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Originally published online as doi:10.1189/jlb.0406253 on September 14, 2006

Published online before print September 14, 2006
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(Journal of Leukocyte Biology. 2006;80:1251-1261.)
© 2006 by Society for Leukocyte Biology

Effects of LPS-mediated bystander activation in the innate immune system

Rajwardhan Yadav, David J. Zammit, Leo Lefrancois and Anthony T. Vella1

Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, USA

1 Correspondence: Department of Immunology, University of Connecticut Health Center, 263 Farmington Ave., MC1319, Farmington, CT 06030, USA. E-mail: vella{at}uchc.edu

ABSTRACT

LPS induces dendritic cell (DC) activation, but the precise in vivo mechanism is unclear since DCs express low levels of TLR4. Here, it is shown that DCs can be activated in response to LPS through a bystander mechanism. This result was obtained using chimeric mice reconstituted with LPS-responsive and nonresponsive bone marrow cells. Thus, after indirect in vivo conditioning by LPS, bystander-activated DCs (LPS nonresponsive) up-regulated CD86. This up-regulation occurred even when LPS-responsive cells were MyD88 deficient. Functional analysis demonstrated that in vivo LPS conditioning endowed both the LPS-responsive and bystander cells with the ability to produce IFN-{gamma} in response to TLR9 stimulation in vitro. IFN-{gamma} production was also shown to be important for enhanced T-bet gene expression but not important for up-regulation of CD86. To investigate aspects of the mechanism, we used intracellular cytokine staining and found that NKDCs were responsible for at least some of the IFN-{gamma} production. Thus, our in vivo results demonstrated that bacterial LPS can bridge activation of various cellular populations of the innate immune system through a bystander mechanism.

Key Words: dendritic cells • cytokines

INTRODUCTION

Specialized families of pattern recognition receptors (PRRs), expressed predominantly by hematopoetic cells, bind highly conserved pathogen associated molecular patterns (PAMPs) and activate the innate immune system. The outcome of a microbial infection depends on the efficiency of the innate immune response to combat microbes and induce adaptive immune responses against the invading pathogen. DCs are believed to be critical cellular mediators of this process that serve to link the innate and adaptive immune systems, resulting in robust and long-lasting immunity [1 ].

LPS, the most studied and well-characterized PAMP, mediates much of its pleiotropic effects on APCs. In vivo coadministration of LPS, along with soluble proteins, results in lasting antigen-specific T cell memory [2 ]. LPS enhances antigen presentation and costimulatory molecule expression by DCs and induces the synthesis and release of proinflammatory mediators [3 ]. The molecular mechanisms by which LPS activates DCs are well studied. A great deal is known concerning how LPS binds and stimulates through the TLR4 pathway, but precisely which cells are directly or indirectly stimulated by LPS in vivo is still being actively pursued [4 ]. For example, LPS may mediate its effects by directly binding TLR4 expressed on DCs. However, human and mouse DCs express low levels of TLR4 mRNA and surface molecules [5 6 7 8 ]. Notwithstanding the very low levels of surface TLR4, it has been demonstrated that after LPS exposure, DCs attain an activated phenotype and hence prime T cells in vivo with enhanced efficiency [9 ]. Currently, there is no in vivo experimental evidence that explains the profound maturation of DCs, in spite of low TLR4 expression, following LPS exposure. Injection of LPS in the mouse footpad results in accumulation of LPS in the subcapsular and medullary sinuses of draining lymph nodes [10 ]. Compared with DCs, macrophages are the predominant immune cells in the subcapsular sinus. Interestingly, macrophages express very high levels of TLR4 compared with DCs [7 ]. These anatomical and physiological constraints could limit the accessibility of DCs to LPS. It is logical that LPS would, therefore, be encountered by macrophages, endothelial and epithelial cells, which upon activation may initiate a cascade of events resulting in the release of inflammatory mediators and tissue factors. Consistent with this idea are reports suggesting that DC maturation can be induced after release of inflammatory mediators [11 , 12 ]. Specifically, IFN-{gamma} and IFN-{alpha} have been demonstrated to be potent stimuli of DC maturation [13 ] and the adjuvanticity of CFA, which contains several TLR ligands, has been shown to be dependent on IFN receptors [14 ].

Thus, considering these important data, we tested whether LPS was capable of indirectly activating DCs using an in vivo system and began to investigate the critical molecules involved in this process. A mixed bone-marrow chimera mouse model was developed, and results showed that LPS activated LPS nonresponsive DCs, but only in the presence of LPS-responsive cells. In vivo LPS conditioning of the LPS-responsive and bystander population endowed them both with the ability to produce IFN-{gamma} in response to TLR9 stimulation in vitro. Our data indicate that natural killer DCs (NKDCs) were a source of IFN-{gamma} after LPS stimulation, suggesting that they may play a role in conditioning cells of the innate immune system in a bystander fashion.

