High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists

Petar J. Popovic, Richard DeMarco, Michael T. Lotze, Steven E. Winikoff, David L. Bartlett, Arthur M. Krieg, Z. Sheng Guo, Charles K. Brown, Kevin J. Tracey and Herbert J. Zeh III
J Immunol December 15, 2006, 177 (12) 8701-8707; DOI: https://doi.org/10.4049/jimmunol.177.12.8701

Abstract

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-α, IL-6, TNF-α, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-γ-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.

Plasmacytoid dendritic cells (PDCs)3 are the natural type I IFN-producing cells, secreting high levels of IFN-α in response to viral infection. They serve an additional important role as APCs, regulating other immune cell function at sites of chronic inflammation. Their precise biologic role has not yet been fully defined, but it is clear that microbial mediators, so-called pathogen-associated molecular pattern (PAMP) molecules, tissue- derived factors or damage-associated molecular pattern (DAMP) molecules, and cytokines present in the inflammatory microenvironment alter the effector function of PDCs, thereby shaping the adaptive immune response (1, 2). For example, PDCs, exposed to the CpG oligodeoxynucleotide (ODN), a TLR9 agonist, and CD40 ligation secrete IL-12 and preferentially drive Th1 T cell responses (3, 4). Conversely, when exposed to CpG alone, PDCs secrete very little IL-12 but rather drive the development of CD4+CD25+ regulatory cells (5). PDCs preferentially drive Th2 T cell responses when cultured in IL-3 alone or following exposure to histamine, an endogenous inflammatory mediator released by mast cells and basophils (6, 7). Adenosine, a DAMP, released from activated or stressed cells promotes migration of immature PDCs but suppresses function of mature cells (8). Together these findings suggest that PDCs: 1) have significant functional plasticity, 2) are capable of responding to a wide range of endogenous and exogenous danger signals, and 3) are able to integrate these environmental signals and modify the quality of the evolving adaptive immune response.

High mobility group B1 (HMGB1) is an evolutionarily ancient DNA-binding protein that within the nucleus enhances access to transcriptional regulatory factors, nuclear hormones/hormone receptors (9), transposons, and recombinases. HMGB1 also serves as an extracellular cytokine, with an acetylated form secreted by activated macrophages, NK cells, PDCs, and mature myeloid dendritic cells (MDCs) (9, 10). HMGB1 is also passively released by cells following unscheduled (necrotic) cell death (11, 12), linking this to the subsequent recruitment of inflammatory cells. HMGB1 released in the presence of other mediators results in an inflammatory cascade characterized by endothelial activation, recruitment of inflammatory cells, mesangioblast recruitment and proliferation, and MDC maturation (9). HMGB1 also serves as a DAMP, delivering signals that tissue damage has occurred possibly priming the immune system to identify and then eradicate pathogens that might accompany such tissue damage (13). The impact of HMGB1 on PDC function is largely unexplored. In this study, we demonstrate that HMGB1 suppresses the PDC response to TLR9 agonists including viruses and CpG ODNs. HMGB1 inhibits the release of proinflammatory cytokines and down-regulates costimulatory and adhesion molecules on PDCs stimulated with CpG ODNs. Pretreatment of PDCs with HMGB1 inhibits their ability to promote a Th1 response.

Materials and Methods

Cells and reagents

Human PDCs were purified from PBMC of healthy Central Blood Bank of Pittsburgh donors using a direct magnetic labeling and positive selection kit for BDCA-4 (Miltenyi Biotec). PDCs were >95% pure, based upon positive expression of CD123, BDCA-2, and HLA-DR, and lack of expression of CD11c, CD3, CD19, CD14, and CD56. PDCs (0.5–5 × 105/ml) were cultured in complete medium supplemented with 10 ng/ml IL-3 (PeproTech). CD4+CD45RA+, naive T cells were obtained from PBMC of healthy donors using a human naive CD4+ T cell enrichment mixture (StemSep; StemCell Technologies). CD40L-transfected J558 cells (J558/PDC ratio, 1:2; provided by Dr. P. Kalinski, University of Pittsburgh, Pittsburgh, PA) were added to the culture when IL-12 production was assessed. All cultures were maintained at 37°C in a humidified 5% CO2 atmosphere in flat-bottom 96-well plates (Costar).

