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Originally published online as doi:10.1189/jlb.0806504 on July 11, 2007

Published online before print July 11, 2007
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(Journal of Leukocyte Biology. 2007;82:906-914.)
© 2007 by Society for Leukocyte Biology

Tolerization with BLP down-regulates HMGB1—a critical mediator of sepsis-related lethality

J. Calvin Coffey*,1, Jiang Huai Wang*,1,2, Ray Kelly{dagger}, Laszlo Romics, Jr.*, Adrian O'Callaghan*, Carmen Fiuza{ddagger} and H. Paul Redmond*

Departments of
* Academic Surgery and
{dagger} Medicine, University College Cork (UCC)/National University of Ireland (NUI), Cork University Hospital, Cork, Ireland; and
{ddagger} Department of Surgery, Queen's University Hospital, Nottingham, United Kingdom

2 Correspondence: Department of Academic Surgery, University College Cork (UCC)/National University of Ireland (NUI), Cork University Hospital, Cork, Ireland. E-mail: jh.wang{at}ucc.ie


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ABSTRACT
 
Tolerization with bacterial lipoprotein (BLP) affords a significant survival benefit in sepsis. Given that high mobility group box protein-1 (HMGB1) is a recognized mediator of sepsis-related lethality, we determined if tolerization with BLP leads to alterations in HMGB1. In vitro, BLP tolerization led to a reduction in HMGB1 gene transcription. This was mirrored at the protein level, as HMGB1 protein expression and release were reduced significantly in BLP-tolerized human THP-1 monocytic cells. BLP tolerance in vivo led to a highly significant, long-term survival benefit following challenge with lethal dose BLP in C57BL/6 mice. This was associated with an attenuation of HMGB1 release into the circulation, as evidenced by negligible serum HMGB1 levels in BLP-tolerized mice. Moreover, HMGB1 levels in peritoneal macrophages from BLP-tolerized mice were reduced significantly. Hence, tolerization with BLP leads to a down-regulation of HMGB1 protein synthesis and release. The improved survival associated with BLP tolerance could thus be explained by a reduction in HMGB1, were the latter associated with lethality in BLP-related sepsis. In testing this hypothesis, it was noted that neutralization of HMGB1, using anti-HMGB1 antibodies, abrogated BLP-associated lethality almost completely. To conclude, tolerization with BLP leads to a down-regulation of HMGB1, thus offering a novel means of targeting the latter. HMGB1 is also a mediator of lethality in BLP-related sepsis.

Key Words: monocytes/macrophages • proinflammatory cytokines • DNA microarray • real-time RT-PCR • human • mice


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INTRODUCTION
 
The defining features of severe sepsis remain multi-organ dysfunctions, which leads, almost inexorably, to significant morbidity and often mortality. Sepsis generates a systemic, inflammatory response, which may culminate in coagulopathy, multiple organ failure, and death. The mortality associated with severe sepsis remains as high as 30% [1 ]. These facts have prompted an ongoing search for novel mediators of lethality in sepsis, as well as the identification of therapeutic modalities aimed at targeting such mediators.

Bacterial lipoprotein (BLP), the most abundant protein in the outer membrane of gram-positive and gram-negative bacteria [2 , 3 ], is characterized by a unique, NH2-terminal, lipo-amino acid, N-acyl-S-diacylglyceryl cysteine. BLP leads to lethal shock in endotoxin-responsive (C3H/HeN) and hyporesponsive (C3H/HeJ) mice. Until the present, this effect has been regarded as mainly a result of the production of proinflammatory cytokines such as TNF-{alpha} and IL-6 [4 5 6 ]. As is the case for endotoxin/LPS, BLP also can be released from proliferating, gram-negative bacteria, e.g., Escherichia coli. The latter is augmented significantly by treatment with antibiotics [7 ].

High mobility group box protein 1 (HMGB1), formerly known as amphoterin and sulfoglucoronyl carbohydrate-binding protein, is a 30-kDa member of the HMGB family, which was identified recently as a proinflammatory cytokine [8 , 9 ]. HMGB1 is a delayed and potent mediator of systemic inflammation, which plays a critical role during sepsis [8 , 9 ]. Serum HMGB1 levels are known to increase at 8–12 h after mice challenged with endotoxin [8 ] or over a 24- to 48-h period following cecal perforation in mice [9 ]. The role of HMGB1 in sepsis is supported by the finding that targeting HMGB1, using anti-HMGB1-neutralizing antibodies [8 9 10 ] or ethyl pyruvate [11 ], leads to a significantly enhanced, long-term survival. Such benefits were not apparent for anti-TNF-{alpha}-based treatment modalities.