MATERIALS AND METHODS

Reagents
LPS was purchased from Sigma-Aldrich (St. Louis, MO) and ultra-pure LPS (upLPS) from Alexis Pharmaceuticals (San Diego, CA). Collagenase D was purchased from Roche Laboratories (Indianapolis, IN). C3H/HeJ (H2k), C57BL/6 (H2b) mice were purchased from Charles River–National Cancer Institute (Frederick, MD). IFN-{gamma}–/– and IFN-{gamma}R–/– mice (C57BL/6 background) were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88–/– mice (Shizuo Akira, Osaka University, Osaka, Japan via Ruslan Medzhitov, Yale University, New Haven, CT) were backcrossed to C57BL/6 (H2b) until the 9th generation. Mice were between 4 and 20 wk of age and were maintained in the Central Animal Facility at the University of Connecticut Health Center in accordance with federal guidelines.

FITC-conjugated anti-MHC class I H2Kb (AF6-88.6), anti-MHC class I H2Kk (36-7-5), anti-MHC class II IEk (14-4-4S), anti-MHC class II IAb (AF6-120.1), APC-conjugated anti-CD11c (HL3), anti-CD49b (DX5), PE-conjugated anti-CD86 (GL1), anti-CD80 (16-10A1), anti-MHC class I H2Kb (AF6-88.5), anti-MHC Class I H2Kk (36-7-5), anti-rat IgG2a (RG7/1.30), anti-IFN-{gamma} (XMG1.2), anti-Rat IgG2b, and anti-mouse IgG2a were purchased from BD Biosciences (Mountain View, CA), or eBiosciences (San Diego, CA).

Generation of chimeric mice
C3H/HeJ recipient mice were {gamma}-irradiated twice ~3 h apart with 550 rads and reconstituted with ~2 x 106 bone-marrow cells of each relevant donor strain. Bone-marrow cells were depleted of T cells in most of the mixed bone-marrow chimeras made, but it was noteworthy that there was a no significant difference in the mortality of mice in depleted as compared with the nondepleted chimeras (data not shown). Eight weeks after reconstitution, chimerism was ascertained by analysis of blood leukocytes by surface staining and flow cytometric analysis of the respective MHC class I/II molecules. When necessary chimeric mice were matched between groups, such that a comparable level of chimerism was present in the mice.

Cell fractionation and flow cytometry
Density gradient fractionation of DCs was followed as previously published [15 ]. Briefly, spleens were treated with 1 ml of 3.3 mg/ml 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 of 0.1 M EDTA for 5 min at room temperature, spleen tissue was passed through cell strainers and rinsed with Ca++/Mg++-free BSS. Red blood cells were lysed, and cells were resuspended in 3 ml of 35% BSA in PBS (4:1, vol/vol), added to ultra-clear centrifuge tubes (Beckman, Palo Alto, CA), with an additional 3 ml Ca++/Mg++ free BSS layered on top. After centrifugation at 9500 g for 25 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 staining buffer (BSS, 3% FCS, 0.1% NaN3) and counted on a Z1 particle counter (Beckman Coulter, Miami, FL). Surface staining of cells for flow cytometry was done as described before [16 , 17 ]. Briefly, cells were resuspended in 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 (Sigma-Aldrich, St. Louis, MO). Cells were incubated on ice with antibodies for 30 min and then washed and resuspended in staining buffer before analysis on a FACSCalibur flow cytometer. Data were analyzed using Flow jo software (Tree Star Inc., Ashland, OR).

Cell purification and culturing
Cells were fractionated from whole spleens of PBS and upLPS-treated chimeric mice using CD11c MACS beads and columns (Miltenyi Biotech, Auburn, CA). We consistently obtained 90–95% pure CD11c+ cells after MACS bead purification. We further separated these cells using anti-IAb (C57BL/6) PE followed by anti-PE (Miltenyi Biotech, Auburn, CA) on MACS columns. To obtain CD11c+- IEk cells by MACS columns the flow through from the CD11c+-IAb cells was washed twice with MACS buffer and incubated with anti-IEk (C3H/HeJ) PE mAbs followed by anti-PE (Miltenyi Biotech, Auburn, CA). In an initial experiment, we stained the CD11c+ MHC class II+ cells with anti-H2Kb (C57BL/6 cells) and anti-H2Kk (C3H/HeJ) mAbs and detected ~97% purity. Our cell yields (shown in Table 1 ) were proportional to the level of chimerism, suggesting that contamination was negligible. We cultured the C57BL/6 and C3H/HeJ cells at a concentration of 1 x 106/well (except in the 99:1 bone marrow chimeric mouse where 1x105 C3H/HeJ cells were added/well) of a 96-well flat bottom plate and stimulated with CTM alone, 100 ng of upLPS, LPS, CpG (The Midland Certified Reagent Company, Midland, TX), poly I:C (Sigma) and Flagellin (Alexis Biochemical’s, San Diego, CA). After 24-h supernatants were harvested and assayed for IFN-{gamma} using an ELISA kit (BD Biosciences), according to the manufacturer’s recommendation.


