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Originally published online as doi:10.1189/jlb.0306171 on October 11, 2006

Published online before print October 11, 2006
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(Journal of Leukocyte Biology. 2007;81:84-91.)
© 2007 by Society for Leukocyte Biology

The secretion of HMGB1 is required for the migration of maturing dendritic cells

Ingrid E. Dumitriu*, Marco E. Bianchi{dagger}, Monica Bacci*, Angelo A. Manfredi* and Patrizia Rovere-Querini*,1

* Clinical Immunology Unit and
{dagger} Chromatin Dynamics Unit, H San Raffaele Scientific Institute and Università Vita-Salute San Raffaele, Milan, Italy

1Correspondence: Cancer Immunotherapy and Gene Therapy Program, Clinical Immunology Unit, H. San Raffaele Scientific Institute, via Olgettina 58, Milano 20132, Italy. E-mail: rovere.patrizia{at}hsr.it

ABSTRACT

Chemokines regulate the migration and the maturation of dendritic cells (DC) licensed by microbial constituents. We have recently found that the function of DC, including their ability to activate naïve, allogeneic CD4+ T cells, requires the autocrine/paracrine release of the nuclear protein high mobility group box 1 (HMGB1). We show here that human myeloid DC, which rapidly secrete upon maturation induction their own HMGB1, remodel their actin-based cytoskeleton, up-regulate the CCR7 and the CXCR4 chemokine receptors, and acquire the ability to migrate in response to chemokine receptor ligands. The events are apparently causally related: DC challenged with LPS in the presence of HMGB1-specific antibodies fail to up-regulate the expression of the CCR7 and CXCR4 receptors and to rearrange actin-rich structures. Moreover, DC matured in the presence of anti-HMGB1 antibodies fail to migrate in response to the CCR7 ligand CCL19 and to the CXCR4 ligand CXCL12. The blockade of receptor for advanced glycation end products (RAGE), the best-characterized membrane receptor for HMGB1, impinges as well on the up-regulation of chemokine receptors and on responsiveness to CCL19 and CXCL12. Our data suggest that the autocrine/paracrine release of HMGB1 and the integrity of the HMGB1/RAGE pathway are required for the migratory function of DC.

Key Words: cell trafficking • cell death

INTRODUCTION

The high mobility group box 1 protein (HMGB1) is a prototypic intracellular molecule released in the environment as a consequence of tissue necrosis [1 , 2 ]. Moreover, monocytes and macrophages challenged with TLR ligands actively secrete HMGB1 [3 ]. Active secretion of HMGB1 involves the acetylation of the protein and its transfer to secretory endolysosomes [3 4 5 ].

Extracellular HMGB1 is an important mediator of inflammation elicited by the presence of dying cells and/or infectious agents [6 7 8 ]. Exogenous HMGB1 activates in vitro the most potent APC, the dendritic cells (DC) [9 , 10 ]. HMGB1 also controls DC function in vivo, with adjuvant effects on the immunogenicity of soluble or corpuscolate antigens [9 ]. In response to microbial constituents, including LPS, DC undergo a complex differentiation program, referred to as maturation. DC maturation includes the up-regulation of surface markers involved in T cell activation and costimulation such as MHC, CD40, CD80, and CD86 molecules and is involved in the productive activation of naïve T cells [11 ]. In certain conditions, necrotic cells provide HMGB1 for DC maturation, an event that may result in immune surveillance after traumatic tissue death [6 , 9 ]. Other potentials sources of environmental HMGB1 are human myeloid and plasmacytoid DC, which express HMGB1 in the nucleus and actively secrete it during maturation in response to inflammatory stimuli [12 13 14 ].

Secreted HMGB1 is necessary for proliferation, survival, and polarization of naïve CD4+ T cells after activation by allogeneic DC [13 ]. These effects abate in the presence of blocking antibodies specific for the best-characterized membrane receptor for HMGB1, the receptor for advanced glycation end products (RAGE) [15 , 16 ], which is expressed by DC. These results suggest a paracrine/autocrine role of released HMGB1, which sustains via the activation of the RAGE receptor, the maturation of DC primed by microbial constituents [13 ].