Tolerance to bacterial antigens such as endotoxin, first described in the 1960s, involves a transient state of hyporesponsiveness to LPS [12 ]. Endotoxin or LPS tolerance manifests in reduced, proinflammatory cytokine production as well as in protection against endotoxin lethality [13 , 14 ]. Recently, we have demonstrated that BLP induces tolerance to the stimulatory effects of BLP in vitro [15 ] and more importantly, to polymicrobial sepsis in vivo [16 ]. Tolerance is thought to represent an adaptive host response, which results from a down-regulation of specific cellular responses (i.e., reduced TNF-{alpha}, IL-1ß, and IL-6 production) rather than from a general cellular hyporesponsiveness (i.e., levels of IL-10 production are conserved) [13 14 15 16 ]. Proinflammatory cytokine production represents the end-point of complex intracellular signaling pathways. We have demonstrated recently that BLP tolerance involves a down-regulation of TLR2 expression [15 ] and attenuation in MyD88–IL-1R-associated kinase (IRAK) immunocomplex formation [17 ].

Given that tolerization with BLP leads to survival benefits in polymicrobial sepsis, we determined whether tolerization with BLP was a novel means of down-regulating HMGB1 production. HMGB1 was down-regulated significantly in vitro and in vivo following BLP tolerization. This was associated with a transcriptional reduction in HMGB1 gene expression, reduced protein production, and secretion. The identification of a relationship between BLP and HMGB1 prompted an evaluation of the role of the latter in BLP-related sepsis. In keeping with this, it was found that targeting HMGB1 abrogated lethality associated with BLP-induced sepsis almost completely.


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MATERIALS AND METHODS
 
Survival studies
Pyrogen-free, male C57BL/6 mice (8- to 10-weeks old and 18–22 g) were purchased from Harlan (Oxon, UK). Mice were housed in barrier cages under controlled environmental conditions (12/12 h light/dark cycle, 55±5% humidity, 23°C) and had free access to standard laboratory chow and water. Animals were fasted 12 h before experiments and allowed water ad libitum. All animal procedures were conducted in the University Biological Services Unit under a license from the Department of Health and Children (Republic of Ireland).

Mice (n=24) underwent tolerization via i.p. injection of 10 mg/kg BLP, dissolved in 200 µl PBS as described previously [16 , 18 ]. A separate cohort (n=24) remained as nontolerized controls and received an equal volume (200 µl) of PBS. Twenty-four hours later, all mice received a lethal i.p. BLP challenge (45 mg/kg). Survival was monitored in BLP-tolerized and nontolerized groups at 12-h intervals for at least 14 days. BLP, a synthetic, bacterial lipopeptide (Pam3-Cys-Ser-Lys4-OH), was purchased from EMC Microcollections (Tubingen, Germany), which was LPS-free, as confirmed by the Limulus amoebocyte lysate assay (Charles River Endosafe, Charleston, SC, USA).

In a separate experiment, mice challenged with a lethal dose of BLP (45 mg/kg, i.p.) received two i.p. injections of nonimmune rabbit IgG (500 µg/mouse per injection; n=18) or anti-HMGB1-neutralizing antibody (500 µg/mouse per injection; n=16) at 30 min before and 12 h after lethal BLP challenge. Survival was monitored in both groups at 12-h intervals for at least 14 days. The anti-HMGB1-neutralizing antibody was raised from rabbits with >80% inhibition of HMGB1-induced macrophage TNF-{alpha} release and kindly provided by Dr. K. J. Tracey (North Shore-Long Island Jewish Research Institute, Great Neck, NY, USA). Additional experiments in mice as described above were performed, and blood samples from nonimmune rabbit IgG-treated or anti-HMGB1-neutralizing, antibody-treated mice were collected at different time-points after lethal BLP challenge. Serum TNF-{alpha} and IL-6 were determined by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.

Cell culture and induction of BLP tolerance in vitro
A human monocytic cell line, THP-1, was obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI-1640 medium, supplemented with 10% heat-inactivated FCS, penicillin (100 units/ml), streptomycin sulfate (100 µg/ml), and glutamine (2.0 mM) at 37°C in a humidified, 5% CO2 atmosphere. All culture medium and reagents used for cell cultures were purchased from Invitrogen Life Technologies (Paisley, Scotland, UK). All other chemicals, unless indicated, were from Sigma-Aldrich (St. Louis, MO, USA).