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  		Table 1. Cell Counts Obtained After MACS Bead Purification

 
Intracellular IFN-{gamma} cytokine staining
Low-density cells were fractionated from whole spleens of PBS and upLPS-treated C57BL/6 mice by density-gradient centrifugation for initial experiments and cultured with or without CpG for 5 h. For later experiments, CD11c+ cells were enriched from density gradient fractionated spleen cells using CD11c MACS beads and columns (Miltenyi Biotech, Auburn, CA). CD11c+ cells were then cultured, for a total of 5 h, in a 96-well flat-bottom plate and stimulated with CTM alone or 1 µg of CpG. Brefeldin A was added to all cultures at a concentration of 10 µg/ml. Cultured cells were washed and surface stained on ice with anti-CD49b (pan-NK cell marker) conjugated to APC; IAb, or IEk conjugated to FITC. We added 1 µg/ml of brefeldin A to the FACS staining buffer, which was used for washing the cells, to prevent leaching of IFN-{gamma} during the intracellular staining. Surface-stained cells were fixed with 2% paraformaldehyde and later washed with ice-cold FACS staining buffer containing 2.5% saponin followed by the addition of anti-IFN-{gamma} conjugated to PE and incubated on ice for 30 min. Cells were washed three times with FACS staining buffer containing 2.5% saponin and once with ice-cold PBS and analyzed by flow cytometry. Isotype controls were also run to establish appropriate gate settings.

Real-time PCR
T-bet gene expression was analyzed by quantitative real-time PCR. Total cellular RNA was extracted from splenic C3H/HeJ (CD11c+ and IEk) cells using RNeasy mini-kit (Qiagen Sciences, Germantown, MD). RNA was reverse transcribed using SuperScript II RNase H (Invitrogen Life Technologies, Carlsbad, CA). 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, and eightfold dilutions were also completed to ensure quality control of the real-time assay. Primers specific for the amplification of murine HPRT and T-bet were synthesized by Invitrogen Life Technologies (Carlsbad, CA). HPRT: sense strand 5'-CTCCTCAGACCGCTTTTTGC-3' and anti-sense strand 5'-TAACCTGGTTCATCATCGCTAATC-3'. T-bet: sense strand 5'-CGGGAGAACTTTGAGTCCATGT-3' and anti-sense strand 5'-GCTGGCCTGGAAGGTCG-3' [18 , 19 ]. 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 nonspecific amplification of cDNA. Amplification of HPRT acted as a control. The fold difference in cDNA levels were quantified using the ddCT method [20 ]. Real-time PCR was performed using the Bio-Rad iCycler.

RESULTS

Effect of LPS on DCs in vivo
In vivo administration of LPS activates cells through the well-characterized TLR4 pathway. We used an in vivo system to test whether activation of DCs by LPS was direct or indirect. We compared ultrapure LPS (upLPS) obtained from Alexis Pharmaceuticals to LPS (Sigma Aldrich) and assayed for DC activation. An array of molecules (CD40, CD80, CD86, MHC class II and 41BB ligand) considered as phenotypic activation markers for DCs were monitored by flow cytometry. Results revealed that CD86 was an ideal, consistent, and representative biomarker to assess the phenotypic activation status of DCs. As measured by flow cytomtery, CD86 was increased on DCs from LPS-responsive (C57BL/6) mice after stimulation with both types of LPS ranging from a dose of 0.01 to 100 µg (Fig. 1A ). It was unexpected and important to note that after administration of higher concentrations (1–100 µg) of LPS, DCs in C3H/HeJ mice demonstrated a marked up-regulation of CD86 (Fig. 1A , right panel and B, left panel). Nevertheless, the DCs in C3H/HeJ mice as identified by MHC class II (IEk) and CD11c+ did not respond to upLPS at any given concentration, which is consistent with previous reports [21 ]. Time kinetic evaluation of DC phenotypic activation revealed that up-regulation of activation markers peaked at 5 h post-LPS administration, and by 24 h, a significant number of DCs expressed less CD86, as shown in Fig. 2A and 2B .


