IgG1 and 10% human IgG was used to prevent cross detection of the rat anti-Fc (2.4G2) with Sa15-21. Cells were incubated on ice with primary antibodies for ~30 min and then washed and resuspended in staining buffer before analysis. If a secondary incubation was necessary, cells were washed and resuspended in staining buffer, and incubated on ice with streptavidin–PE, -APC, or -FITC for 30 min. The cells were analyzed on a FACSCalibur flow cytometer and data were analyzed using CELLQuest software (BD Biosciences, Mountain View, CA).

For intracellular cytokine staining, cells were taken directly ex vivo, processed as above, stained for surface markers and treated as previously described (18). Briefly, cells were washed with cold BSS without phenol red. Cytofix (BSS without phenol red and 2% formaldehyde) was added dropwise and vortexed to prevent clumping of cells. Cells were incubated for 5 min at 37°C and then centrifuged at 4°C for 4 min at 2000 g. After washing with 1 ml of permwash (FACS staining buffer and 2.5% Saponin), the cells were resuspended in permwash containing anti-cytokine mAbs and incubated at room temperature for 20 min. After several washes the cells were analyzed by flow cytometry.

Fluorescence activated cell sorting and histology
Cells were stained and resuspended in PBS with 2% FBS. Sorting was completed using a FacsVantage SE Cell Sorter (BD Biosciences, Mountain View, CA).

After sorting, 1 x 104 sorted cells were loaded onto glass slides by centrifugation at 3500 r.p.m. for 5 min using a Shandon Cytospin 2 centrifuge (Shandon, Scientific Products, Astmoor, England). The samples were air dried, fixed with methanol for 1 min and then submersed in Quick-Dip staining reagents (Mercedes Medical Inc, Sarasota, FL). The slides were rinsed in deionized water and air-dried. Digital pictures were taken using an Olympus Camedia E-10 digital camera attached to an Olympus BX51 microscope (Olympus America, Melville, NY).

Real-time PCR
TLR2, TLR4 and TLR5 mRNA expression were analyzed by quantitative real-time PCR. Total cellular RNA was extracted from splenic low-density cells using RNeasy mini-kit (Qiagen Sciences, Maryland). RNA was reverse transcribed using SuperScript II RNase H (Invitrogen Life Technologies, Carlsbad, California). Real-time PCR assays used a final volume of 40 µl containing 20 µl iQ SYBR Green Supermix (Bio-Rad Laboratories), 4 µl gene-specific primers (640 nmol), 5 µl cDNA and 11 µl H2O. The starting concentration of cDNA was 30 ng but 8- and 64-fold dilutions were also completed to ensure quality control of the real-time assay. Primers specific for the amplification of murine HPRT, TLR2, TLR4 and TLR5 were synthesized by Invitrogen life technologies (Carlsbad, CA). TLR2, TLR4 and TLR5 primers were obtained using the Beacon Designer software (Premier Biosoft International, Palo Alto, CA). HPRT: sense strand 5'-CTCCTCAGACCGCTTTTTGC-3' and anti-sense strand 5'-TAACCTGGTTCATCATCGCTAATC-3' (19). TLR2: sense strand 5'-TGGTTCTTTTCCCAAACTGG-3' and anti-sense strand 5'-GCTTTCTTGGGCTTCCTCTT-3'. TLR4: sense strand 5'-ATTGCTTGGCGAATGTTTCT-3' and anti-sense strand 5'-GACCCATGAAATTGGCACTC. TLR5: sense strand 5'-GCTCGCTTAGACCTATCTGGC-3' and anti-sense strand 5'-TACGTCGCTTAAGGAATTCAGTTC. The cycling protocol was as follows: 95°C 3 min, followed by 45 cycles of 95°C denaturation for 15 s and gene-specific annealing and extension at 59°C for 70 s. A melt-curve was generated at the end of each real-time assay to check for non-specific amplification of cDNA. Amplification of Hypoxanthine PhosphoRibosylTransferase (HPRT) acted as a control. The fold difference in cDNA levels were quantitated using the ddCT method (20). Real-time PCR was performed using the Bio-Rad iCycler.

