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Originally published online as doi:10.1189/jlb.0404242 on August 26, 2004

Published online before print August 26, 2004
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(Journal of Leukocyte Biology. 2004;76:994-1001.)
© 2004 by Society for Leukocyte Biology

Bacterial endotoxin stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms

Guoqian Chen*, Jianhua Li{dagger}, Mahendar Ochani{dagger}, Beatriz Rendon-Mitchell{dagger}, Xiaoling Qiang{dagger}, Seenu Susarla{dagger}, Luis Ulloa{dagger}, Huan Yang{dagger}, Saijun Fan{dagger}, Sanna M. Goyert{dagger}, Ping Wang{dagger}, Kevin J. Tracey{dagger}, Andrew E. Sama* and Haichao Wang*,{dagger},1

* Department of Emergency Medicine, North Shore University Hospital, New York University School of Medicine, and
{dagger} Center of Immunology and Inflammation, North Shore–LIJ Research Institute, Manhasset

1 Correspondence: Department of Emergency Medicine, North Shore University Hospital, New York University School of Medicine, 350 Community Drive, Manhasset, NY 11030. E-mail: hwang{at}nshs.edu


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ABSTRACT
 
Bacterial endotoxin [lipopolysaccharide (LPS)] stimulates macrophages to sequentially release early [tumor necrosis factor (TNF)] and late [high mobility group box 1 (HMGB1)] proinflammatory cytokines. The requirement of CD14 and mitogen-activated protein kinases [MAPK; e.g., p38 and extracellular signal-regulated kinase (ERK)1/2] for endotoxin-induced TNF production has been demonstrated previously, but little is known about their involvement in endotoxin-mediated HMGB1 release. Here, we demonstrated that genetic disruption of CD14 expression abrogated LPS-induced TNF production but only partially attenuated LPS-induced HMGB1 release in cultures of primary murine peritoneal macrophages. Pharmacological suppression of p38 or ERK1/2 MAPK with specific inhibitors (SB203580, SB202190, U0126, or PD98059) significantly attenuated LPS-induced TNF production but failed to inhibit LPS-induced HMGB1 release. Consistently, an endogenous, immunosuppressive molecule, spermine, failed to inhibit LPS-induced activation of p38 MAPK and yet, still significantly attenuated LPS-mediated HMGB1 release. Direct suppression of TNF activity with neutralizing antibodies or genetic disruption of TNF expression partially attenuated HMGB1 release from macrophages induced by LPS at lower concentrations (e.g., 10 ng/ml). Taken together, these data suggest that LPS stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.

Key Words: lipopolysaccharide • MAP kinases • spermine


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INTRODUCTION
 
Gram-negative bacterial infection and sepsis are the most common cause of death in intensive care units, claiming ~225,000 victims annually in the U.S. alone. The high mortality of sepsis is in part mediated by bacterial endotoxin [lipopolysaccharide (LPS); ref. 1 ], which activates macrophages and monocytes to sequentially release early [e.g., tumor necrosis factor (TNF) and interleukin (IL)-1; refs. 2 , 3 ] and late [e.g., high mobility group box 1 (HMGB1); ref. 4 ] proinflammatory cytokines. To ensure a sensitive response to endotoxin, mammals have evolved an effective innate recognition system consisting of LPS-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4). Covalently anchored to cells via glycosylphosphatidylinositol, the membrane-bound CD14 lacks intrinsic signaling capabilities as a result of lack of transmembrane and intracellular domains [5 ]. When presented to CD14 by LBP, LPS is delivered to high-affinity transmembrane receptors such as TLR4 [6 ], leading to activation of mitogen-activated protein kinases [MAPK; e.g., p38, extracellular signal-regulated kinase (ERK)1/2, and c-jun NH2-terminal kinase (JNK)] and production of early proinflammatory cytokines. The important role of CD14 in LPS-mediated TNF production is supported by studies showing that transgenic mice overexpressing CD14 become hypersensitive to LPS [7 ], whereas CD14-deficient mice become resistant to endotoxin-induced shock [8 ]. In addition to CD14 and TLR4, downstream p38 or ERK1/2 MAPK signaling pathways are also critical for LPS-mediated production of TNF and IL-1 [9 ].