BLP tolerance in THP-1 cells was induced as described previously [15 ]. In brief, cells were preincubated with culture medium (nontolerance) or 10 ng/ml BLP (BLP tolerance) for 24 h. Cells were then washed twice in PBS, incubated in fresh culture medium for 2 h, and stimulated with BLP at 1000 ng/ml for 6, 12, 18, and 24 h.

Cytokine measurements
THP-1 cells (2x105 cells/well) were incubated in 24-well plates (Falcon, Lincoln Park, NJ, USA) and subjected to BLP tolerization and subsequent stimulatory challenges as described above. Following BLP stimulation, cell-free supernatants were collected by centrifugation, transferred to new tubes, and stored at –80°C until analysis. TNF-{alpha} and IL-6 concentrations in the supernatants collected from BLP-tolerized and nontolerized cells were determined by ELISA (R&D Systems).

Western blot analysis
In vitro, following tolerization with BLP (10 ng/ml) for 24 h, BLP-tolerized and nontolerized THP-1 cells were stimulated with a high-dose BLP (1000 ng/ml) for 6, 12, 18, and 24 h. The culture supernatants were collected and ultrafiltered with Centricon 10 (Millipore, Bedford, MA, USA) as described previously [8 , 9 ]. The cells, after being washed in cold PBS, were lysed in ice for 30 min to extract proteins with lysis buffer containing 1% Triton X-100, 20 mM Tris, 137 mM NaCl, 1 mM PMSF, 2 mM Na3VO4, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. In vivo serum, peritoneal macrophages, and visceral organs such as spleen and liver were harvested at 12, 18, and 24 h after lethal BLP (45 mg/kg) challenge in BLP-tolerized and nontolerized C57BL/6 mice. Serum samples were ultrafiltered with Centricon 100 (Millpore) as described previously [8 , 9 ]. Peritoneal macrophages were isolated by peritoneal lavage as described previously [18 ] and lysed with the lysis buffer to extract proteins. Spleen and liver were homogenized and lysed in the lysis buffer at 4°C for a further 30 min.

Western immunoblotting was performed as described previously [15 , 19 , 20 ]. In brief, protein concentration in each sample was determined using a Micro bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL, USA). The proteins were denatured at 95°C for 10 min in loading buffer (60 mM Tris, 2.5% SDS, 10% glycerol, 5% ME, and 0.01% bromophenol blue). Aliquots, which contained equal amounts of total proteins from each sample, were separated in SDS-polyacrylamide gels and transferred electrophoretically onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Following a 2-h blocking period in TBS containing 0.1% Tween-20 and 6% nonfat milk, the nitrocellulose membranes were subsequently probed overnight at 4°C with an anti-HMGB1 polyclonal antibody (gift from Dr. K. J. Tracey). Blots were incubated further for 1 h with a secondary HRP-conjugated, anti-rabbit IgG (Dako, Cambridgeshire, UK) and then developed using an ECL detection system (Santa Cruz Biotechnology, Santa Cruz, CA, USA), according to the manufacturers' instructions. Recombinant human HMGB1 (gift from Dr. C. Fiuza, Queen's University Hospital, Nottingham, UK) was used as the positive control. The intensities of HMGB1 signals were quantified using the Scion Image analysis software (Scion Inc., Frederick, MD, USA).

DNA microarray analysis
To characterize HMGB1 gene expression in BLP-tolerized and nontolerized THP-1 cells, following a subsequent BLP stimulation, total RNA from both cell cohorts was extracted and purified according to the method described by Chomczynski and Sacchi [21 ] with slight modifications. RNA was stored in TRIzol reagent (Invitrogen Life Technologies; 5x106 cells/ml TRIzol). Human 10K cDNA microarrays (MWG Biotech, Ebersberg, Germany) were used for the generation of gene expression profiles from BLP-tolerized and nontolerized cells. Amplification and labeling were performed according to the manufacturer's protocol. Arrays were hybridized overnight and then washed and dried as described. Scanning of the arrays was performed on the GeneArray scanner (Affymetrix, Santa Clara, CA, USA) at three different fluorescent values per channel. MAVI (MWG Biotech) was used for background correction, normalization, as well as to increase the dynamic range by linear regression analysis of each result. The Biodiscovery software package (MWG Biotech), Imagene and Genesight, was used to analyze the scanner tiff images.