Dendritic cell purification and culturing
DCs were fractionated from whole spleens of untreated and 20 h 0.3 µg SEA-treated B10.A mice or 1 µg SEA in C57BL/6 mice using MACS columns (Miltenyi Biotec, Auburn, CA). Typically the purified cells were ~65% CD11c+ and possessed excellent viability (data not shown). One hundred thousand purified cells were seeded into wells of a 96-well plate and separately stimulated in duplicate cultures with media alone, 0.5 µg/ml LPS, 0.5 µg/ml poly I:C (Sigma), 500 ng/ml Flagellin (Alexis Biochemicals, San Diego, CA) and 0.5 µg/ml CpG DNA (The Midland Certified Reagent Company, Midland, TX). After an overnight culture the supernatants were harvested and cytokine analyses were completed using TNF, IFN{gamma} (BD Biosciences) and IL-12 (R&D Systems, Minneapolis, MN) specific ELISAs according to the manufacturer's recommendation.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effects of PAMPs and SAg on TLR4
The idea that murine DCs express TLRs has been established in the literature, but this has largely been based on gene expression experiments rather than cell-surface protein expression (69). We examined this idea by staining various cell populations from the innate immune system for TLR4 surface expression. Based on many preliminary experiments we found that innate APCs could be generally categorized into cells possessing high versus low side scatter.

Our data show that CD11c or CD11b bearing DCs have low to intermediate levels of side scatter and that macrophages possess high side scatter (Figs 1 and 4A, respectively). CD11c or CD11b MHC class IIhi spleen cells possess relatively low levels of TLR4 on their surface (Fig. 1A). In contrast, the highest expressers of TLR4 were cells possessing lower levels of MHC class II, but high levels of side scatter (Fig. 1B). We suspected that these spleen cells were macrophages and therefore sorted the TLR4+ side scatterhi population and examined them histologically. The data show that compared to CD8+CD11c+ DCs, the sorted TLR4+ side scatterhi population possessed a granular cytoplasmic phenotype and did not present long dendrite morphology, confirming our suspicion that these cells were at least a mixed population of macrophages but not classical DCs.



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Fig. 1. TLR4 expression is much greater on macrophages than on DCs. (A) DCs were fractionated from whole spleen, stained for surface expression of CD11c or CD11b, MHC class II, and TLR4 or Cont. mAb. CD11c or CD11b, MHC class II double positive populations were gated and analyzed for TLR4 (or control mAb) expression. Data are from one representative experiment of three performed. (B) Splenocytes from a density gradient were stained for MHC class II, TLR4, or control mAb. MHC class II+ (based on unstained cells as a negative control), side scatterhi cells were gated and expression of TLR4 was analyzed (upper panels). Cells expressing high levels of TLR4 were sorted and histologically stained as described in the Methods, and compared to CD8+ CD11c+ DCs. These data are from one experiment similar to two others, but are based on many preliminary experiments.

 
Because of the high levels of TLR4 on the macrophages at time 0 h (Fig. 1B), we wanted to test the response of this population to different stimuli. LPS was injected into mice and the response of splenic macrophages was monitored over time (Fig. 2A). We show that by 1 h TLR4 was completely downregulated using the MTS510 mAb and did not appear again until 24 h later, where the levels were still very low. One striking feature of this population was the increase in autofluorescence in response to LPS over time, as can be seen by examining the shift in control mAb stain and by analysis of side scatter (data not shown). SEA is not a PAMP but a potent activator of adaptive immunity (21,22), and we show that macrophages respond in the same way to SAg as LPS, although not to the same degree (Fig. 2A). Using the TLR4 mAb Sa15-21, Akashi et al. (13) demonstrated an absence of TLR4 downregulation after LPS treatment of gene transfected Ba/F3 cells, which was in direct contrast to simultaneous testing with the MTS510 mAb. Their data show that LPS binding inhibits MTS510 mAb but not Sa15-21 mAb binding. We examined this idea in our in vivo system and the results show very little downregulation of TLR4 when using the Sa15-21 mAb in contrast to the MTS510 mAb (Fig. 2B).



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Fig. 2. Surface TLR4 expression on splenic macrophages is rapidly downregulated after treatment with LPS or SEA. (A) Two groups of mice were treated in vivo with either LPS (left column) or with SEA (right column). At the times indicated after treatment, side scatterhi MHC class II+ macrophages (as described in Fig. 1B) were gated and expression of TLR4 (black line) was monitored and compared to a control mAb stain (gray line). These data are from one experiment and each timepoint has been assessed between three and seven times. (B) Mice were treated with 250 µg of LPS or given nothing; 4 h later innate APCs were fractionated from spleen and stained for TLR4 expression. The top panel shows data from the MTS510 mAb and the bottom from the Sa15-21 mAb with the dotted line representing no treatment and the thick line LPS treatment. This is data is from one of three comparable experiments.