CpG ODNs class A (2336), B (2006), and C (2395) were created on a fully or partially protected phosphorothioate backbone (Coley Pharmaceutical Group). Recombinant human HMGB1 (0.1–10 μg/ml) was provided by K. J. Tracey (North Shore-Long Island Jewish Research Institute, Manhasset, NY) or was purchased from Sigma-Aldrich. HSV was the gift of Dr. F. Jenkins (University of Pittsburgh). Adenoviruses were the gifts of Dr. A. Gambatto (University of Pittsburgh).

HMGB1 purification from cells

Cells at a density of 1 × 107 cells/ml were lysed in buffer containing PBS with 1% igepal, 1 mM n-ethylmaleimide, 5 mg/ml 6-aminohexanoic acid, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 500 nM aprotinin, 50 μM leupeptin, 100 μM E64, 36 μM phosphoramidon, 40 μM bestatin, 1 mM benzamidine, 16 μM antipain, 10 μg/ml trypsin inhibitor, and 1 mM EDTA (Sigma-Aldrich). The lysate was centrifuged for 15 min at 16,000 × g to pellet nuclei and insoluble material. The supernatant was collected and filtered through a 0.45-μm filter. HMGB1 polyclonal Ab was obtained from New Zealand White rabbits immunized with the peptide sequence KSEAGKKGPGRPTGS corresponding to amino acids 166–181 of HMGB1 by Sigma-Aldrich under contract. The affinity purification of the polyclonal anti-HMGB1 Ab was performed following standard procedures. To affinity purify, HMGB1 10 ml of freshly prepared and filtered cell lysate was loaded onto the rabbit polyclonal anti-HMGB1 affinity column and recirculated for 30 min. The column was then washed with roughly 5 volumes of PBS, then the bound protein was eluted using three-column volumes of 3 M potassium thiocyanate (pH 7.0). The eluted protein was dialyzed overnight at 4°C in 2 L of PBS with magnetic stirring. The dialyzed protein was sterile filtered through a 0.2-μm filter and concentrated to a final volume of 1 ml using an Amicon Ultra 15 centrifugal concentrator (10,000 molecular mass). Final protein concentration was determined using the BCA reagent (Pierce Endogen).

Cytokine assays

Both IFN-α and IL-12 levels were determined by commercially available ELISA kits according to the manufacturer’s directions. The IFN-α ELISA (human IFN-α Module Set; Bender MedSystems) detects all species of human IFN-α at a lower detection limit of 10 pg/ml. The assay does not recognize IFN-β or IFN-γ. The IL-12 ELISA (Endogen Matched Ab Pairs; Pierce Biotechnology) detects human IL-12 p70 heterodimer at a lower detection limit of 10 pg/ml and does not cross-react with human p35 or p40 subunits. IL-6, IL-10, TNF-α, and inducible protein-10 (IP-10) were quantified by Luminex immunoassay within the University of Pittsburgh Cancer Institute Proteomics Core.

Cell surface staining of PDCs

After culture, PDCs were harvested, washed with PBS containing 2% FCS and 0.1% NaN3, and stained using a panel of directly conjugated mAb. mAbs used for staining were CD3-PC5, CD14-PC5, CD16-PC5, CD19-PC5, CD20-PC5, CD56-PC5, HLA-DR-ECD, CD40-PE, CD54-PE, CD58-PE, CD80-PE, CD83-PE, CD86-PE, HLA-ABC-PE (Immunotech; Beckman Coulter) and CD123-FITC (Miltenyi Biotec), TLR2-PE, TLR4-PE (eBioscience), and biotinylated human receptor for advanced glycation end products (RAGE; R&D Systems). PDCs were defined as CD3−14−19−20−56−16− (lineage mixture negative), HLA-DR+CD123+. For surface staining, cells were light fixed with 0.5% paraformaldehyde and analyzed using four-color flow cytometer (Epix XL; Beckman Coulter).