Proinflammatory cytokines have been implicated in mediating lethal, systemic inflammation, as inhibition of their release or activity attenuates the development of tissue injury in animal models of septic shock or sepsis [10 ]. For instance, neutralizing antibodies to TNF, the first cytokine elaborated in an inflammatory cascade, reduce lethality in the animal model of endotoxemic/bacteremic shock [10 ]. However, the early kinetics of TNF production makes it difficult to target in a clinical setting. It is thus important to identify late proinflammatory cytokines that may offer a wider therapeutic window for the treatment of lethal, systemic inflammation diseases.

We have discovered that a ubiquitous protein, HMGB1, is released by endotoxin-activated macrophages/monocytes [4 , 11 ] and functions as a late mediator of lethal endotoxemia and sepsis [4 , 12 13 14 ]. Circulating HMGB1 levels are elevated in animals between 16 and 32 h after onset of endotoxemia or sepsis [4 , 12 ] and in patients with sepsis [4 ] or hemorrhagic shock [15 ]. In vitro, HMGB1 can induce production of TNF and IL-1 by macrophage/monocytes and neutrophils in a p38 MAPK-dependent mechanism [16 , 17 ]. In vivo, HMGB1 causes allodynia [18 ], derangement of intestinal barrier function [19 ], lung injury [20 ], fever [21 ], and even lethality [4 ]. Administration of anti-HMGB1 antibodies or inhibitors (e.g., ethyl pyruvate) significantly protects mice against LPS-induced acute lung injury [20 ], lethal endotoxemia [4 ], and sepsis [12 ], even when the initial treatments are delayed by 24 h after onset of inflammation.

The mechanisms underlying regulation of endotoxin-mediated HMGB1 release, however, have not been investigated previously. These studies were therefore undertaken to determine whether LPS-induced HMGB1 release is dependent on expression of CD14 or TNF. Here, we demonstrated that LPS stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.


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MATERIALS AND METHODS
 
Cell culture
Murine macrophage-like RAW 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum and 2 mM glutamine. At 80–90% confluence, RAW 264.7 cells were washed twice with and subsequently cultured in serum-free OPTI-MEM I medium (Gibco BRL) before LPS stimulation.

CD14-deficient (CD14–/–) mice were generated by gene-targeting in embryonic stem cells as described previously [8 ] and were back-crossed on a BALB/c background to the 10th generation. Age- and sex-matched wild-type BALB/c mice (CD14+/+) were obtained from the Jackson Laboratory (Bar Harbor, ME) and used as control for CD14–/– mice. Wild-type C57BL/6J mice (TNF+/+, Stock #000664, the Jackson Laboratory) were used as control for the TNF-deficient mice (TNF–/–, C57BL/B6x129S6, Stock #003008, the Jackson Laboratory). Primary peritoneal macrophages were isolated from mice (male, 7–8 weeks, 20–25 g) at 3 days after intraperitoneal injection of 2 ml thioglycollate broth (4%). After several extensive washings, thioglycollate-elicited macrophages were resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum (for culturing TNF+/+ and TNF–/– macrophages), 1% wild-type mouse serum (for culturing CD14+/+ macrophages), or 1% CD14-deficient mouse serum (for culturing CD14–/– macrophages) and were immediately transferred onto six-well tissue-culture plates (4x106 cells/2 ml/well). CD14-deficient mouse serum was used to culture CD14–/– macrophages, as soluble CD14 in serum of wild-type mice may restore LPS responsiveness to CD14 null macrophages. After preculture for 12 h, the adherent cells were gently washed with and cultured in serum-free OPTI-MEM I medium 2 h before LPS stimulation.