Quantitative real-time RT-PCR
Using avian myloblastosis virus RT (Promega, Madison, WI, USA) and random primers (Promega), cDNA was synthesized from RNA extracted and purified from BLP-tolerized and nontolerized THP-1 cells as described above. To control for variations in cDNA synthesis, cDNA was generated in quadruplicate, after which replicates were pooled. Samples evaluated thus represented an average of four cDNA synthesis reactions. Primers were designed for HMGB1 (sense-GGACAAGGCCCGTTATGAAAGAGAAATGA and antisense-AGCAGAAGAGGAAGAAGGCCGAAGGAG), IFN-{gamma}R1 (IFNR1; sense-ATTATGATCCCGAAACTACCTGT and antisense-ATACTGGAATCGCTAACTGGCACTGAATCT), and ß-actin (sense-GGCTACAGCTTACCACCAGG and antisense-GCCAGACAGCAGTGTGTTGGC) using the DNA Lasergene PrimerSelect program (DNAstar Inc., Madison, WI, USA). Real-time RT-PCR was performed using QuantiTech SYBR Green (Qiagen, Hilden, Germany) by the LightCycler (Roche Diagnostics, Mannheim, Germany) to determine the relative expression of HMGB1 and IFNR1 in BLP-tolerized and nontolerized cells. Relative expression was determined based on the ratio of gene expression:a reference gene, ß-actin. As PCR primers may also detect genomic DNA, control samples underwent evaluation in which the RT was replaced by water during cDNA synthesis. As these samples did not show a PCR amplification product, this pointed to the absence of contaminating, genomic DNA. All analysis was performed using the "relative expression software tool" (REST) [22 ], which makes use of a mathematical model based on PCR efficiencies and the mean crossing-point deviation between the control and the treated group [22 ]. Expression ratios were then assessed for statistical significance using a Pair-Wise Fixed Reallocation Randomization [22 ].

Statistical analysis
All data are presented as the mean ± SD. Statistical analysis was performed using the log rank test for survival studies and the Student's t-test or the Mann-Whitney U-test for all parametric and nonparametric data where appropriate. Differences were judged statistically significant when P < 0.05.


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RESULTS
 
BLP tolerization is associated with reduced HMGB1 protein expression and release in vitro
Given that tolerization with BLP is known to afford protection against BLP-induced lethality, it was determined next if BLP tolerization led to changes in the HMGB1 axis. Attenuated, proinflammatory cytokine production is a characteristic end-point of BLP tolerization [15 , 16 ]. To confirm the induction of BLP tolerization in vitro, human THP-1 monocytic cells were preincubated with 10 ng/ml BLP for 24 h to induce BLP tolerance. Tolerized and nontolerized cells were stimulated with BLP, after which TNF-{alpha} and IL-6 release were assessed and compared. BLP tolerization abrogated the increases significantly in TNF-{alpha} and IL-6 release, which normally followed BLP stimulation in nontolerized cells (P<0.01 vs. nontolerized cells; Fig. 1 ). The reduction in TNF-{alpha} was more pronounced, as 44-fold and 39-fold reductions in TNF-{alpha} and IL-6 were observed in BLP-tolerized cells, respectively.


Figure 1
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  		Figure 1. Attenuated, proinflammatory cytokine release in BLP-tolerized human THP-1 cells, which were preincubated with culture medium (nontolerized cells, open bars) or 10 ng/ml BLP (BLP-tolerized cells, solid bars) for 24 h. Cells were then washed twice, incubated in fresh medium for 2 h, and stimulated with 1000 ng/ml BLP for 24 h. Culture supernatants were collected and assayed for TNF-{alpha} and IL-6 levels by ELISA. Data represent mean ± SD of five independent experiments. *, P < 0.05, versus nontolerized cells.

As shown in Figure 2a , in nontolerized THP-1 cells, HMGB1 protein was present in the absence of BLP stimulation, and stimulation of nontolerized THP-1 cells with BLP resulted in increased total cellular HMGB1 protein, which was also detectable in BLP-tolerized, THP-1 cells in the absence of BLP stimulation. However, the expression of HMGB1 protein remained at constitutive levels following 6-, 12-, and 24-h BLP stimulation in tolerized cells (Fig. 2a and 2c) . In contrast, significant HMGB1 release was observed in nontolerized THP-1 cells following 12-, 18-, and 24-h BLP stimulation. This response was abrogated almost totally in BLP-tolerized THP-1 cells (Fig. 2b and 2c) . These findings indicate that in nontolerized cells, HMGB1 protein expression and secretion increased following BLP stimulation. However, this response was absent in BLP-tolerized cells.