T cell polarization assays

After purification, PDCs were first cultured in IL-3-containing medium for 24 h, and then treated for an additional 48 h with CpG ODN (5 μg/ml), HMGB1 (5 μg/ml), or both. CD4+CD45RA+ naive T cells were isolated from an allogeneic donor using a negative selection kit (StemSep; StemCell Technologies). T cells were then added at a 5:1 or 1:1 ratio to the PDCs with addition of IL-2 (100 IU/ml) and incubated for 7–10 days. After coculture, T cells were stimulated for an additional 4 h with PMA (10 ng/ml) and ionomycin (1 μg/ml) in the presence of brefeldin A (10 μg/ml). Cells were washed, stained for surface Ag (CD3, 30 min on ice), fixed with 2% paraformaldehyde (20 min at room temperature), permeabilized by treatment with saponin (10 min at room temperature), and stained for intracellular expression of IL-5, IL-10, or IFN-γ (30 min at room temperature).

Statistical analysis

Data were analyzed using a two-tailed Student’s t test. All analyses were performed using Prism software (GraphPad Software). Differences were considered significant (∗) at a p value <0.05 and highly significant (∗∗) at a p value <0.01.

Results

rHMGB1 inhibits type I IFN production from PDCs stimulated with the TLR9 agonist, CpG ODN

We hypothesized that HMGB1, an inflammatory cytokine released by activated macrophages, mature MDCs, NK cells, and necrotic cells (11, 12), would modulate the immune function of PDCs. To characterize PDC function, we assessed its ability to produce IFN-α in response to TLR9 agonists such as CpG. We first established that CpG ODNs classes A (2336) and C (2395) could induce significant IFN-α secretion from freshly purified PDCs. Significant IFN-α production was noted with CpG concentration <1 μg/ml, with maximal release following stimulation with 5 μg/ml. As presented, CpG B failed to promote IFN-α secretion from PDCs in culture (Fig. 1⇓A). The presence of HMGB1 in culture inhibited CpG-induced IFN-α secretion from PDCs in a dose-dependent fashion (Fig. 1⇓B). This pattern of inhibition was reproduced in over 20 separate donors, and over a broad range of concentrations of CpG ODNs. In most donors, significant inhibition of IFN-α secretion was observed with rHMGB1 at concentrations of 0.2 μg/ml for CpG A and 0.5 μg/ml for CpG C. Complete inhibition was observed with 2 μg/ml and 5 μg/ml rHMGB1 for CpG A and CpG C, respectively. We did not observe direct effects of rHMGB1 on PDCs viability as assessed by trypan blue exclusion, tetrazolium salt conversion (MTT assay), or flow cytometric evaluation even at the highest concentration of rHMGB1 used (data not shown). Because HMGB1 is a DNA-binding protein, we wanted to exclude the possibility that the CpG ODNs were neutralized. Therefore, we first incubated PDCs with either CpG or rHMGB1 alone followed by washing and reincubation. As presented (Fig. 1⇓C), we observed similar inhibition of IFN-α secretion under these culture conditions. Stimulation with CpG ODNs followed by washing and rHMGB1 treatment demonstrated complete blocking of IFN-α production. Inhibition with rHMGB1 preincubation followed by washing and CpG ODN stimulation was not as complete. Comparable results were obtained when experiments were performed in the presence of polymyxin B, making contaminating LPS as an etiology of suppression less likely (data not shown).