LPS stimulation
After preincubation for 2 h, cell cultures were stimulated with LPS (Sigma Chemical Co., St. Louis, MO) at indicated concentrations, and the culture medium was assayed for levels of TNF and HMGB1 at 16 h after LPS stimulation. Various inhibitors specific to p38 MAPK (SB203580, Catalog no. 559389; SB202190, Catalog no. 559388, Calbiochem, La Jolla, CA) or MAPK (MEK)1/2 MAPK (PD98059, Catalog no. 513000; U0126, Catalog no. 662005, Calbiochem) or TNF-neutralizing antibodies (AF-410-NA, R&D Systems, Minneapolis, MN) were used to evaluate the roles of MAPK or TNF in LPS-induced HMGB1 release.

Assay of phosphorylation state of p38 MAPK
Macrophage cells were lysed in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue) at 30 min after LPS stimulation. The concentrations of phosphorylated p38 MAPK were determined by Western blot analysis using the p38 (Thr180/Tyr182) MAPK antibody kit (Catalog no. 9210, Cell Signaling Technology, Beverly, MA) as described previously [11 ]. To verify equal loading for different samples, the samples were probed with a different antibody specific to total p38 MAPK.

TNF enzyme-linked immunosorbent assay (ELISA)
The levels of TNF in the culture medium were determined using a commercially available ELISA kit (Catalog no. MTA00, R&D Systems) as described previously [22 , 23 ]. The levels of TNF were calculated with reference to standard curves of purified recombinant (r)TNF at various dilutions.

HMGB1 Western blot analysis
The levels of HMGB1 in the culture medium were assayed by Western blot analysis using rabbit polyclonal antibodies as described previously [11 ]. Western blots were scanned with a silver image scanner (Silverscaner II, Lacie Ltd., Beaverton, OR), and the relative band intensity was quantified by using the NIH Image 1.59 software. The levels of HMGB1 were calculated with reference to standard curves generated with purified rHMGB1.

HMGB1 immunostaining
Cellular HMGB1 was immunostained with antigen affinity-purified anti-HMGB1 polyclonal antibodies following a protocol described previously [11 ]. Briefly, macrophage cultures were transferred onto adhesion slides (Catalog no. 62407335, Lab-Tek chamber slides, VWR) and incubated at 37°C for 1 h to allow adherence. The adherent cells were fixed with phosphate-buffered formaldehyde (4%, pH 7.4, 15 min) and permeabilized with Triton X-100 (0.3%, pH 7.4, 10 min). After blocking with 10% bovine serum albumin (37°C, 1 h), cells were sequentially incubated with antigen affinity-purified anti-HMGB1 antibodies and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G (IgG; Catalog no. F9887, Sigma Chemical Co.). Following several extensive washings, the slides were mounted immediately with Vectashield (Catalog no. H-1000, Vector Laboratories, Burlingame, CA), and images were acquired using a confocal microscope (OLYMPUS 1X70, Fluoroview).

Statistical analysis
Values in the bar graphs were expressed as mean ± SEM of three independent experiments in duplicates or triplicates (n=6–9). Student’s two-tailed t-test was used to compare means between groups. A P value less than 0.01 or 0.05 was considered statistically significant (# or *, respectively).


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RESULTS
 
LPS induces HMGB1 release partly through a CD14-dependent mechanism
Previous studies have established a critical role of CD14 in endotoxin-induced TNF release [8 ]. To evaluate the potential role of CD14 in endotoxin-mediated HMGB1 release, we examined cytokine production from wild-type or CD14-deficient peritoneal macrophages. In wild-type peritoneal macrophages, LPS induced the release of TNF (Fig. 1A ) and HMGB1 (Fig. 1B) in a dose-dependent manner. At a relative low concentration (e.g., 1 ng/ml), LPS induced significant release of TNF (TNF=1600±300 pg/ml vs. control background, TNF=50±25 pg/ml, P<0.01, Fig. 1A ) but did not significantly induce HMGB1 release (HMGB1=1000±750 pg/ml vs. control background, 500±250 pg/ml, P>0.05, Fig. 1B ). At a higher concentration (10 ng/ml), LPS started to induce significant HMGB1 release (HMGB1=24,500±2500 pg/ml vs. control background, 500±250 pg/ml, P<0.01). Thus, the minimal effective LPS concentrations for inducing significant TNF release (LPS=1 ng/ml) were markedly lower than that for inducing HMGB1 (LPS=10 ng/ml), implicating that TNF can be induced at early stages of infection while LPS levels are still low.