HMGB1 therefore has activating effects on APC and is released in response to cell and tissue injury and in response to microbial constituents. DC are mobile cells, and this feature is crucial for their action in vivo. After detection and capture of microbes in peripheral tissues, DC disassemble the actin-based cytoskeleton [17 ] and up-regulate the expression of receptors to chemokines, which are constitutively expressed in secondary lymphoid organs such as lymph nodes and spleen [18 , 19 ], where they meet and activate (or tolerize) antigen-specific T cells. In this study, we concentrated on the effect of HMGB1 on DC migration. We show that HMGB1 is indeed a crucial regulator of DC chemotaxis and that this action involves its membrane receptor, RAGE.

MATERIALS AND METHODS

Media and reagents
DC were propagated in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 100 U/ml penicillin (BioWhittaker, Walkersville, MD), 100 µg/ml streptomycin (BioWhittaker), 1.5 mM L-glutamine (BioWhittaker), and 10% heat-inactivated FCS (EuroClone, Italy). Affinity-purified rabbit polyclonal anti-HMGB1 antibody raised against peptide 166–181 was purchased from BD Biosciences PharMingen (San Jose, CA). Goat polyclonal antibody against human RAGE was purchased from Chemicon International (El Segundo, CA). Expression and purification of recombinant HMGB1, box A, and GST protein (used as control) were performed as described previously [20 ]. Endotoxin was removed by passage through Detoxy-Gel columns (Pierce Chemical, Rockford, IL) following the manufacturer’s instructions. Endotoxin contamination of recombinant proteins was below 0.1 EU/ml. All reagents, including the culture medium, were tested for endotoxin contamination using the chromogenic Limulus amebocyte lysate assay (BioWhittaker) and were found negative.

Cells
Human PBMC of healthy donors were isolated from buffy coats (kindly provided by the Blood Transfusion Department of San Raffaele Scientific Institute, Milano, Italy) by Ficoll (BioChrome, Berlin, Germany) density gradient centrifugation. To generate immature DC, PBMC were resuspended in RPMI containing 5% heat-inactivated human serum (BioWhittaker) and allowed to adhere to six-well plates (Corning, Corning, NY) for 1 h at 37°C in a humidified 5% CO2 atmosphere. Nonadherent cells were discarded, and the monocytes were incubated with 800 U/ml GM-CSF and 800 U/ml IL-4 (R&D Systems, Minneapolis, MN) for 6 days. Cytokines were replenished on Days 3 and 5. LPS (1 µg/ml, Sigma Chemical Co., St. Louis, MO), alone or in the presence of rabbit anti-HMGB1 antibodies (1:200 dilution), goat anti-RAGE antibodies (1:200 dilution), recombinant box A (10 µg/ml), control Igs (1:200 dilution), or control GST protein (10 µg/ml), was added as indicated to induce DC maturation. In selected experiments, TNF-{alpha} (200 ng/ml, R&D Systems) or soluble human CD40 ligand (CD40L; 0.5 µg/ml, Alexis, San Diego, CA) was also used.

Flow cytometry
Surface antigen staining was performed using mouse mAb against human CD1a, CD14, CD40, CD80, CD83, CD86, and HLA-DR (BD Biosciences PharMingen). Appropriate fluorochrome-conjugated isotype control antibodies (BD Biosciences PharMingen) were used. Cells were washed with PBS, and then, 2–5 x 105 cells were incubated with mAb for 30 min at 4°C in PBS containing 1% FCS. Then, cells were washed and resuspended in cold PBS. Cellular debris was excluded from analysis using the forward- and side-scatter parameters. The samples were analyzed on a FACScan (BD Biosciences PharMingen) flow cytometer. At least 5000 events were collected for each sample. Results were processed using CellQuest software (BD Biosciences).

Expression of chemokine receptors and chemotaxis assay
The expression of chemokine receptors was detected by indirect immunofluorescence. To this aim, cells were incubated with mAb to human CCR7 (MBL, Tokyo, Japan) and CXCR4 (BD Biosciences PharMingen, San Diego, California) for 30 min on ice, followed by incubation with fluorochrome-conjugated secondary antibodies. The samples were analyzed by flow cytometry as above. Before chemotaxis, DC were washed and resuspended in migration medium (RPMI supplemented with 0.5% BSA). The migration of DC was measured by chemotaxis through a 5-µm pore polycarbonate filter in 24-well transwell chambers (Corning), as described previously [21 ]. Three replicate wells were used for each tested chemokine as well as for the medium control. Briefly, 1 x 105 cells were placed into the transwell inserts, and 500 ng/ml CCL19 (R&D Systems) or 100 ng/ml CXCL12 (R&D Systems) was added to the lower wells. After incubating for 2 h at 37°C, the cells were recovered and mixed with a fixed number of 10 µm polystyrene fluorospheres (Flow-Count fluorospheres, BD Biosciences PharMingen). Migrated cells were quantified by acquisition of a fixed number of polystyrene fluorospheres using a FACScan (BD Biosciences PharMingen) flow cytometer. The number of cells in the starting population and the migrated population was measured, and the percent migration was calculated using these values.