Figure 1
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  		Figure 1. Dose-response of DC activation in response to LPS. (A) C57BL/6 and C3H/HeJ mice were treated with either PBS (gray line) or varying doses of upLPS (solid line) or LPS (dotted line). Five hours later, DCs were enriched from collagenase-digested spleens that were subjected to density-gradient fractionation. DCs were stained for CD11c, MHC class II (IAb/IEk), and CD86. CD11c+ and MHC class II+ cells were gated and analyzed for CD86 expression by flow cytometry (top). (B) Data are from mice injected with varying amounts of upLPS (top) and LPS (bottom). Mean fluorescence intensity (MFI) of CD86 expression after 5 h is shown, and open circles indicate C3H/HeJ, and closed circles indicate C57BL/6.

 

Figure 2
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  		Figure 2. Time kinetic of DC activation in response to LPS. (A) Mice were treated with 100 µg of upLPS and killed at various time points as indicated. DCs were enriched from spleens after collagenase digestion and density gradient fractionation and analyzed for CD86 expression after gating on CD11c+ and IAb+ cells as indicated (top). Histograms in gray represent PBS, and solid line represents upLPS. (B) MFI of CD86 expression at different time points analyzed post-LPS treatment. Line connecting solid circles denotes LPS treatment, and gray line represents PBS treatment. These data are from one of the 3 experiments performed, all yielding comparable results.

 
Bystander activation of DCs in response to LPS occurs in vivo
To test whether LPS induced direct and indirect activation of DCs, we generated mixed bone-marrow chimeras, in which irradiated C3H/HeJ mice (recipients) were reconstituted with C57BL/6 and C3H/HeJ bone-marrow cells in differing proportions (Fig. 3A ). After ~8 wk of bone-marrow reconstitution, upLPS was administered, and 5 h later, splenic DCs were analyzed for their activation status and compared with a PBS control from the chimeric mice (60:40). DCs originating from C57BL/6 and C3H/HeJ bone-marrow cells up-regulated CD86 in response to in vivo injection of upLPS (Fig. 3B) . We postulated that this in vivo activation of C3H/HeJ DCs occurred as a consequence of a bystander effect, as C3H/HeJ DCs were not activated in response to upLPS unless bone marrow cells from C57BL/6 background were present (Fig. 3B) . Further documentation of this bystander activation was re-enforced as the quantitative increase of CD86 on C3H/HeJ DCs was directly proportional to the level of chimerism with C57BL/6 bone marrow cells (Fig. 3C) . Specifically, the expression of CD86 by C3H/HeJ DCs declined as the proportion of C57BL/6 cells decreased (Fig. 3C) . It is noteworthy, however, that the DCs in the chimeric mouse with ~99% reconstitution from C3H/HeJ bone marrow cells were activated by less pure LPS (data not shown). Thus, bystander activation of LPS nonresponsive DCs can occur in vivo in response to LPS-induced inflammatory mediators.


Figure 3
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  		Figure 3. UpLPS-induced activation of LPS nonresponsive DCs. (A) Bone marrow was harvested from C57BL/6 (open) and C3H/HeJ (solid) mice indicated in parenthesis. Bone marrow cells, devoid of RBCs and T cells, were injected i.v. into irradiated recipient C3H/HeJ mice. After 8 wk, mice were screened for percentage of reconstitution of C57BL/6 and C3H/HeJ bone marrow that yielded varying percentages of reconstitution as denoted in the contour plots. Screened mice were matched for the percentage of bone marrow reconstitution before injection of PBS or upLPS. (B) Five hours later, DCs were stained for CD11c, IEk, and CD86 expression. (C) MFI of CD86 expression on DCs (C3H/HeJ origin) represented in a bar graph. These data are from one of 5 experiments that yielded comparable results.

 
A hallmark of LPS-induced responses is the release of inflammatory mediators by a variety of cell types [22 ]. It is, therefore, plausible that LPS-mediated bystander activation of DCs could occur as a consequence of release of soluble inflammatory mediators and/or via cell-to-cell adhesion and/or interaction. Recent in vitro experiments using LPS suggest that cell adhesion/interaction is unlikely to make a major contribution to bystander activation [12 ], but other studies have shown evidence for both cell-to-cell contact and soluble mediators [4 , 13 ]. Thus, we decided to focus on the contribution of soluble mediators in bystander activation of DCs such as those dependent on MyD88 [23 , 24 ].