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  		Figure 1. LPS stimulates macrophages to release HMGB1 partly through a CD14-dependent mechanism. Primary peritoneal macrophages were isolated from normal BALB/c (CD14+/+) or CD14-deficient (CD14–/–) BALB/c mice, stimulated with LPS at indicated concentrations, and the culture medium was assayed for TNF (A) or HMGB1 (B) at 16 h after LPS stimulation. The detection limits were 50 ± 25 pg/ml for TNF ELISA and 500 ± 250 pg/ml for HMGB1 Western blot analysis. Shown in the bar graphs were levels of TNF (A) or HMGB1 (B) expressed as mean ± SEM of three independent experiments. (B) Shown in the lower portion was a representative Western blot. #, Statistically significant at P < 0.01 as compared with controls (CD14+/+); *, statistically significant at P < 0.05 as compared with controls (CD14+/+).

In CD14-deficient macrophages, the LPS-induced TNF release was almost completely impaired (P<0.01, Fig. 1A ), confirming the important role of CD14 in LPS-mediated TNF production [8 ]. In contrast, the LPS-stimulated HMGB1 release was only partially reduced in CD14-deficient peritoneal macrophages (Fig. 1B) . At a wide range of concentrations (10, 100, 500 ng/ml), LPS induced significantly less (30–50%) HMGB1 release in cultures of CD14-deficient macrophages as compared with normal, wild-type peritoneal macrophages (P<0.05, Fig. 1B ). It suggests that LPS can induce HMGB1 release partly through a CD14-dependent mechanism.

LPS induces HMGB1 translocation in CD14-deficient macrophages
Accumulating evidences suggest that LPS-stimulated macrophages and monocytes actively translocate nuclear HMGB1 to the cytoplasm prior to its release [11 , 24 , 25 ]. To evaluate the importance of CD14 in LPS-induced HMGB1 translocation, cultures of wild-type and CD14-deficient peritoneal macrophage were immunostained with antigen affinity-purified anti-HMGB1 antibodies. Quiescent primary peritoneal macrophages of wild-type (BALB/c) or CD14-deficient mice similarly maintained an intracellular "pool" of HMGB1 in the nucleus (Fig. 2B 2E ). Following LPS stimulation, nuclear HMGB1 was translocated to the cytoplasm in wild-type (Fig. 2C) and CD14-deficient (Fig. 2F) macrophages, indicating that CD14-deficient macrophages similarly translocate nuclear HMGB1 to the cytoplasm after LPS stimulation.



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  		Figure 2. LPS induces nuclear HMGB1 translocation in CD14-deficient macrophages. Primary peritoneal macrophages were isolated from normal BALB/c (CD14+/+) or CD14-deficient (CD14–/–) mice, stimulated with LPS (100 ng/ml) for 16 h, and immunostained with antigen affinity-purified, anti-HMGB1 antibodies as described previously [11 ]. (A, D) Light translucent microscopic photographs for wild-type (CD14+/+) or CD14–/– peritoneal macrophages in B and E, respectively. HMGB1 staining appeared to be diffusely distributed in the nuclear region of unstimulated (Control) macrophages of wild-type (CD14+/+; B) and CD14–/– mice (E) but was observed predominantly in the cytoplasm of LPS-stimulated (+ LPS) wild-type (CD14+/+; C) and CD14-deficient (CD14–/–; F) macrophages. The arrows point to nuclear regions of representative cells.