 
To further study TLR4 expression we examined an early (4 h) and a late (24 h) time point for gene expression of three important TLRs: TLR2, TLR4 and TLR5 (Table 1). The data show that expression of the TLR genes examined are either minimally changed like TLR2 at 4 h, or massively reduced by LPS stimulation such as TLR5 at 24 h. Specifically, LPS downregulated TLR4 gene expression as well as other TLRs, which do not signal via LPS. Thus, even though LPS binding hampers accurate estimation of surface TLR4 protein as measured by the MTS510 mAb (Fig. 2B), these collective data nevertheless demonstrate that LPS downregulates TLR4 gene expression at the time points examined.


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Table 1. The relative change in TLR2, 4 and 5 gene expression after in vivo exposure to LPS

 
Since TLR4 staining on macrophages in response to SEA was less dramatic compared to LPS (Fig. 2A), we injected varying amounts of SEA and examined multiple time points. Spleen macrophages were examined for TLR4 expression after 6 h of in vivo SEA stimulation (Fig. 3). A low dose of SEA had no effect, but our standard dose of 0.3 µg SEA was effective at inducing a partial TLR4 effect. Similar data were obtained for a 3 h time point, but no difference was observed at 10 h, which is consistent with the data in Fig. 2(A). Also, SEA did not induce changes in TLR4 gene expression like LPS did (data not shown). Therefore, SEA induces an early effect on TLR4 in macrophages in the apparent absence of LPS, but these cells recover within hours.



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Fig. 3. Titration of SEA-induced TLR4 alteration on macrophages. Density-gradient fractionated cells from 0 µg (Nothing), 0.012 µg, 0.3 µg or 7.5 µg SEA-injected mice were tested for TLR4 expression with mAb MTS510 6 h after injection. The cells were gated as in Fig. 2 and the unfilled histogram is data from the control mAb stain. Similar data were obtained from a 3 h time point (not shown).

 
T cell-dependent DC activation
Next we wanted to investigate the effects of these reagents on DCs. LPS responses can occur independently of activating the adaptive immune system and thus, we were interested in testing whether SAgs would activate innate immunity in the absence of the adaptive immune system. To answer this question the levels of MHC class II and ICAM-1 (or CD54) on the surfaces of DCs in response to SEB within SCID mice were examined. The data show that wild-type DCs upregulated MHC class II and ICAM-1, but this effect was inhibited in SCID mice (Fig. 4A).

The effect of SAgs on T cells in vivo is well documented and we tested whether DC responses to SAgs were dependent on T cells by comparing wild-type to nude mice. DCs upregulated MHC class II and CD54 in response to SEB only in the presence of T cells (Fig. 4B). Further, we demonstrated that upregulation of the key costimulatory molecule CD86 was also dependent on the presence of T cells. Thus, our data show that SAgs can induce the adaptive immune system to activate the innate immune system in a T cell-dependent manner. To examine a mechanism for T cell directed activation of DCs, we inhibited CD28 costimulation using the chimeric molecule CTLA4-Ig. In the past we found that CTLA4-Ig inhibited SEA-mediated T cell activation (23), and therefore reasoned that DCs would be dependent upon T cell activation. Our data clearly show that inhibiting CD28 ligation substantially blocked the upregulation of CD86 on DCs (Fig. 5). This is a diametrically opposed mechanism to that of PAMPs, which activate the innate immune system in order to instruct adaptive immunity (24).



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Fig. 5. Optimal SEA-induced DC activation is dependent on T cell costimulation. After overnight in vivo stimulation density-gradient fractionated cells from normal, SEA (3 µg) + Control Ig, or SEA + CTLA4-Ig treated mice were stained for CD11c, MHC II and CD86. Cells were gated on CD11c low side scatter (data not shown) and then analyzed for MHC II CD86 expression. The percentage indicates the fraction of double positive cells in the upper left panel. These data are from one of three comparable experiments.

 
SAg conditioning of DCs
SAgs can activate DCs via T cells, and therefore we reasoned that those DCs may become conditioned. To test this hypothesis we designed an ‘in vivo/ex vivo’ model where SEA is injected into mice, followed by DC isolation from spleen. After purification, DCs from SEA treated versus untreated mice were stimulated overnight with either nothing, LPS, poly I:C, flagellin, or CpG DNA. Supernatants were analyzed for the presence of TNF (data not shown), IFN{gamma} and IL-12. We found that LPS and CpG DNA induced comparable levels of IFN{gamma} (Table 2). A greater than 10-fold induction of IFN{gamma} release was detected when either PAMP was used to stimulate SEA-treated cells compared to non-conditioned cells. Poly I:C and flagellin induced low to moderate levels of IFN{gamma}, and virtually no detectable IL-12. In both experiments LPS, but more so with CpG DNA, potently induced IL-12 only when SEA-primed cells were stimulated. Thus, the purified cells taken from SEA-treated mice responded with greater vigor to the selected PAMPs, which all signal through different TLRs. Although this does not formally prove that DCs produced the cytokines analyzed, it is convincing evidence that SEA can condition cells to hyper-respond to PAMPs.