Immunofluorescence
Cellular cytoskeleton was assessed as described [17 ]. Shortly, immature DC and DC induced to mature by LPS in the presence or in the absence of anti-HMGB1 antibodies (see above) were transferred on polylysine-coated coverslips and incubated at 37°C for 60 min. Adherent cells were fixed with phosphate-buffered formaldehyde (4%, pH 7.4) for 15 min and permeabilized with saponin after blocking with BSA 10%. To visualize F-actin, DC were incubated for 30 min at room temperature with tetramethylrhodamine isothiocyanate-labeled phalloidin. Nuclei were counterstained with Hoechst 33342 (Molecular Probes, Junction City, OR). Samples were mounted using Moviol medium (Calbiochem, San Diego, CA). Confocal microscopy was performed as described [17 ] using a Leica instrument.

RESULTS

DC depend on secreted HMGB1 for CCR7 and CXCR4 up-regulation and for migration to chemokine receptor ligands, CCL19 and CXCL12
We derived DC by culturing peripheral monocytes in the presence of recombinant human (rh)IL-4 and GM-CSF. After 1 week, we analyzed the expression of various membrane receptors. DC did not express the monocyte markers CD14. In contrast, they expressed, as expected, the CD1a marker. They also expressed low levels of the costimulatory molecules CD80 and CD86 and of molecules involved in T cell activation such as CD40 and MHC Class II (HLA-DR) and negligible amounts of the chemokine receptors CCR7 and CXCR4 (Fig. 1 ). After stimulation with LPS, mature DC up-regulated CD40, CD80, CD83, CD86, and HLA-DR and expressed CCR7 and CXCR4 (Fig. 1) . In parallel, they relocated HMGB1 from the nucleus to cytosolic vesicles and actively secreted HMGB1 in the extracellular environment (not shown; see ref [13 ]).


Figure 1
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  		Figure 1. Phenotype of in vitro-generated DC. Immature DC (w/o) did not express the monocyte marker CD14, but they expressed CD1a and low levels of molecules involved in the costimulation and activation of T cells such CD40, CD80, CD86, and MHC Class II molecules (DR). Stimulation with LPS induced the expression of the activation marker CD83 and the up-regulation of CD40, CD80, CD86, and MHC Class II molecules. Treatment with LPS resulted in up-regulation of chemokine receptors CCR7 and CXCR4, which endow mature DC with the ability to migrate to the lymph nodes. Black histograms represent the staining with isotype control antibodies. Results are from representative, routine experiments.

 
Extracellular HMGB1 has a well-characterized role in migration and proliferation of cells involved in tissue repair in vitro and in vivo [22 , 23 ]. We therefore verified whether bioactive HMGB1 at the site of DC maturation is necessary for the expression of receptors for lymph node chemokines. To this aim, we challenged DC with LPS in the presence or the absence of antibodies that antagonize secreted HMGB1.

After LPS stimulation, DC up-regulated the CCR7 and CXCR4 chemokine receptors (Fig. 2 ). This event abated when DC maturation was elicited in the presence of HMGB1-blocking antibodies (P<0.05; Fig. 2 ). HMGB1 blockade per se did not influence the expression of chemokine receptors by DC not committed to HMGB1 secretion (Fig. 2) , suggesting that the latter event is necessary for up-regulation of CCR7 and CXCR4. HMGB1 blockade also interferes with the up-regulation of CCR7 and CXCR4 (Fig. 2) and of CD40, CD83, and CD86 (Fig. 3 ) induced by rhTNF-{alpha} or soluble CD40L and on the associated production of IL-12 p70 (P<0.05).