LPS induces HMGB1 release in a MAPK-independent manner
The impairment of LPS-induced TNF production in CD14 null macrophages has been associated with impaired activation of MAPK signaling pathways [26 , 27 ]. We thus examined the potential roles of MAPK in the regulation of LPS-mediated HMGB1 release. A specific p38 MAPK inhibitor, SB203580, dose-dependently suppressed TNF production from murine macrophage-like RAW 264.7 cells (Fig. 3A ), indicating that SB203580 effectively attenuated LPS-induced activation of p38 MAPK under these experimental conditions. In contrast, the LPS-induced HMGB1 release was not reduced by SB203580 (Fig. 3B) or other p38-specific inhibitors (such as SB202190, data not shown), even at concentrations up to 5–20 µM. The role of p38 MAPK in LPS-induced HMGB1 release was further confirmed in primary murine peritoneal macrophages. At concentrations up to 20 µM, neither SB203580 nor SB202190 significantly inhibited LPS-induced HMGB1 release by primary BALB/c peritoneal macrophages (data not shown), indicating that LPS stimulates HMGB1 release in a p38 MAPK-independent mechanism.



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  		Figure 3. Effects of p38 or ERK1/2 MAPK inhibitors on LPS-induced HMGB1 release. Murine macrophage-like RAW 264.7 cells were stimulated with LPS alone or in the presence of inhibitors for p38 (A, B) or ERK1/2 (C, D) MAPK, and the culture medium was assayed for TNF or HMGB1 at 16 h after LPS stimulation. Shown in the bar graphs were the levels of TNF (A, C) or HMGB1 (B, D), expressed as mean ± SEM of three independent experiments. (B, D) Representative Western blots were shown. *, Statistically significant at P < 0.05 as compared with positive controls (+ LPS alone).

To examine the dependence of HMGB1 release on the ERK signaling pathway, specific inhibitors (U0126 and PD98059) of MEK, an upstream activator of ERK1 and ERK2, were used in parallel experiments. U0126, at concentrations up to 5 µM, significantly inhibited LPS-induced TNF production (Fig. 3C) but did not affect LPS-induced HMGB1 release in cultures of RAW 264.7 cells (Fig. 3D) or primary BALB/c peritoneal macrophages (data not shown). Similarly, PD98059, even at concentrations up to 20 µM, did not affect LPS-induced HMGB1 release in primary peritoneal macrophages (data not shown), indicating that LPS induces HMGB1 release through an ERK1/2-independent mechanism.

In parallel experiments, a lower dose of LPS (10 ng/ml) was used to stimulate murine peritoneal macrophages and to examine the effect of p38 and ERK1/2 inhibitors on HMGB1 release. At concentrations up to 20 µM, SB203580 significantly suppressed LPS-induced TNF production (TNF=18.1±5.2 ng/ml, LPS alone vs. TNF=4.2±1.3 ng/ml, LPS+20 µM SB203580; n=6, P<0.01) but failed to significantly reduce LPS-induced HMGB1 release (HMGB1=60.5±12.1 ng/ml, LPS alone vs. HMGB1=58.7±10.2 ng/ml, LPS+20 µM SB203580; P>0.05). Similarly, at concentrations up to 5 µM, U0126 effectively suppressed LPS-induced TNF production (TNF=18.1±5.2 ng/ml, LPS alone vs. TNF=8.1±2.2 ng/ml, LPS+5 µM U0126; n=3, P<0.05) but also failed to significantly reduce LPS-induced HMGB1 release (HMGB1=60.5±12.1 ng/ml, LPS alone vs. HMGB1=60.8±15.0 ng/ml, LPS+5 µM U0126; P>0.05). Taken together, these data suggest that LPS stimulates macrophages to release HMGB1 through p38- and ERK1/2-independent mechanisms.