Figure 2
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  		Figure 2. Down-regulated HMGB1 protein expression and release in BLP-tolerized human THP-1 cells, which were preincubated with culture medium (nontolerized cells) or 10 ng/ml BLP (BLP-tolerized cells) for 24 h. Cells were then washed twice, incubated in fresh medium for 2 h, and stimulated with 1000 ng/ml BLP for different time-points. Cellular extracts (a) prepared at 0, 6, 12, and 24 h and supernatants (b) collected at 0, 12, 18, and 24 h after BLP stimulation were subjected to Western blot analysis for detecting HMGB1 protein expression as described in Materials and Methods. Results shown represent one experiment from a total of three separate experiments at each time-point. (c) The intensity of HMGB1 signals was analyzed using a digital imaging analysis system. Data represent mean ± SD of three separate experiments. *, P < 0.05, versus nontolerized controls.

Induction of BLP tolerance in vivo attenuates HMGB1 protein expression and release
As demonstrated above, BLP tolerization was associated with reduced responsiveness in terms of HMGB1 production and secretion in vitro. To determine if a similar phenomenon occurred in vivo, BLP-tolerized and nontolerized C57BL/6 mice were subjected to a lethal BLP challenge. Following this, survival was monitored and correlated with HMGB1 protein levels in serum, peritoneal macrophages, and visceral organs. After lethal BLP challenge, survival in BLP-tolerized mice greatly exceeded that of nontolerized mice (92% in BLP-tolerized mice vs. 34% in nontolerized mice, P=0.000317; Fig. 3 ). Differences in survival emerged within 24 h and reached a steady-state 48 h after lethal BLP challenge. These differences were maintained over the long-term period (up to 14 days; Fig. 3 ). Splenic and hepatic HMGB1 protein levels were determined in BLP-tolerized and nontolerized mice, 12, 18, and 24 h after lethal BLP challenge. At each time-point, no differences were observed for hepatic HMGB1 levels (Fig. 4a ). Of note, in nontolerized mice, hepatic HMGB1 levels did not change during the interval examined. An identical pattern was also observed for splenic HMGB1 levels in BLP-tolerized and nontolerized mice (Fig. 4b) .


Figure 3
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  		Figure 3. Long-term survival benefit observed in BLP-tolerized mice following a lethal BLP challenge. C57BL/6 mice received i.p. injection with 10 mg/kg BLP (BLP-tolerized mice) or PBS (nontolerized mice), and 24 h later, all mice were challenged with a lethal dose of BLP (45 mg/kg). Survival was monitored for at least 14 days. The Kaplan-Meier survival graph shows that BLP-tolerized mice (n=24, •) were conferred with a statistically significant, long-term survival benefit over the nontolerized mice (n=24, {circ}), and *, P = 0.000317.


Figure 4
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  		Figure 4. Attenuated HMGB1 protein expression and release in BLP-tolerized mice. Liver (a), spleen (b), peritoneal macrophages (c), and serum (d) were harvested at 12, 18, and 24 h post-lethal BLP challenge (45 mg/kg) in BLP-tolerized and nontolerized C57BL/6 mice. HMGB1 protein expression was detected by Western blot analysis as described in Materials and Methods. Results shown represent one experiment from a total of three to five independent experiments at each time-point. (e) The intensity of HMGB1 signals was analyzed using a digital imaging analysis system. Data represent mean ± SD of three to five separate experiments. *, P < 0.05, versus nontolerized controls.

Peritoneal macrophages were harvested from BLP-tolerized and nontolerized mice at 12, 18, and 24 h post-lethal BLP challenge. HMGB1 protein levels in macrophages from BLP-tolerized mice were clearly reduced relative to their nontolerized counterparts (Fig. 4c) . This difference persisted over the interval examined. Serum HMGB1 protein levels were assessed in BLP-tolerized and nontolerized mice. HMGB1 protein was evident in the sera of nontolerized mice, at significant levels, throughout the interval examined (Fig. 4d) . In contrast, the serum HMGB1 level in BLP-tolerized mice was negligible 12 h following lethal BLP challenge (Fig. 4d) . Although the serum HMGB1 level in BLP-tolerized mice increased by 18 h, this remained significantly reduced relative to that seen in the nontolerized counterparts at 18 h. A further marginal increase in serum HMGB1 was evident in BLP-tolerized mice at 24 h. However, this was again reduced significantly relative to that seen in the nontolerized mice 24 h after lethal BLP challenge (Fig. 4d) . A greater-than-threefold difference was evident when serum HMGB1 levels were compared in BLP-tolerized and nontolerized mice (P<0.05; Fig. 4e ).

HMGB1 gene transcription is reduced following BLP tolerization
The above findings indicated that BLP tolerization was associated with reduced levels of serum HMGB1 in vivo, as well as with reduced HMGB1 protein expression and release following BLP challenge in vitro. The intracellular basis for such differences remained to be evaluated. A comparison of gene expression profiles from BLP-tolerized and nontolerized monocytes was used as a platform to screen for transcriptional differences associated with BLP tolerization.