Figure 2
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  		Figure 2. Blockade of HMGB1 prevents the up-regulation of CCR7 and CXCR4 by maturing DC. Immature DC were cultured alone (–) or in the presence of LPS, TNF-{alpha}, or CD40L for 48 h. Anti-HMGB1 (anti HMG) or isotype control (ctrl. Ig) antibodies were added to the culture medium as indicated. Up-regulation of CXCR4 receptor induced by LPS (A) or by TNF-{alpha} and CD40L (B). Up-regulation of CCR7 receptor induced by LPS (C) or by TNF-{alpha} and CD40L (D). In the presence of anti-HMGB1 antibodies, the up-regulation of CXCR4 and CCR7 by mature DC was decreased, and no effect was observed on immature DC. Control antibodies did not alter the expression of chemokine receptors by DC. Results are depicted as mean ± SD of three independent experiments performed with DC from different donors. *, Statistically significant values (P<0.05). MFI, Mean fluorescence intensity; a.u., arbitrary units.

 

Figure 3
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  		Figure 3. Blockade of HMGB1 prevents the up-regulation of CD40, CD83, and CD86 but not of HLA-DR by maturing DC. Immature DC were cultured in the presence of TNF-{alpha} or CD40L for 48 h. Anti-HMGB1 (anti HMG) or anti-RAGE (anti RAGE) antibodies were added to the culture medium as indicated. In the presence of anti-HMGB1 and anti-RAGE antibodies, the up-regulation of CD40 (A), CD83 (B), and CD86 (C) by maturing DC was decreased, and no effect was observed on the up-regulation of HLA-DR (D). Results are depicted as mean ± SD of three independent experiments performed with DC from different donors. * and **, Statistically significant values (*, P<0.05; **, P<0.005). n.d., Not determined.

 
Next, we checked if the decreased expression of chemokine receptors is accompanied by an alteration of DC migration in response to CCL19 or CXCL12, the chemokine ligands for CCR7 and CXCR4, respectively [21 , 24 ]. As expected, LPS-stimulated DC migrated in response to both chemokines (Fig. 4 ). DC stimulated with LPS in the presence of anti-HMGB1 antibodies had a significantly reduced migration capacity to CCL19 and CXCL12 (P<0.005 and P<0.05, respectively; Fig. 4 ).


Figure 4
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  		Figure 4. Blockade of HMGB1 inhibits the migration of mature DC to CCL19 and CXCL12 chemokines. Immature DC were stimulated with LPS alone or in the presence of anti-HMGB1 antibodies for 16 h (LPS antiHMGB1). The migration of 105 cells/well (input), alone (A) or in the presence of the indicated chemokines (B, CCL19, 500 ng/ml; and C, CXCL12, 100 ng/ml), was measured. Mature DC migrated to CCL19 and CXCL12. DC stimulated with LPS in the presence of antibodies antagonizing secreted HMGB1 migrated less efficiently to CCL19 and CXCL12. Representative results from one out of three experiments performed are expressed as mean percent migration of input cells ± SD of triplicate wells. * and **, Statistically significant values (*, P<0.05; **, P<0.005).

 
Blockade of HMGB1 influences the cytoskeletal reorganization of maturing DC
Cell migration requires a coordinate series of events, which includes a sequential rearrangement of surface adhesion molecules and of the actin-based cytoskeleton. Immature DC are characterized by numerous membrane expansions, which terminate in adhesive structures (Fig. 5 ). These podosome structures, whose assembly reflects the cytoskeletal integrity in immature DC, concentrate ß2-integrins around a core of actin surrounded by a ring containing vinculin, talin, and paxillin and represent sites of rapid actin turnover [25 ]. As expected, TLR4 activation by LPS resulted in a profound shape alteration with substantial podosome disassembly: DC lost the large membrane expansions and adhesiveness, and polymerized F-actin and adhesive structures were no longer detectable (Fig. 5) . Functional blockade of extracellular HMGB1 by mean of specific antibodies during DC maturation strikingly prevented the cytoskeleton modifications (Fig. 5) .


Figure 5
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  		Figure 5. Blockade of HMGB1 inhibits the cytoskeleton modifications in DC after treatment with LPS. Confocal laser-scanning microscopy of immature DC (A) or DC cultured in the presence of LPS, alone (B) or with anti-HMGB1 antibodies for 48 h (C, D). Cells were stained with phalloidin (red color, A, B, and D); nuclei were counterstained with Hoechst 33342 (blue color, A–D). Untreated cells appear to be characterized by subcortical actin aggregates (A). Treatment with LPS induces loss of subcortical actin organization (B). Cytoskeleton modifications associated to DC maturation abate in the presence of anti-HMGB1 antibodies (D).