FIGURE 1.
FIGURE 1.

rHMGB1 dose dependently inhibits IFN-α secretion from PDCs activated with CpG. A, PDCs (0.2 × 106/ml) were incubated in IL-3 (10 ng/ml) plus varying concentrations of CpG ODNs class A (2336), B (2006), and C (2395). B, PDCs (0.2 × 106/ml) were incubated in IL-3 (10 ng/ml) plus CpG ODNs (5 μg/ml) in the presence of the indicated rHMGB1 concentrations (micrograms per milliliter). C, PDCs (0.2 × 106/ml) were incubated with either CpG A (5 μg/ml) (▦) or increased doses of rHMGB1 (▪) for 1 h, washed, and incubated for 48 h in the presence of either rHMGB1 (▦) or CpG A (▪). Following 48 h of culture, supernatants were collected and assayed for IFN-α production. Data are presented as mean IFN-α concentration ± SD from triplicates in nanograms per milliliter from one representative of three independent experiments. ∗ correspond to p < 0.05.

Purified mammalian HMGB1 inhibits type I IFN production from PDCs stimulated with the TLR9 agonist, CpG ODN

Because it is possible that posttranslational modifications such as acetylation may be critical to the function of HMGB1 in mammalian systems, we next examined whether purified mammalian HMGB1 would result in similar inhibition of PDC function. HMGB1 was purified from human liver cell homogenates (total or nuclear fraction) or the HeLa cell line. Using PDCs from five different donors, we observed the same pattern of IFN-α inhibition with recombinant and purified mammalian HMGB1 (Fig. 2⇓). Although the pattern of inhibition was the same, purified HMGB1 was slightly less potent. In no instance did we observe enhancement of IFN-α production.

FIGURE 2.
FIGURE 2.

Purified mammalian HMGB1 dose dependently inhibits IFN-α secretion from PDCs activated with CpG. PDCs (0.2 × 106/ml) were incubated in IL-3 (10 ng/ml) plus either CpG C (A) or CpG A (B) (5 μg/ml) in the presence of the indicated concentrations of purified mammalian HMGB1 (μg/ml). Following 48 h of culture, supernatants were collected and assayed for IFN-α production. Data are presented as mean IFN-α concentration ± SD from triplicates in nanograms per milliliter from one representative of three independent experiments. ∗ correspond to p < 0.05.

rHMGB1 inhibits type I IFN production from PDCs stimulated with viral pathogens

CpG ODNs are synthetic molecules that mimic natural viral/bacterial PAMPs. We next examined whether HMGB1 could similarly inhibit endogenous viral CpG-induced IFN-α release from PDCs. In preliminary experiments, we established that the effective optimal multiplicity of infection (MOI) for IFN-α production for several representative viral strains fell between 1 and 5 MOI (data not shown). We observed that HSV induced the highest level of IFN-α production from infected PDCs. Interestingly, the dsDNA Western Reserve vaccinia virus (WRvv) failed to elicit any IFN-α secretion from infected purified PDCs, while the effect of ssDNA adenovirus strain 35 (Adeno 2) depended on the presence of IL-3 (Fig. 3⇓). Similarly to its effect on synthetic CpG-stimulated PDCs, we observed that rHMGB1 (5 μg/ml) was able to significantly inhibit PDC secretion of IFN-α in response to the HSV and the adenoviral preparations used (Fig. 3⇓). These data also support our observations in Fig. 1⇑C, as the viral-encoded CpG would be inaccessible for ex vivo recombination with our exogenously added HMGB1.

FIGURE 3.
FIGURE 3.

rHMGB1 inhibits IFN-α secretion from virus activated PDCs. PDCs (0.2 × 106/ml) were stimulated with CpG ODNs (5 μg/ml) or different viral vectors (Adeno 1, adenovirus strain 33; Adeno 2, adenovirus strain 35; vaccinia virus, WRvv) (the most effective MOI), with or without rHMGB1 (5 μg/ml) (A) in the presence of IL-3 (10 ng/ml) or (B) without IL-3. Data are presented as a mean ± SD from triplicates in picograms per milliliter from one representative of three independent and comparable experiments. ∗ correspond to p < 0.01.