BLP-tolerized and nontolerized THP-1 cells were stimulated with BLP for 12 h, following which gene expression profiles were compared between the two cell cohorts. In response to BLP stimulation, HMGB1 gene expression in BLP-tolerized cells was reduced by a factor of 1.2 when compared with nontolerized cells (Fig. 5a ). Transcripts for high-mobility group 20b and high-mobility group protein 14 were also down-regulated (Table 1 ). Although according to the array data, the reduction in HMGB1 mRNA transcript level in BLP-tolerized cells did not reach statistical significance, it was assessed further by real-time RT-PCR. As shown in Figure 5a , HMGB1 mRNA levels, following a second BLP stimulation for 12 h in BLP-tolerized cells, were 59% that of nontolerized cells. This reduction was statistically significant when evaluated by REST (P=0.0255 vs. nontolerized cells). Hence, in response to BLP stimulation, HMGB1 gene transcription is reduced in BLP-tolerized THP-1 cells when compared with nontolerized cells.


Figure 5
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  		Figure 5. Altered HMGB1 and INFR1 gene expression in BLP-tolerized human THP-1 cells, which were preincubated with culture medium (nontolerized cells, open bars) or 10 ng/ml BLP (BLP-tolerized cells, solid bars) for 24 h. Cells were then washed twice, incubated in fresh medium for 2 h, and stimulated with 1000 ng/ml BLP for 12 h. Total RNA was extracted, purified, and subjected to DNA microarray and real-time RT-PCR for detecting HMGB1 and INFR1 gene expression. Data are expressed as percent gene expression (n=3). *, P = 0.0255, versus nontolerized cells; **, P = 0.02, versus nontolerized cells.


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  		Table 1. Select Genes Induced in BLP-Tolerized Human THP-1 Cells, As Outlined Using DNA Microarray

HMGB1 gene expression is regulated partly by IFN-{gamma} [23 ], which binds to its cognate receptor IFNR1, dimerizing with IFNR2 to initiate an intracellular signaling cascade. This culminates in increased HMGB1 gene expression. As demonstrated above, BLP tolerization was associated with reduced HMGB1 gene expression. Such a difference may relate to changes in IFNR1 gene and/or protein expression. A comparison of gene expression profiles demonstrated that in response to BLP stimulation, INFR1 gene expression in BLP-tolerized cells was up-regulated by 1.25-fold, relative to that seen in nontolerized cells (Fig. 5b) . This finding was confirmed further by real-time RT-PCR, in which IFNR1 mRNA levels in BLP-tolerized cells were increased significantly (P=0.02 vs. nontolerized cells) following BLP stimulation (Fig. 5b) . Hence, reduced HMGB1 gene expression occurs following BLP tolerization, despite an up-regulation in IFNR1 gene expression.

Anti-HMGB1-neutralizing antibody protects mice against BLP-induced lethality
No studies to date have evaluated the role of HMGB1 in BLP-induced sepsis. This is somewhat surprising, given that BLP is the most abundant protein in the cell wall of gram-positive and -negative bacteria. Given the above findings, i.e., that BLP tolerization is associated with a survival benefit as well as with reduced HMGB1 release, we speculated that HMGB1 may also play a role in BLP-related sepsis. To determine whether an increased HMGB1 release contributes to BLP-associated lethality, we thought to block HMGB1 in mice challenged with lethal BLP using a specific HMGB1-neutralizing antibody. Male C57BL/6 mice were administered i.p. twice with control IgG (500 µg/mouse per injection) or anti-HMGB1-neutralizing antibody (500 µg/mouse/injection) at 30 min before and 12 h after lethal BLP challenge (45 mg/kg). As shown in Figure 6a , neutralization of HMGB1 with anti-HMGB1 antibody conferred a statistically significant survival benefit against BLP-induced lethality, from 28% (5/18) in mice treated with control IgG to 75% (12/16) in mice treated with anti-HMGB1-neutralizing antibody (P=0.0019). This improved survival in anti-HMGB1-neutralizing, antibody-treated mice was maintained over a 14-day monitoring period. Of note, blocking HMGB1 did not affect BLP-stimulated, proinflammatory TNF-{alpha} and IL-6 release (Fig. 6b) .