We used the same strategy to generate chimeric mice wherein the C3H/HeJ recipient mice were reconstituted with bone marrow cells either from C57BL/6 or MyD88–/– mice along with C3H/HeJ bone marrow cells. Reconstituted mice were i.p. injected with upLPS, and 5 h later, DCs analyzed by flow cytometry. Results from the wild-type chimeric mice revealed optimal up-regulation of CD86 on C57BL/6 and C3H/HeJ DCs, although CD86 up-regulation on the C3H/HeJ DCs was less than on the C57BL/6 DCs (Fig. 4A ). The same trend was also observed in the chimeric mice reconstituted with MyD88–/– bone marrow cells arguing for the minimal role of MyD88 in inducing CD86 through bystander activation (Fig. 4B) . Another study demonstrated even more up-regulation of CD86 on MyD88–/– DCs in response to LPS [25 ]. Ultimately, we found that CD86 up-regulation by the bystander cells was largely proportional to the LPS-responsive cells regardless of whether the responsive cells expressed MyD88.


Figure 4
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  		Figure 4. Testing a role for MyD88 during bystander DC activation in response to LPS. (A) Recipient C3H/HeJ mice having chimerism of ~1:1 were treated with PBS or 100 µg of upLPS. Five hours later, DCs were enriched from collagenase-digested splenocytes by density-gradient fractionation and stained for CD11c, MHC class II (IEk), and CD86 expression. Histograms represent surface expression of CD86 in CD11c and IEk positive (C3H/HeJ) and negative (C57BL/6). (B) MFI of CD86 expression on DCs (C3H/HeJ origin) represented in a bar graph as means ± SEM. These data are representative of 3 experiments performed with one mouse in each group per experiment.

 
Contribution of IFN-{gamma} as a mediator of bystander activation
IFN-{gamma} is expressed in variable quantities secondary to intracellular infections by MyD88–/– macrophages [26 ]. Release of IFN-{alpha} and β can also occur in a MyD88-independent manner in response to LPS. IFN-{alpha} is known to activate STAT1, which is critical for the expression of IFN-{gamma} [26 ]. These results, and other data showing effects on T cells [4 ], suggest that IFN-{gamma} can be released in response to LPS, and hence, we decided to evaluate the role of IFN-{gamma} in bystander DC activation.

IFN-{gamma} is a signature cytokine of Th1 responses [27 ], but it has been shown that cells of the innate immune system produce IFN-{gamma} in response to LPS [28 ] and that IFN-{gamma} enhances the antigen-presenting capabilities of DCs and macrophages and promotes the killing of intracellular pathogens in macrophages [29 ]. The role of IFN-{gamma} in mediating bystander activation of DCs in response to LPS in vivo is unknown; hence, we evaluated the role of IFN-{gamma} and IFN-{gamma}R in this process. We made chimeric mice such that C3H/HeJ recipient mice were reconstituted with bone marrow cells from either C57BL/6, IFN-{gamma}–/–, and IFN-{gamma}R–/–, mice along with bone marrow from C3H/HeJ mice. In response to upLPS administration, C3H/HeJ DCs up-regulated CD86 in chimeric mice reconstituted with bone marrow from C57BL/6, IFN-{gamma}–/–, or IFN-{gamma}R–/– mice (Fig. 5 ).


Figure 5
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  		Figure 5. Testing a role of IFN-{gamma} or IFN-{gamma}R in bystander activation in response to LPS. Bone marrow was harvested from C57BL/6, IFN-{gamma}–/–, IFN-{gamma}R–/–, and C3H/HeJ mice as indicated in parentheses. Bone marrow cells were injected i.v. into irradiated recipient C3H/HeJ mice. After 8 wk, mice were screened for a percentage of bone marrow reconstitution. Recipient C3H/HeJ mice having reconstitution of ~1:1 were treated with PBS or upLPS. After 5 h, spleens were collagenase digested, and DCs were enriched by density-gradient fractionation. Spleen cells were stained for CD11c, MHC class II (IEk), and CD86 expression. Histograms represent surface expression of CD86 on CD11c+ and IEk+ (C3H/HeJ) and IEk negative (C57BL/6) DCs. These data are from one of the 3 experiments performed that yielded comparable results.

 
Thus, IFN-{gamma} and the IFN-{gamma}R were dispensable in LPS mediated bystander phenotypic activation. However, it is unclear from these data whether bystander-activated cells would have similar functional capabilities compared with their wild-type counterparts.