Spermine inhibits HMGB1 release in a p38 MAPK-independent manner
An endogenous molecule, spermine, occupies an important role in the regulation of the innate immune response by down-regulating the synthesis and release of various proinflammatory cytokines such as TNF, IL-1, macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), and MIP-1ß from activated macrophages/monocytes [28 ]. It is thus interesting to determine if spermine can inhibit LPS-mediated HMGB1 release in macrophage cultures. Consistent with earlier report [28 ], spermine dose-dependently attenuated LPS-induced TNF secretion in murine macrophages (Fig. 4A ). Similarly, at a wide range of concentrations (0, 10, and 100 µM), spermine promoted a dose-dependent suppression of LPS-induced HMGB1 release, with a maximal of >80% reduction of HMGB1 release (Fig. 4B) .



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  		Figure 4. An endogenous, immunosuppressive molecule, spermine, inhibits HMGB1 release in a p38 MAPK-independent manner. Murine macrophage-like RAW 264.7 cells were stimulated with LPS alone or in the presence of spermine at indicated concentrations. The concentrations of TNF (A) and HMGB1 (B) in the culture medium were assayed at 16 h after LPS stimulation by ELISA or Western blotting analysis. In parallel experiments, the concentrations of cellular phospho-p38 (pp-p38) MAPK were determined at 30 min after LPS stimulation by Western blotting analysis using specific antibodies (C).

The suppression of spermine on LPS-induced HMGB1 release was not a result of cell toxicity, as cell viability assessed by trypan blue exclusion assay, was unaffected by spermine even at concentrations up to 100 µM [control cell viability=94–96% vs. spermine-treated cells (100 µM), cell viability=95–97%, 16 h after stimulation]. At higher concentrations (e.g., 1000 µM), spermine exhibited a noticeable cytotoxicity to macrophage cultures (cell viability=80–85%) and consequently increased (rather than decreased) LPS-stimulated HMGB1 release (data not shown). This is not unexpected, as it has been demonstrated that necrotic cells passively release HMGB1 [29 ]. However, even at concentrations up to 100 µM, spermine failed to inhibit LPS-induced activation of p38 MAPK (Fig. 4C) , suggesting that spermine suppresses HMGB1 release in a p38 MAPK-independent mechanism.

LPS induces HMGB1 release partly through a TNF-dependent mechanism
LPS stimulates macrophages to sequentially release TNF and HMGB1 in a time-dependent manner. As TNF itself can induce HMGB1 release [4 ], it may contribute to LPS-induced HMGB1 release. To test this possibility, we first examined the effect of TNF-neutralizing antibodies on LPS-mediated HMGB1 release in RAW 264.7 cell cultures. When stimulated by LPS at low concentrations (10 ng/ml), TNF-neutralizing antibodies dose-dependently attenuated LPS-induced HMGB1 release, with a significant, maximal suppression of HMGB1 release by 40–50% (P<0.05, Fig. 5A ). In contrast, TNF-neutralizing antibodies failed to attenuate HMGB1 release if macrophages were stimulated by LPS at overwhelming high concentrations (100 ng/ml, Fig. 5B ).



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  		Figure 5. Effects of anti-TNF antibodies on LPS-induced HMGB1 release. RAW 264.7 cells were stimulated with LPS (100 ng/ml) alone or in the presence of anti-TNF antibodies (Anti-TNF IgG) or irrelevant (Control IgG) antibodies for 16 h. The levels of HMGB1 in the culture medium were determined by Western blot analysis. Shown in the upper panel was mean ± SEM of three independent experiments; shown in the lower panel was a representative Western blot. Note that TNF-neutralizing antibodies only partially attenuated HMGB1 release induced by LPS at low (A) but not high concentrations (B). *, Statistically significant at P < 0.05 as compared with positive controls (LPS).