Figure 6
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  		Figure 6. Anti-HMGB1-neutralizing antibody protects mice against BLP-induced lethality but does not affect BLP-stimulated, proinflammatory cytokine release. Male C57BL/6 mice received nonimmune rabbit IgG (500 µg per mouse, i.p.) or anti-HMGB1-neutralizing antibody (500 µg per mouse, i.p.) at 30 min before and 12 h after lethal BLP challenge (45 mg/kg). (a) The Kaplan-Meier survival graph shows that anti-HMGB1-neutralizing, antibody-treated mice (n=16, •) had a statistical survival advantage over control, IgG-treated mice (n=18, {circ}); *, P = 0.0019. (b) Data shown here are peak serum levels of TNF-{alpha} at 90 min and IL-6 at 4 h from mice treated with anti-HMGB1-neutralizing antibody (solid bars) or control IgG (open bars) after lethal BLP challenge and expressed as the mean ± SD of five independent experiments.


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DISCUSSION
 
The phenomenon of endotoxin tolerance, first reported in 1963, results in a transient hyporesponsiveness against bacterial antigen LPS [12 ]. This may represent a protective state, whereby the body exerts a break-like effect on "self-destructive" host-response mechanisms when faced with a lethal bacterial challenge. As tolerance may be protective, intensive efforts are ongoing to elucidate its underlying molecular mechanisms with a view to exploiting these beneficially during sepsis. To date, most mechanistic studies have focused on LPS-induced tolerance, which leads to a suppression of the MAPKs including ERKs, p38, and JNKs [24 25 26 ]. In addition, LPS-induced tolerance is associated with reduced NF-{kappa}B activation as well as decreased degradation of I{kappa}B{alpha} and I{kappa}Bß [24 , 27 , 28 ].

Although BLP is the most ubiquitous cell wall protein in gram-positive and -negative bacteria, less is known about the cellular and molecular mechanisms involved in BLP-induced tolerance. Recently, we [15 ] and others [27 ] have shown that BLP tolerance inhibits phosphorylation of MAPKs and NF-{kappa}B activation. BLP tolerance does not affect cell surface expression of the TLR4–myeloid differentiation protein-2 complex [27 ]. However, it does correlate to suppressed TLR2 protein expression [15 ] and the MyD88–IRAK association [17 ]. Furthermore, we demonstrated that BLP-induced tolerance is associated with enhanced bacterial recognition and bactericidal activity in wild-type [16 ] and TLR4-deficient [18 ] mice. These effects relate partly to the activation in complement receptor type 3 and Fc{gamma}RIII/II on neutrophils and peritoneal macrophages [16 , 18 , 29 , 30 ].

HMGB1 was identified recently as a late mediator of systemic inflammation [8 , 9 ]. Originally described as an intracellular transcription factor, HMGB1 is released from endotoxin-stimulated macrophages after a significant delay, beginning 8–12 h after the release of the early cytokines TNF-{alpha} and IL-1ß [8 , 31 ]. The identification of HMGB1 as a critical mediator of late lethality in sepsis has led to a search for antagonists of HMGB1. To date, three modalities have been identified. Although the systemic administration of HMGB1 is lethal, anti-HMGB1-neutralizing antibodies confer a significant protection [8 9 10 ]. Ethyl pyruvate, an experimental, anti-inflammatory agent, inhibits systemic HMGB1 release and rescues animals from the sequelae of systemic inflammation [11 ]. More recently, it was noted that the specific inhibition of endogenous HMGB1 using the DNA-binding A box, reduces the lethality associated with established sepsis [9 ]. However, no studies to date have characterized the effects of BLP tolerization on the HMGB1 proinflammatory axis. Given the pivotal role of HMGB1 in sepsis-related lethality and also given the long-term survival benefits associated with BLP tolerance, it was reasonable to speculate on an interaction between the two.

We first investigated whether BLP tolerization was associated with changes in HMGB1, and HMGB1 protein was readily detectable in nontolerized THP-1 monocytes in vitro. When these cells were stimulated with BLP, cellular HMGB1 protein expression was increased. However, a BLP-induced increase in HMGB1 levels in naïve cells was considerably attenuated in BLP-tolerized cells. Furthermore, BLP stimulation of naïve THP-1 monocytes resulted in marked increases in HMGB1 release. This was almost abrogated totally by BLP tolerization of THP-1 cells. These results indicate that nontolerized monocytes respond to BLP stimulation by increasing HMGB1 protein expression and release, whereas BLP-tolerized cells exhibit a reduced response in this regard.