rHMGB1 suppresses IL-6, TNF-α, and IP-10 production from PDCs stimulated with the TLR9 agonist, CpG ODN

Following stimulation with virus or CpG ODNs, PDCs produce various cytokines and chemokines (IL-6, TNF-α, and IP-10) in addition to IFN-α (2). We next examined the rHMGB1 effects on CpG-induced secretion of these cytokines. As presented in Fig. 4⇓, CpG treatment of PDCs induced >20-fold increases in IL-6, TNF-α, and IP-10 production when compared with IL-3 alone. CpG activation of PDCs alone did not result in consistent production of either IL-10 or IL-12. The presence of rHMGB1, however, decreased the induction of all three of these cytokines. The level of inhibition varied from ∼35% for IL-6, 59% for IP-10, and up to 75% inhibition of TNF-α production (Fig. 4⇓). Such inhibition of individual cytokines suggests that rHMGB1 interferes with PDC activation and/or maturation, rather than simply with cytokine (IFN-α) secretion alone.

FIGURE 4.
FIGURE 4.

rHMGB1 inhibits IL-6, TNF-α, and IP-10 secretion from CpG-stimulated PDCs. PDCs (106/ml) were cultured in IL-3 (10 ng/ml) ± CpG C ODN (5 μg/ml) in the presence of rHMGB1 (2 μg/ml). After 48 h, supernatants were collected for cytokine detection. Data are presented in picograms per milliliter as a mean ± SD from triplicates from one representative experiment of three performed. ∗ correspond to p < 0.05.

rHMGB1 inhibits IL-12 production from PDCs stimulated by TLR9 and CD40 ligation

Both human and murine PDCs produce IL-12 (both p35/p40 as well as p40) in response to CpG and CD40 ligation in the presence of IL-3 (2, 14). We next examined the effect of rHMGB1 on this function. PDCs were incubated with IL-3 and CpG for 48 h. Irradiated CD40L-expressing J558 cells were then added for an additional 48 h and supernatants were assayed for the presence of IL-12. Although the observed level of IL-12 production was not as high as in other cell types, it was consistent and reproducible in all tested donors. Importantly, we observed a significant decrease in IL-12 production in the presence of rHMGB1 (Fig. 5⇓). As presented, similar levels of suppression of IL-12 production were observed when rHMGB1 was present at the beginning of the 4-day culture (rHMGB1 early) as well as when it was added after the first 48 h, together with the CD40L-expressing J558 cells (rHMGB1 late). The level of IL-12 secretion corresponded directly with the level of CD40 expression on PDCs, which increased coordinately when incubated in the presence of IL-3 and CpG (Fig. 6⇓A). However, IL-12 was not detected from PDCs following CD40 ligation alone. This suggests that rHMGB1 could influence not only the behavior of immature PDCs in early stages of activation but also cytokine secretion by fully stimulated and more mature cells. We also examined IL-10 production in this system. We observed consistent increase in IL-10 following CD40 ligation in all samples tested. rHMGB1 failed to mediate a consistent effect on IL-10 production (data not shown).

FIGURE 5.
FIGURE 5.

rHMGB1 inhibits IL-12 production from CpG plus CD40-stimulated PDCs. PDCs (106/ml) were cultured in IL-3 (10 ng/ml) ± CpG C ODN (5 μg/ml) in the presence of rHMGB1 (2 μg/ml). After 48 h, irradiated CD40L-transfected J558 cells were added (ratio 1:1). IL-12 production was measured after an additional 48 h of culture. rHMGB1 were not present (▦), added at the start (▪) or concurrently with J558 cells (□). Data are presented in picograms per milliliter as a mean ± SD from triplicates from one representative experiment of five performed. ∗ correspond to p < 0.01.