 
DC depend on RAGE for HMGB1-mediated control of chemokine receptor up-regulation and for migration to lymph node chemokines
DC express RAGE, a well-characterized membrane receptor for HMGB1, which plays a role in LPS-induced, DC maturation [10 , 12 , 13 , 26 ]. Upon LPS-elicited maturation, DC significantly down-regulated RAGE expression (Fig. 6 ). RAGE down-regulation occurred contextually to the secretion of HMGB1 and may suggest ligand-induced down-regulation [11 ]. We therefore assessed the expression of chemokine receptors when DC matured in the presence of RAGE-blocking antibodies. Similar to the effects observed with neutralizing anti-HMGB1 antibodies, blockade of RAGE hindered the up-regulation of CCR7 and CXCR4 (Fig. 6) . Anti-RAGE antibodies did not influence the expression of chemokine receptors by DC, which were not committed to HMGB1 secretion and maturation (Fig. 6) . Moreover, the migration of LPS-stimulated DC to CCL19 and CXCL12 was inhibited substantially by RAGE blockade (P<0.005 and P<0.05, respectively; Fig. 6 ). These results suggest that RAGE is required for the up-regulation of CCR7 and CXCR4 by HMGB1-secreting, maturing DC and for their ability to migrate to the lymph node chemokines.


Figure 6
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  		Figure 6. Blockade of the HMGB1 receptor RAGE prevents chemokine-receptor modifications and migration shifts associated to DC maturation. (A) Immature DC were cultured alone (–) or in the presence of LPS for 48 h. Staining was performed with anti-RAGE antibodies followed by incubation with fluorochrome-conjugated antimouse antibodies (A). Treatment with control isotype-matched antibodies provided the fluorescence background levels. LPS-treated, mature DC express significantly lower levels of RAGE. Results are depicted as the MFI ± SD, as assessed by flow cytometry of five independent experiments. (B, C) Immature DC were cultured alone (–) or in the presence of LPS for 48 h. Anti-RAGE (anti RAGE) or isotype control (ctrl. Ig) antibodies were added to the culture medium as indicated. LPS induced the up-regulation of CXCR4 (B) and CCR7 (C). In the presence of anti-RAGE antibodies, the up-regulation of CCR7 and CXCR4 was decreased. No effect was observed on immature DC. Control antibodies did not alter the expression of chemokine receptors by DC. Results are depicted as mean ± SD of three independent experiments performed with DC from different donors. (D–F) Immature DC were stimulated with LPS alone or in the presence of anti-RAGE antibodies for 16 h. The migration of 105 cells/well (input) in the absence of chemotactic stimuli (D) to CCL19 (500 ng/ml, E) or to CXCL12 (100 ng/ml, F) is indicated. Mature DC migrate to CCL19 and CXCL12. DC stimulated with LPS in the presence of anti-RAGE antibodies displayed a significantly reduced migration to both chemokines. Representative results from one out of two experiments performed are expressed as mean percent migration of input cells ± SD of triplicate wells (y-axis). *, **, and ***, Statistically significant values (*, P<0.05; **, P<0.005; ***, P<0.0001).

 
DISCUSSION

HMGB1 in resting cells associates and dissociates rapidly from the chromatin and traffics between the nucleus and cytosol. After leukocyte activation, lysine residues are acetylated, the import into the nucleus abates, and HMGB1 localizes to secretory lysosomes, a specific population of vesicles present in hematopoietic cells, whose content is released extracellularly [4 , 5 ]. Once released, HMGB1 activates innate immune cells to secrete cytokines and chemokines, thereby sustaining and prolonging inflammation. HMGB1 in the extracellular environment regulates the maturation and function of the most potent APC, the DC [8 9 10 , 12 13 14 , 26 27 28 29 ]. Conversely, HMGB1 controls the migration and proliferation of cells involved in tissue repair in vitro and in vivo [22 , 30 31 32 33 ].