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Table 2. SEA induces potential for hyperresponsiveness to various PAMPs

 
Our goal was to directly measure DC cytokine production without in vitro stimulation. We found that DCs produced TNF and IL-12 in an exquisitely timed fashion as indicated by intracellular cytokine staining. Our attempts at examining macrophage cytokine production were equivocal since we had difficulty identifying these cells using this method, potentially because the fixation process may have distorted cell morphology. Mice were stimulated with either LPS or SEA, and DCs were examined directly ex vivo without any in vitro stimulation (Fig. 6). As an example, DCs were fractionated over a density gradient and then gated on forward scatter to mark viable cells. CD11c cells possessing low side scatter were analyzed for TNF levels in comparison to a control mAb (Fig. 6A). Using this approach we analyzed the time points shown in Fig. 2 and found that blasting DCs produced TNF and IL-12 very early after stimulation in vivo, but perhaps surprisingly not at later time points (Fig. 6B). In fact, we did not detect much DC cytokine production after 4 h of in vivo stimulation with LPS. Thus, DCs can produce cytokines within 1 h of in vivo LPS stimulation even though they bear very little surface TLR4.



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Fig. 6. Peak production of TNF and IL-12p70 by CD11c DCs occurs 1 h after LPS injection. An example of our intracellular cytokine staining analysis 1 h after in vivo LPS stimulation is given in (A). Spleen cells taken from density gradients were determined to be viable by forward scatter (upper left) and CD11c+ cells (upper right) were analyzed for intracellular TNF production by staining with a negative control mAb (lower left) versus anti-TNF mAb (lower right). (B) Cumulative data for 1 h (left panel) and 4 h (right panel) time points are shown after mice were treated with nothing, SEA or LPS. All data are given as mean ± SEM from individual mice for TNF (upper panel) or IL-12p70 (lower panel). The 0 (normal) and 1 h data are combined from at least five separate experiments, and the 4 h data are from at least three.

 
Nevertheless, what was most striking was the fact that SEA did not induce DC cytokine production at any of the time points; this was unexpected since it has been shown here and by others that SAgs can potently activate DCs in vivo (25). Additionally, SAg and LPS combined responses induce lethal shock in a TNF dependent manner (26). Therefore, we hypothesized that SEA stimulation may increase the sensitivity of TLR4 signaling in DCs. To test this idea, we performed in vivo primary stimulations with either SEA or LPS and then 24 h later challenged the mice with either SEA or LPS (Fig. 7). Two in vivo stimulations with SEA still did not enhance DC production of TNF or IL-12, nor did SEA enhance DC cytokine production when given after LPS. Although one injection of LPS was sufficient to induce cytokine production (Fig. 6), two LPS injections were totally inhibitory (Fig. 7). In sharp contrast, LPS stimulation after SEA priming resulted in the greatest levels of cytokine production over that of any other combination. The proportion of cytokine producing DCs taken from mice stimulated with SEA then LPS was much greater than for DCs from mice treated with a single injection of LPS: 1.5-fold more TNF and 2.2-fold more IL-12 (compare Fig. 6B with 7). These data show that SAgs condition DCs differently than LPS and that DC cytokine production is incredibly rapid and transitory.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our data show that macrophages possess much more surface TLR4 than DCs (Fig. 1). Although this may seem contra-dogmatic since many studies routinely use LPS to stimulate DCs, this is consistent with the anatomical location of macrophages and the locale of where LPS collects in draining LN. Specifically, unlike small molecular weight molecules, LPS is largely excluded from the cortex of the LN (27). Since the location of DCs is in the T cell areas of the cortex in LNs (28), it is possible that DCs would have little access to LPS under physiological conditions. On the other hand, macrophages are detected in the LN subcapsular sinus (28), precisely where LPS collects (27). Therefore, from a physiological perspective these data are consistent with the notion that macrophages may be the crucial sentinel for LPS detection compared to DCs.