FIGURE 6.
FIGURE 6.

rHMGB1 diminishes expression of costimulatory and adhesion molecules following stimulation with PDCs. A, PDCs were cultivated for 72 h in illustrated different experimental conditions or (B) in IL-3 (10 ng/ml) ± CpG C ODN (5 μg/ml) and stained for membrane expression of individual maturation molecules with or without rHMGB1 (5 μg/ml). Only viable (forward scatter/side scatter gate) and HLA-DR+CD123+ cells were gated and analyzed. Data are presented as mean fluorescence intensity (m.f.i.) and histograms fluorescence from one of five experiments with similar results.

rHMGB1 inhibits the maturational phenotype of PDCs

Following exposure to synthetic TLR9 agonists, PDCs undergo a maturation and differentiation program characterized by up-regulation or de novo expression of the costimulatory (CD40, CD80, CD86), adhesion (CD54, CD58), and homing (CCR7) molecules as well as maturation markers (CD83) (3, 4, 15). In preliminary experiments, we observed, as have others (2), that CpG A was less effective than CpG C in up-regulating the relevant cell surface maturation markers and these changes were maximal at 48–72 h (Fig. 6⇑A). PDCs cultured in medium alone demonstrated no apparent changes in these cell surface molecules, while PDCs cultured in low-dose IL-3 alone demonstrated a small but consistent up-regulation of CD40 only. As presented, the presence of IL-3 additionally increased the effect of CpGs (Fig. 6⇑A). When rHMGB1 was added to CpG C-activated PDCs, we consistently observed diminished cell surface expression of CD40, CD80, CD83, CD86, CD54, and CD58 molecules (Fig. 6⇑B). Interestingly, there was no change in cell surface expression of either class I or class II MHC molecules. It is important to note that the relative low level of HLA-DR fluorescence intensity depicted is a consequence of compensation for multicolor flow analysis.

rHMGB1 inhibits the generation of Th1 cells

Our findings suggest that the HMGB1 as a DAMP molecule, in addition to altering secretion of inflammatory cytokines, also significantly alters the ability of PDCs to promote T cell activation. We therefore next directly examined the effect of HMGB1 on PDC-driven maturation of naive T cells. We pretreated PDCs with IL-3 plus CpG with or without rHMGB1 and then cocultured them with allogeneic naive CD4+CD45RA+ T lymphocytes. Following priming and expansion in low concentrations of IL-2, T cells were activated and the patterns of IL-5, IL-10, and IFN-γ production were analyzed using intracellular staining and flow cytometry. We did not observe any difference in the total number of T cells by trypan blue exclusion or flow cytometric evaluation between these culture conditions. PDCs treated with CpG increased the number of T cells producing IFN-γ (Fig. 7⇓) as previously described by others (16). Pretreatment of PDCs with rHMGB1 led to potent and sometimes complete abrogation of T cell secretion of IFN-γ (Fig. 7⇓). Although the effect of IL-3 was significantly weaker by itself, rHMGB1 suppression was also evident in PDCs cultured in IL-3 alone. We did not observe significant increases in the number of T cells capable of either IL-5 or IL-10 secretion (<0.5%; data not shown). PDCs that encountered rHMGB1 during the maturation process potently inhibited generation of Th1 cells producing IFN-γ. Overall, these findings demonstrate that rHMGB1 in addition to suppressing proinflammatory cytokine production, also modulates the ability of PDCs to communicate directly with naive T cells.

FIGURE 7.
FIGURE 7.

rHMGB1 inhibits allogenic PDC-driven IFN-γ production from naive T cells. PDCs were cultured in IL-3 (10 ng/ml) ± CpG C ODN (5 μg/ml) with or without rHMGB1 (5 μg/ml) for 48 h. PDCs were then added to allogeneic naive CD4+CD45RA+ T cells, at 1:1 ratio. After 9 days, T cells were activated with PMA (10 μg/ml) and ionomycin (1 μg/ml) in the presence of brefeldin A. Cells were then stained for CD3 and IFN-γ. Data are presented as a percentage of IFN-γ-positive cells in CD3 gate. Results are representative of five independent experiments using cells obtained from five different donors.