Bystander conditioning favors enhanced responsiveness to PAMPs
To assess the functional capacity of bystander-activated innate cells, we examined the response of direct and bystander-activated cells to various PAMPs. Chimeric mice were injected with upLPS, 5 h later C57BL/6 and C3H/HeJ CD11c+ cells were enriched using anti-IAb or –IEk (respectively) by magnetic bead separation, as described in the Materials and Methods. Cell yields from these bone-marrow chimeric mice were proportional to the level of chimerism (Table 1) . These primary cultures are representative of innate immune cells since multiple cell populations can up-regulate CD11c or even MHC class II. Nevertheless, the fractionated cells were cultured in vitro in the presence of various PAMPs (Table 2 ). Measurement of IFN-{gamma} in the supernatants revealed that LPS conditioned the fractionated cells to respond much better to CpG than to other PAMPs. The bulk amount of IFN-{gamma} detected in the supernatant from the bystander population (C3H/HeJ) was lower than compared with their C57BL/6 counterparts, but the fold increase over no PAMP (compare None to CpG, Table 2 ) was profoundly enhanced in both groups of cells. For example, chimeric mouse number 3 showed <2 picograms of IFN-{gamma} with no PAMP, but after CpG treatment, 165 pg were detected, and similar data were obtained for C57BL/6 cells (Table 2) . In contrast to upLPS conditioning, C3H/HeJ cells from PBS control mice did not make detectable amounts of IFN-{gamma} in response to PAMPs (data not shown). These observations suggest LPS-induced inflammatory mediators can activate CD11c+ cells in a direct and indirect manner, resulting in enhanced responsiveness to CpG and poly I:C.


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  		Table 2. LPS Conditioned Cells Respond to Certain PAMPs With Enhanced Magnitude

 
Role of IFN-{gamma} in mediating functional responsiveness to bystander-activated innate cells
We showed that IFN-{gamma}–/– cells can respond to LPS and lead to activation of C3H/HeJ DCs as measured by CD86 up-regulation (Fig. 5) . To ascertain the functional status of the C3H/HeJ cells in the absence of IFN-{gamma}, we injected chimeric mice with upLPS and compared the functional responsiveness of C3H/HeJ (CD11c+ IEk+) cells to CpG and poly I:C from IFN-{gamma}+/+vs. IFN-{gamma}–/– chimeric mice. C3H/HeJ cells from IFN-{gamma}+/+ chimeric mice produced large quantities of IFN-{gamma} when stimulated with CpG, thus reproducing the previous observation shown in Table 2 , and consistent with the data in Fig. 6 . It was interesting to note that C3H/HeJ cells from IFN-{gamma}–/– chimeric mice, which had up-regulated CD86 in response to upLPS (Fig. 5) , did not produce any detectable levels of IFN-{gamma} when stimulated with CpG and poly I:C in vitro (Table 3 ). Thus, these in vitro data suggest that to attain a functionally competent state to produce IFN-{gamma}, CD11c+ IEk+ cells required IFN-{gamma} and LPS-induced inflammatory mediators void of IFN-{gamma} couldn’t substitute for IFN-{gamma}.


Figure 6
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  		Figure 6. Intracellular IFN-{gamma} staining revealed that NKDCs produced IFN-{gamma}. Mice were injected with PBS or LPS, and 5 h later, low-density cells were obtained from spleens by gradient centrifugation. Fractionated cells were further enriched using magnetic CD11c beads separation and then cultured in the presence or absence of CpG along with brefeldin A. Five hours later, DCs were surface stained for CD49b (DX5), MHC class II, along with respective isotype controls, and intracellular IFN-{gamma} staining performed on these surface-stained cells. (A) Cells from C57BL/6 mice were gated and analyzed for CD49b and IFN-{gamma}. Positive cells were further gated and analyzed for expression of IAb and compared with the isotype controls (top right). These data are from one experiment based on 3 experiments without CD11c bead separation. (B) Cells from mixed bone marrow chimeric mice (C3H/HeJ and C57BL/6 BM into irradiated C3H/HeJ mice) were gated and analyzed for CD49b and IFN-{gamma} (bottom left) and positive cells were analyzed for IAb or IEk expression as compared with isotype control (mIgG2a) (bottom middle and right, respectively). Data are similar to 3 other experiments.

 

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  		Table 3. Role of IFN-{gamma} in LPS Conditioning of Cells to Respond to PAMPs