The effects of BLP tolerization on HMGB1 in vivo were next evaluated. A similar down-regulation of HMGB1 was observed in vivo in BLP-tolerized mice. HMGB1 protein levels were reduced markedly in peritoneal macrophages following the induction of BLP tolerance. In contrast with macrophages, splenic and hepatic HMGB1 levels were not reduced following BLP tolerance. The absence of major differences in HMGB1 protein levels in visceral organs such as spleen and liver may reflect a dilutional effect, given the diversity and number of resident cell types. It is important, however, that it suggests that reductions in macrophage HMGB1 protein levels do not arise as a result of increases in the tissue release of the latter. Together, these results indicate that in vivo, macrophages undergo a reduction in HMGB1 protein synthesis in response to BLP tolerance. The survival benefit of BLP tolerance emerged within 24 h of lethal BLP challenge. This was preceded by massive reductions in serum HMGB1 levels in BLP-tolerized mice. Although at 24 h, serum HMGB1 began to increase, levels remained reduced still significantly relative to those seen in nontolerized mice. Hence, the survival benefits of BLP tolerance are preceded by a marked attenuation in serum HMGB1 levels.

Next, we investigated the intracellular basis for changes in HMGB1 levels in BLP-tolerized and nontolerized monocytes in vitro. A comparison of gene expression profiles from BLP-tolerized and nontolerized monocytes served as a platform to screen for transcriptional differences between the two cell populations. The latter identified a modest reduction in HMGB1 transcription. However, when this was characterized further using real-time RT-PCR, a significant reduction in HMGB1 gene transcription was noted in BLP-tolerized cells. Little is known regarding the intracellular events that regulate HMGB1 gene expression. Events at the plasma membrane are better characterized. For example, IFN-{gamma} is thought to play a role after binding to its cognate receptor IFNR1. It is interesting that reduced HMGB1 gene and protein expression occurred, despite an up-regulation in the IFNR1 component of the HMGB1 signal transduction machinery. The above observations suggest that tolerization with BLP leads to a transcriptional down-regulation in HMGB1 gene expression. This may explain the reductions of HMGB1 protein levels seen in serum and peritoneal macrophages and in this manner, explain enhanced and prolonged survival observed in BLP-tolerized mice.

The above findings point to a relationship between BLP and HMGB1. BLP tolerization leads to a survival benefit in BLP-related sepsis as well as in polymicrobial sepsis. BLP tolerization is also associated with a reduction in HMGB1 production and release. Hence, it may be hypothesized that in BLP-induced sepsis, the survival benefits that follow BLP tolerization relate to altered HMGB1 levels. For this to be the case, HMGB1 would have to be a critical mediator of lethality in BLP-induced sepsis. We next demonstrated that the targeting of HMGB1, using the anti-HMGB1-neutralizing antibody, almost abrogated the lethality associated with BLP challenge. This indicates that although BLP-associated lethality involves a number of factors, HMGB1 occupies a primacy in this regard. This suggestion is strengthened further by the observation that of those animals that received anti-HMGB1-neutralizing antibody and survived, they did so into the long-term. TNF-{alpha} and IL-6 levels were identically similar in BLP-challenged mice in both groups (i.e., those receiving anti-HMGB1-neutralizing antibody and those receiving control IgG). Hence, the survival benefit observed in the anti-HMGB1-neutralizing antibody group could not be associated with changes in each of these proinflammatory mediators. A role for HMGB1 is suggested further in the fact that survival differences following BLP tolerization emerge within 24 h, plateau after 48 h, and remain stable thereafter. HMGB1 release generally follows a similar pattern [8 , 9 ].

Such observations reinforce the role of HMGB1 in sepsis in general and emphasize further the need for therapeutic regimens, which aim to down-regulate HMGB1 or its sepsis-associated activities. To confirm definitively that HMGB1 is a critical mediator of BLP-related lethality, one would have to challenge HMGB1-knockout mice with BLP and demonstrate enhanced survival relative to wild-type mice. However, HMGB1-knockout mice die soon after birth as a result of lethal hypoglycemia [32 ]. As such, it is not possible to compare survival following septic challenges in such mice. Nevertheless, tolerization with BLP represents a novel and readily achieved means of down-regulating HMGB1 production and release.

In conclusion, BLP tolerization represents a further novel means of reducing HMGB1 protein levels. This effect relates to a transcriptional down-regulation of HMGB1 gene expression, reduction in protein synthesis, and attenuation of HMGB1 secretion. In addition, HMGB1 is a critical factor involved in BLP-associated lethality, as the targeting of HMGB1 affords almost complete protection against BLP-associated lethality.


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ACKNOWLEDGEMENTS
 
This work was supported in part by the Basic Research Grants Scheme from the Irish Research Council for Science, Engineering and Technology (SC/2003/27 to J. H. W.).


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FOOTNOTES
 
1 These authors contributed equally to this work and share first authorship. Back

Received August 8, 2006; revised May 22, 2007; accepted June 2, 2007.


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