To further evaluate the role of TNF in LPS-induced HMGB1 release, primary peritoneal macrophages were isolated from TNF-deficient C57BL/B6 mice or control littermates and were stimulated with LPS at indicated concentrations. As shown in Figure 6 , LPS triggered a dose-dependent HMGB1 release in wild-type and TNF-deficient peritoneal macrophages. At lower concentrations (10 ng/ml), LPS induced significantly less HMGB1 release in TNF-deficient (TNF–/–) macrophages as compared with normal (TNF+/+) macrophages (P<0.05, Fig. 6 ). At higher concentrations (100 and 500 ng/ml), however, LPS induced indistinguishable amounts of HMGB1 release in TNF-deficient and wild-type peritoneal macrophages. It confirms that LPS, especially at physiologically relevant concentrations, can stimulate HMGB1 release partly through a TNF-dependent mechanism.



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  		Figure 6. Effect of genetic disruption of TNF expression on LPS-induced HMGB1 release. Primary peritoneal macrophages were isolated from normal C57BL/6J (TNF+/+) or TNF-deficient (TNF–/–) C57BL/B6 mice and were stimulated with LPS at indicated concentrations. The levels of HMGB1 in the culture medium were determined at 16 h after LPS stimulation and expressed as mean ± SEM of three independent experiments; shown in the lower panel was a representative Western blot. *, Statistically significant at P < 0.05 as compared with controls (TNF+/+).


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DISCUSSION
 
Bacterial endotoxin activates macrophages to sequentially release early (e.g., TNF) and late (e.g., HMGB1) proinflammatory cytokines. However, the mechanisms underlying the regulation of early and late cytokines are quite different. TNF is produced in vanishingly small amounts (if any at all) in quiescent macrophages, but its transcription and translation are rapidly up-regulated by LPS, leading to TNF synthesis and secretion within 1–2 h [2 ]. Containing a leader signal sequence, TNF is secreted via a classical endoplasmic reticulum (ER)-Golgi secretory pathway. In contrast, HMGB1 is constitutively expressed in quiescent cells, and a large pool of preformed HMGB1 is stored in the nucleus, owing to the presence of two lysine-rich nuclear localization sequences [25 ]. Upon LPS stimulation, HMGB1 is released by macrophages/monocytes in a delayed manner (by 8–16 h) [4 ]. Lacking a leader signal sequence, HMGB1 cannot be released via the classical ER-Golgi secretory pathway. Instead, activated macrophages/monocytes acetylate HMGB1 at lysine-rich nuclear localization sequences, leading to translocation of nuclear HMGB1 into cytoplasmic vesicles and subsequent release into the extracellular milieu [11 , 24 , 25 ].

The critical role of CD14 in LPS-mediated TNF production has been suggested by a previous notion that CD14-deficient cells become significantly less responsive to LPS [30 ]. Genetic disruption of CD14 expression almost completely abrogated LPS-mediated TNF production in macrophage cultures. However, depletion of CD14 expression only partially attenuated LPS-mediated HMGB1 release, suggesting that bacterial endotoxin stimulates macrophage to release HMGB1 partly through a CD14-dependent mechanism. Despite of the fact that CD14 only partly contributes to endotoxin-mediated HMGB1 release, a downstream LPS receptor, TLR4, is still critically important in endotoxin-mediated HMGB1 release, as LPS fails to induce HMGB1 release in TLR4-defective, C3H/HeJ murine macrophages (Fig. 7 ) [4 , 6 ]. It is thus plausible that other accessory LBP may also contribute to LPS-mediated HMGB1 release.



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  		Figure 7. Conceptual relationships among LPS, CD14, TNF, and HMGB1 release in macrophage cultures. When LPS is presented to CD14, it is delivered to high-affinity transmembrane receptors such as TLR4, leading to activation of MAPK (e.g., p38, ERK1/2, and JNK) and sequential release of early (e.g., TNF and IL-1ß) and late (e.g., HMGB1) proinflammatory cytokines. In this study, we demonstrated that LPS stimulates macrophages to release HMGB1 partly through CD14-, TNF-, but not p38 MAPK-dependent mechanisms. The potential involvement of other LPS receptors (?; such as CD11b, CD55, LBP, scavenger receptors, or others), other signaling molecules (?), or other cytokines [such as IL-1ß or interferon-{gamma} (IFN-{gamma})] in the regulation of LPS-induced HMGB1 release remains to be further investigated in future studies.