 
Because primary cell cultures are likely to contain contaminating cells regardless of attempts to purify them, we used intracellular cytokine staining procedure. To identify IFN-{gamma} producers, LPS was injected into C57BL/6 mice, and 5 h later, BSA density gradient-fractionated cells were cultured in the absence or presence of CpG along with brefeldin A. Surface staining followed by intracellular IFN-{gamma} staining showed that cells producing IFN-{gamma} expressed CD11c and CD49b (data not shown). Stringent analysis of these cells revealed that the majority of these low-side scatter cells were also MHC class II+ when compared with an isotype-matched control antibody. These data suggested that a major proportion of the IFN-{gamma}-producing population were NKDCs [30 31 32 ]. Therefore, LPS or PBS was injected into C57BL/6 mice, and 5 h later, spleen cells were fractionated using a density gradient and further purified by CD11c magnetic bead separation. CD11c+ purified cells were cultured in vitro with CpG in the presence of brefeldin A. Five hours later, the cultured cells were stained for CD49b (DX5), MHC class II, and IFN-{gamma}, and the data show several important points (Fig. 6A) . First, much of the IFN-{gamma} production was derived from MHC class II+ cells making this unlikely to be an activated NK population. Second, consistent with previous reports [4 , 30 ], we observed that NKDCs from PBS-administered mice did not produce IFN-{gamma} when stimulated with CpG in vitro, and this result contrasts our observation that in vivo LPS conditioning was not required for detection of intracellular TNF-{alpha} in response to in vitro CpG stimulation (unpublished observation). Third, and perhaps most importantly, in vivo conditioning with LPS induced these cells to produce IFN-{gamma}, even without PAMP stimulation, which was consistent with IFN-{gamma} detection in the None (no PAMP) column of Table 2 .

Analysis of the IFN-{gamma}-producing cells, when LPS was administered in mixed bone marrow chimeric mice as that in Table 2 , revealed that CD11c+ CD49b+ IAb+ (C57BL/6), or IEk+ (C3H/HeJ) cells produced IFN-{gamma} (Fig. 6B) , and in all experiments, the percentage of IFN-{gamma}-producing cells increased when cultured with CpG as opposed to brefeldin A alone. These finding were consistently reproducible in all four experiments that were performed with chimeric mice, and the data are consistent with those shown in Table 2 . Also, unconditioned cells, those receiving PBS in vivo, were unable to produce IFN-{gamma} even after CpG stimulation (data not shown). Although, it is possible that other cell types, such as activated NK cells were producing IFN-{gamma}, our data clearly suggest that LPS bystander-activated NKDCs (IEk+) produce IFN-{gamma} in appreciable amounts.

LPS induces T-bet up-regulation in an IFN-{gamma}-dependent manner
T-bet, a member of the T-box transcription factor family, controls Th1 responses [33 ]. T-bet is expressed in DCs and is necessary for IFN-{gamma} production, and at the same time, IFN-{gamma} is known to up-regulate T-bet gene expression, thus having autocrine control [34 ]. In a time kinetic study, LPS exposure of myeloid cells revealed that IFN-{gamma} production occurs prior to increase in T-bet mRNA [35 ]. We decided to investigate the role of IFN-{gamma} on T-bet gene expression when the cells are activated in a bystander manner. We injected upLPS in IFN-{gamma}+/+:C3H/HeJ and IFN-{gamma}–/–:C3H/HeJ mice, and 5 h later T-bet mRNA expression in C3H/HeJ (CD11c+ IEk+) cells was assessed by real-time PCR. We detected a twofold up-regulation in T-bet mRNA expression within 5 h in the C3H/HeJ (CD11c+ IEk+) cells obtained from IFN-{gamma} chimeric mice (Fig. 7 , top). On the other hand, we observed a profound decrease in T-bet mRNA expression as compared with HPRT mRNA (Fig. 7 , bottom) in C3H/HeJ (CD11c+ IEk+) cells obtained from IFN-{gamma}–/– chimeric mice. These results suggested that IFN-{gamma} might exert effects on bystander cells through T-bet and that T-bet may regulate hyper-responsiveness of conditioned innate cells such as NKDCs to CpG and poly I:C.


Figure 7
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  		Figure 7. IFN-{gamma} is responsible for induction of T-bet expression in CD11c+ IEk+ cells. Bone marrow was harvested from C57BL/6 (IFN-{gamma}+/+), C57BL/6 (IFN-{gamma}–/–), and C3H/HeJ mice and injected into irradiated recipient C3H/HeJ mice. After 8 wk, chimeric mice were screened for percentage of bone marrow reconstitution. Recipient C3H/HeJ mice having reconstitution of ~1:1 were treated with PBS or upLPS. After 5 h, spleens were collagenase digested and C3H/HeJ cells enriched using CD11c MACS beads and then further purified using anti-IEk PE antibodies, followed by anti-PE. T-bet mRNA expression was assessed by real-time PCR and compared with HPRT. Data of 3 different experiments are given.

 
DISCUSSION

During an ongoing microbial infection, cells of the immune system become activated directly or indirectly after they encounter multiple PAMPs. We have investigated some of these interactions in vivo, and our data suggest that DCs can become activated in a bystander manner through bacterial LPS. An earlier study demonstrated that in response to CpG, TLR9-responsive cells could induce bystander activation of TLR9-deficient DCs, and these bystander-activated DCs had suboptimal T cell priming capabilities [12 ]. In this report, we set out to uncover mechanism(s) of bystander activation. Our in vivo results demonstrated that LPS can activate DCs in a bystander manner, and second, although IFN-{gamma} was dispensable for CD86 up-regulation, it played an important role in the induction of T-bet gene and in conditioning innate cells to synthesize IFN-{gamma}.