PDCs express RAGE, but not TLR2 or TLR4

HMGB1 mediates its biologic effects through several receptors including RAGE, TLR2, and TLR4 (9, 13, 17). We next tested expression of those receptors on PDCs. Consistent with the findings of others (18, 19), we observed no significant expression of TLR2 or TLR4 on either resting or stimulated PDCs (data not shown). We, however, did observe low but consistent expression of RAGE on fresh PDCs obtained from several donors, as well as an increase in expression in culture with IL-3 and following CpG activation (Fig. 8⇓). Interestingly, we also observed that s100β, another putative RAGE ligand, could similarly inhibit type I IFN production from PDCs stimulated with the TLR9 agonist, CpG ODN (data not shown). Together, those results are consistent with the notion that HMGB1/RAGE interaction may be involved in the PDC suppression we observed, although additional experiments need to be done to confirm that hypothesis.

FIGURE 8.
FIGURE 8.

PDCs express RAGE. Freshly isolated and PDCs cultivated for 24 h in IL-3 (10 ng/ml) ± CpG C ODN (5 μg/ml) were stained for membrane expression of RAGE. Results are representative of three independent experiments obtained with different donors.

Discussion

The impact of HMGB1 on PDC function has been largely unexplored. It has been shown previously that other DAMP molecules including adenosine (8) and histamine (16) inhibit IFN-α production from PDCs. Here, we demonstrate for the first time that exogenous HMGB1 can inhibit TLR9-mediated IFN-α secretion from PDCs. This potent inhibitory effect was observed when the activation signal was synthetic CpG ODNs or following viral infection. Purified mammalian HMGB1 behaved similarly to rHMGB1 excluding the possibility that posttranslational modification, not available in bacterial production systems, might explain the observed inhibitory effects. The biologically active range of HMGB1 in our system is similar to that reported in other biologic assays including maturation of MDCs and activation of endothelial cells and macrophages (12, 20, 21).

We also observed that rHMGB1 was able to inhibit the production of several important PDC-mediated proinflammatory cytokines. The ability to inhibit IL-6 and TNF-α further supports the anti-inflammatory effect of HMGB1 on PDC function. Inhibition of IP-10 additionally suggests that HMGB1 could prevent attraction of CXCR3-bearing Th1 T cells and NK cells to the site of inflammation, which normally occurs in response to IP-10. Together with TNF-α, IP-10 production is an important Th1 chemokine secreted by PDCs in response to TLR7 agonists (22, 23, 24).

Consistent with previous reports (25), we observed that CpG activation of PDCs alone did not result in detectable production of either IL-10 or IL-12. However, CD40 ligation induced a low, but reproducible, IL-12 production from PDCs in our system. This IL-12 production by PDC was also inhibited by rHMGB1. rHMGB1-mediated suppression of IL-12 secretion can only be partially explained by lower IFN-α levels in culture, or by decrease of membrane expression of CD40, as postponing addition of rHMGB1 to allow maximal IFN-α production and CD40 expression demonstrated similar inhibition. Others have observed that human and murine PDCs can produce IL-12 (p70 and p40) in response to CpG and CD40 ligation in the presence of IL-3 (8, 14, 25). Some have suggested, however, that IL-12 secretion from BDCA-4-purified PDCs could be from contaminating monocytes or MDCs (26). Addition of LPS to our cultures did not influence IL-12 production, making this explanation less likely in our estimation. In addition, rHMGB1 increases IL-12 production from myeloid DC’s (20).

Although early reports suggested that PDCs preferentially favored the development of IL-4-secreting Th2 cells, later work suggested that PDCs are more flexible in directing T cell responses, depending on the maturation stage, nature, and concentration of the Ag (27) as well as other local factors (2). For example, virus-activated or CpG-activated PDCs exposed to CD40 ligation elicit a potent Th1 polarization, inducing T cells to produce IFN-γ (3). The Th1 polarization mediated by mature PDCs depends on the elicitation of type I IFN (28), or on both type I IFN and IL-12 (3). Consistent with what has been previously reported (5), we observed that PDCs treated with CpG increased the number of T cells producing IFN-γ (29). This Th1 polarization was completely inhibited by maturation of PDC in the presence of rHMGB1.