This raises the important question of how DCs become activated after in vivo stimulation with LPS? Perhaps this subject is initially addressed in this study from the data demonstrating that LPS rapidly activates the innate immune response. Within 5 min surface TLR4 is influenced after LPS stimulation (Fig. 2A), and at 4 h TLR4 gene expression is downregulated by at least 4-fold (Table 1). As TLR4 is altered in macrophages there is a coincident increase in APC activation and DC production of cytokines (Fig. 6). Cytokines are produced rapidly even though DCs bear very little TLR4, and as suggested above probably have little access to LPS in vivo. These data suggest that perhaps DCs are activated by an endogenous means after in vivo LPS stimulation, possibly through macrophages. This idea is supported by the fact that TLR4 is altered on macrophages after in vivo SEA treatment (Figs 2A and 3). This study shows that SEA reduced anti-TLR4 binding using the MTS510 mAb, which does not bind TLR4 when TLR4 is bound to LPS (13). This raises the interesting notion that an endogenous factor(s) binds TLR4 after stimulation with SEA and presumably this could happen after LPS or through some other stimulus. Currently, this theory is speculative, but experiments testing communication between macrophages and DCs will likely provide important information linking the external environment to activation of the innate immune system.

PAMPS and SAgs are important links between invading pathogens and host immune responses. Here, we show that SAgs affect TLR activation in DCs. This connection is physiologically relevant because DC cytokine responses induced by LPS after SAg stimulation were enhanced (Table 2 and Fig. 7). This is consistent with earlier data demonstrating that SAg and LPS can synergize to induce lethal shock in mice (29). In contrast, two injections of LPS yielded little, if any, DC cytokine production resulting in endotoxin tolerance. Perhaps this was not surprising since TLR4 gene expression was downregulated, but pretreatment with SAg actually enhanced DC cytokine production after LPS treatment. Thus, as suggested by others, alteration of TLR4 expression may not be an accurate indication of endotoxin tolerance (30). Additionally, recent experiments bring into question the role of TLR4 as an external binding receptor for LPS (31). This study shows that LPS uptake into a cell occurs even in the absence of TLR4. Thus, the binding of LPS and the role of CD14, MD-2 and TLR4 is clearly a very complex process that leads to potent activation of DCs—perhaps indirectly, but the mechanism remains to be determined.

Studies show that DCs can attain diverse levels of activation depending on the type of stimulus. For example, T cells may activate CD40, pathogens can stimulate TLR pathways and macrophages are capable of releasing cytokines and other chemical mediators. Variation in timing, magnitude and sequence of the stimulus may be processed differently by DCs and result in different physiological responses. A number of reports show that DCs are potent activators of CD4 T cells (32), but relatively few have documented that T cells can activate DCs in vivo. In this study it is demonstrated that DCs rapidly upregulate MHC class II, CD54 and CD86 after injection of SAg. The activation of DCs by SAgs is completely abolished in the absence of T cells (Fig. 4), and largely dependent on CD28 costimulation when T cells are present (Fig. 5). This is in vivo evidence that SAg binding to MHC class II on DCs is not sufficient for DC activation, unless SAg-specific T cells are present and can receive costimulation. Thus, it is possible that DC activation via T cells yields a different physiological response in comparison to DC activation by TLRs. Even by using different PAMPs our data speak to this idea since TLR9 stimulation differed to TLR3 or TLR5 stimulation (Table 2). In this study we present examples of similarities and differences in DC activation when different PAMPs are used as well as non-TLR ligands such as SAg.

Another piece of indirect evidence is the fate of stimulated T cells under these different in vivo conditions. For example, SAg stimulation in mice ultimately results in specific peripheral T cell deletion (33); however, co injection of LPS breaks deletion and conditions the specific T cells to become surviving memory cells (34,35). Therefore, it is possible that the status of DC activation may be the determining factor for the fate of the specific T cells. Perhaps the best evidence is the recent data showing that depletion of DCs during infection results in very weak T cell priming (36). Thus, the status of DC activation is likely to influence the quality of the immune response to pathogens.

We show that DCs are capable of varied responses depending on the type of stimulus. Ultimately, understanding how to control these pathways will allow appropriate conditioning of DCs, which may enhance vaccine development. Perhaps this may be accomplished by manipulating other innate APCs to control DC function.


    Acknowledgements
 
The authors would like to thank Dr T. V. Rajan (UCONN Health Center) for assistance with histological analysis, E. Pizzo and D. Gran for help with cell sorting and C. Aguila for help with ELISA data. This work was supported by NIH Grants R01 AI142858 and P01 AI056172 (A.T.V.).


    Notes
 
Transmitting editor: R. Medzhitov

Received 26 February 2004, accepted 22 September 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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