MyD88 is a key adaptor that is involved in TLR4- and TLR9-mediated signaling [23 , 24 , 36 , 37 ]. The in vivo role of MyD88 in bystander activation in response to LPS has not been dissected, and in this report, we showed that MyD88-deficient cells were able to induce up-regulation of CD86 expression on LPS-nonresponsive (C3H/HeJ) cells (Fig. 4) . It is also important to note that the MyD88-deficient cells did not appear to up-regulate CD86 to the same extent as the wild-type cells, and this was translated in the bystander response as well. Ultimately, we found that CD86 up-regulation by the bystander cells was proportional to the LPS-responsive cells regardless of whether the responsive cells expressed MyD88. However, this may be an effect on CD86 but perhaps not for other molecules or for the function of the bystander cells.

IFN-{gamma} plays a pivotal role in determining the effectiveness of the immune response to pathogens. It has been shown that macrophages produce IFN-{gamma} in response to LPS [28 ], and recently, NKDCs have also been identified as a source of IFN-{gamma} producers [30 31 32 , 38 ]. IFN-{gamma} is also known to activate DCs and enhance antigen-presenting capabilities. We showed that IFN-{gamma} plays a critical role in enhancing responsiveness of bystander-activated cells to PAMPs, and at least one source of IFN-{gamma} producers were NKDCs (Fig. 6) . Certainly, other cells of the innate immune system, such as activated CD11c+ NK cells may contribute to IFN-{gamma} production as well [4 , 39 ]. Second, it is also shown that, in the absence of IFN-{gamma}, C3H/HeJ DCs up-regulated CD86, but the primary cell cultures showed very little capacity to synthesize IFN-{gamma} upon secondary challenge with PAMPs and also down-regulation of T-bet gene.

These data speak to the possibility that during an ongoing microbial infection, cells of the innate immune system can attain variegated activation states due to direct or indirect stimulation, but the precise functional and pathophysiological consequences remain unclear. This idea has been tested in one system by demonstrating that indirect activation of DCs by CpG resulted in their diminished capacity to prime specific T cells [12 ]. Second, it has been shown that DCs up-regulate CD80/86 and MHC molecules in response to {alpha}-galactosylceramide-mediated NKT cell activation, but in the absence of CD40 signaling, they were unable to initiate a CD4 and CD8 T cell immune response [40 ]. In this report, we investigated amechanism of attaining bystander DC activation. In addition to showing a role for IFN-{gamma}, we also discovered enhanced responsiveness through the TLR9 pathway (Table 2) . This was the case for both LPS (non) responsive cells.

LPS triggers a host innate immune response resulting in activation of various cell types and production of multiple cytokines. A number of hematopoetic and nonhematopoetic cells express abundant amounts of TLR4 [22 ]. Macrophages are the most prominent cells of the innate immune system, which express high levels of TLR4 and have been demonstrated to interact with LPS in vivo as compared with DCs or B cells [10 ]. DCs being terminally differentiated hematopoetic cells with a short half-life [41 ], they express low TLR4 levels [5 6 7 8 ] but possess highly specialized functions, such as very efficient processing and presentation of antigens. Also, DC exposure to LPS in vivo has been reported to mediate TLR4 gene down-regulation [7 ] and disappearance from T cell areas [42 ]. Even though the TLR4 levels are low, they may still be sufficient to activate DCs in a direct fashion. More in vivo work is needed to decipher the exact mechanism and contribution of the direct and bystander pathways.

In conclusion, our results demonstrated that enhanced responsiveness of bystander-activated cells to PAMPs was mediated through IFN-{gamma}, and at least one cellular source of IFN-{gamma} producers were the recently described CD11c+ CD49b+ MHC class II+ NKDCs [30 31 32 ]. These data suggest mechanisms of innate immune cell activation, including DCs in response to LPS and how the host may benefit from bystander activation. Alternatively, DC hyper-responsiveness to PAMPs may contribute to morbidity and mortality associated with septic shock.

ACKNOWLEDGEMENTS

We acknowledge Adam Adler and Meixiao Long for providing T-bet mRNA primers and for helpful discussions. This work was supported by grants from the National Institutes of Health AI 42858 (to A. T. V.) and IPO1-AI 56172 (L. L.).

Received April 6, 2006; revised July 27, 2006; accepted August 8, 2006.

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