Recently, Dumitriu et al. (15) have suggested that autocrine secretion of HMGB1 can stimulate IFN-α and is necessary for full maturation of human PDCs. It is important to point out that in this manuscript, the role of exogenous HMGB1 on CpG-induced PDC maturation and IFN-α secretion was abstracted from experiments that used agents purported to block HMGB1 activity. A direct effect of exogenous HMGB1 on PDC maturation was never reported. Both groups have confirmed expression of RAGE receptors on the surface of fresh, unstimulated PDC, suggesting that these cells should be susceptible to RAGE-mediated HMGB1 signaling before CpG activation. In personal communication with this group (P. Rovere-Querini), they confirm that exogenous rHMGB1 inhibited IFN-α production from CpG-activated PDC in their system as well. It is possible that the difference in the systems is due to the alternative in RAGE-mediated signaling between actively secreted and passively released HMGB1. Actively secreted HMGB1 has been reported to be hyperacetylated when compared with passively released (30). Alternatively, some have suggested that HMGB1 may be a chaperone/facilitator molecule for a number of individual PAMPs/DAMPs, and that its biologic activity is dependent upon these other molecules (31). We are actively pursuing technology that will allow us to purify sufficient quantities of actively secreted HMGB1 to test these hypotheses.

In summary, we demonstrate that exogenous HMGB1 can negatively modulate the proinflammatory function of PDCs in response to TLR9 agonists. This observation is interesting in light of the growing number of reports demonstrating proinflammatory properties of HMGB1 on macrophages, NK cells, and myeloid dendritic cells (2, 20, 26). These findings support the notion that PDCs may function as biologic “rheostats” sensing and integrating the level of tissue injury delivered by DAMP molecules. We hypothesize that HMGB1 delivers signals to PDCs indicating ongoing tissue injury or damage. Significant levels of tissue injury or damage would also be expected to be associated with release of a variety of other intracellular proteins that could potentially serve as neo-self Ags. Suppressing PDC-driven Th1 cells may be one mechanism by which DAMP molecules such as HMGB1 limit the development of autoimmunity in the setting of chronic inflammation and coordinate the infectious nonself response and the need to repair local tissue damage. Our observations may be of particular significance in the clinical setting of cancer (32). High or persistent levels of tissue damage or injury could favor diminution of the inflammatory response and limit proinflammatory cytokine secretion from recruited PDCs. Several reports have documented significant PDCs infiltration and nominal dysfunction within tumors from patients with breast, ovarian, melanoma, or head and neck carcinomas (33, 34, 35, 36, 37, 38). We have identified elevated HMGB1 in the serum of patients with a variety of cancer types, as well as significant release from dying tumor cells in vitro (our unpublished observations).

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by an American Society of Clinical Oncology Career Development Award (to H.J.Z.).

  • ↵2 Address correspondence and reprint requests to Dr. Herbert J. Zeh III, Division of Surgical Oncology, University of Pittsburgh Cancer Institute, University of Pittsburgh Medical Center Cancer Pavilion, Suite 440, 5150 Center Avenue, Pittsburgh, PA 15232. E-mail address: zehh{at}upmc.edu

  • ↵3 Abbreviations used in this paper: PDC, plasmacytoid dendritic cell; PAMP, pathogen-associated molecular pattern; DAMP, damage-associated molecular pattern; ODN, oligodeoxynucleotide; HMGB1, high mobility group B1; MDC, myeloid dendritic cell; IP-10, inducible protein-10; RAGE, the receptor for advanced glycation end products; RT, room temperature; MOI, multiplicity of infection; WRvv, Western Reserve vaccinia virus.

  • Received October 20, 2005.
  • Accepted September 29, 2006.

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