This study was aimed at defining a new role of extracellular HMGB1 in controlling the ability of maturing DC to disassemble the actin-based cytoskeleton, to up-regulate receptors to lymph node chemokines, and to respond to these signals directly. These events represent crucial steps for DC, licensed by the recognition of the microbe, to leave the periphery and relocate to secondary lymphoid organs, where they interact with naïve or with central memory T cells [34 35 36 ]. Immature DC swiftly disassembled podosomes upon LPS-induced activation of the TLR4 (Fig. 6) , an event that is correlated inversely with the extent of macropynocytosis [25 ]. Actin-based cytoskeleton rearrangement may be required to disrupt immature DC adhesion to surrounding cells and tissue matrix components [37 ] and to facilitate their entry in the lymph via an initial lymphatic vessel. The latter step may be achieved through gaps between lymphatic endothelia, or it may require a cross-talk between endothelial cells and DC [38 ], which with a rearranged cytoskeleton, once in the lymph, are instructed to migrate to lymph nodes, expressing the ligands for the chemokine receptors they have up-regulated. The interaction between CCR7 (which is up-regulated after TLR4 interaction; see for example, Fig. 1 ) and its ligands CCL19 and CCL21 seems to represent a key factor. Its disruption in CCR7–/– animals indeed is sufficient to strongly impair DC migration to secondary lymphoid organs upon activation [38 , 39 ]. Other chemokines and chemokine receptors including CXCL12 and its ligand CXCR4 [40 ] are, however, involved in the fine regulation of this event, influencing the eventual en route acquisition of full immunostimulatory ability by migrating DC [38 , 39 ].

We have found recently that upon activation of the maturation program, DC secrete their own nuclear HMGB1 and use it to sustain the signal transduction events required for efficient activation proliferation and functional polarization of allogeneic, naïve T cells [13 ]. Indeed, given the relevance of DC in the outcome of immune responses, it is not surprising that autocrine loops are involved [41 , 42 ]. In this study, we show that extracellular HMGB1 plays a nonredundant role as well in controlling the migratory pattern of DC after TLR4 activation by LPS. Indeed, cytoskeleton reorganization, expression of chemokine receptor, and migration to lymph node chemokines abated when DC were committed to maturation in the absence of biologically active HMGB1 in the microenvironment (Figs. 2 and 4) . HMGB1 blockade has a more important effect on CCL19- and CXCL12-induced chemotaxis than on the down-regulation of chemokine receptors. This raises the possibility that other events are involved. In agreement, we observed similar effects on the DC intracellular-free Ca2+ after the challenge with the two chemokines in the presence or in the absence of the N-terminal fragment of HMGB1 box A, which behaves as a HMGB1 antagonist (data not shown).

The activation of chemokine receptors recruits independent signaling modules. These include the MAPK (p38 and ERK1/2) and NF-{kappa}B pathways [43 ]. We found previously that HMGB1 secretion by maturing DC is required for the sustained activation of the MAPK and NF-{kappa}B [13 ]. These data strongly suggest that the autocrine/paracrine release of HMGB1 controls DC maturation and migration to lymph node chemokines at multiple levels.

We observed that during maturation, DC down-regulate the expression of RAGE, the best-characterized receptor for HMGB1, which plays a critical role in the regulation of HMGB1-elicited cell motility and proliferation [16 , 30 , 33 ] and in its inflammatory action [44 ] (Fig. 5) . This event, which is compatible with ligand-induced down-regulation [45 ], occurs only when DC were committed to HMGB1 secretion. We therefore verified whether the blockade of RAGE before eliciting DC maturation had any effect on the DC migratory function. The results depicted in Figure 5 strongly support the involvement of RAGE in the autocrine/paracrine effect mediated by the release of HMGB1 by DC. The inhibition we obtained is not complete, indicating that other putative receptors of HMGB1 (including other TLR, see for example, ref. [46 ]) may possibly contribute.

The fine regulation of the migratory ability of APC is a common feature of alarmins, a group of immunostimulatory, structurally diverse host proteins [47 ]. Alarmins comprise structurally diverse endogenous mediators of innate immunity, which augment innate and adaptive immune responses to cell and tissue injury and to ongoing infection [47 , 48 ]. Alarmins share unique features: On one hand, they are released rapidly in response to infectious agents or to tissue damage, and conversely, they have activating and chemotactic effects on APC. As a consequence, alarmins are endowed with effective in vivo adjuvant activity [47 , 48 ]. Our results support the identification of HMGB1 as a bona-fide alarmin, endowed with the key functions required to alert and mobilize the immune response during infection and tissue damage.

ACKNOWLEDGEMENTS

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC; to A. A. M., P. R-Q., and M. E. B.), by the Ministero della Università e della Ricerca Scientifica (cofinanziamento 2005 to A. A. M.), by the Ministero della Salute (to A. A. M.), and by the E.C. (APOCLEAR project to P. R-Q.).

Received March 5, 2006; revised July 2, 2006; accepted July 13, 2006.

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