For instance, some heat shock proteins (e.g., hsp70 and hsp90) can be found in LPS-inducible cytoplasmic membrane receptor clusters [31 ], implicating a potential involvement of a CD14-independent LPS receptor cluster (lipid rafts) in LPS signaling. Members of the ß-integrin family (CD11c/CD18) and other membrane proteins (e.g., CD55, decay accelerating factor) are capable of binding LPS and subsequently, delivering it to cell-surface LPS receptors [32 ]. Some scavenger receptors [e.g., class B type I (SR-BI) or class A types I and II (MSR-A)] can also bind LPS and consequently, mediate the internalization of monomerized LPS [33 ]. In a similar manner, the LBP disrupts large LPS aggregates and facilitates the uptake and delivery of monomerized LPS to intracellular TLR4 [34 ]. Therefore, it will be important to investigate the potential involvement of various accessory LBP in LPS-mediated HMGB1 release in future studies.

We propose that macrophages rely on CD14 and other accessory LBP to sensitively detect low levels of LPS and to ensure effective release of early (e.g., TNF) and late (e.g., HMGB1) proinflammatory cytokines. TNF has been established as a causative mediator of endotoxemic shock but not septic shock, as anti-TNF antibodies improve survival in animal models of entotoxemic or bacteremic shock [10 , 35 ] but actually worsen survival in animal models of sepsis [36 ]. In contrast, HMGB1 has been established recently as a causative mediator of sepsis [13 ].

Although TNF itself can induce HMGB1 release [4 ], it only partly contributes to endotoxin-mediated HMGB1 release, as inhibition of TNF expression (by gene knockout) or activity (by neutralizing antibodies) only partially attenuates HMGB1 release. In addition to TNF, LPS stimulates macrophages to release numerous other proinflammatory cytokines such as IL-1ß and IFN-{gamma}, which individually or synergistically stimulates macrophage to release HMGB1. In light of the distinct roles of TNF and HMGB1 in septic shock and sepsis [37 ], it is now reasonable to consider the possibility that the inability of anti-TNF antibodies in attenuating LPS-induced HMGB1 release may contribute, at least in part, to the failure of anti-TNF sepsis clinical trials.

The p38 MAPK signaling pathway plays an important role in regulating TNF translation efficiency through activating translation initiation factor eIF-4E [9 , 38 ]. However, it is not important in LPS-induced HMGB1 release, as p38 MAPK inhibitors failed to inhibit LPS-mediated HMGB1 release (Fig. 7) . Consistently, an endogenous, immunosuppressive molecule, spermine, fails to inhibit LPS-induced activation of p38 MAPK and yet still, dose-dependently attenuates LPS-mediated HMGB1 release. It not only reinforces the important role of spermine in the counter-regulation of the innate immune response but also suggests a possibility that a novel class of HMGB1 inhibitors not targeting p38 MAPK may be useful for the treatment of inflammation diseases.

Various p38 MAPK inhibitors (e.g., SB203580) confer protection in animal models of inflammation [39 ] and sepsis [40 ]. These p38 inhibitors may be protective partly through inhibiting endotoxin-mediated production of early proinflammatory cytokines (e.g., TNF and IL-1). Although not critical in endotoxin-mediated HMGB1 release, p38 MAPK occupies an important role in HMGB1-mediated production of proinflammatory cytokines, as inhibition of p38 MAPK abrogates HMGB1-mediated production of proinflammatory cytokines [17 ]. It is thus reasonable to consider the possibility that p38 MAPK inhibitors promote a protective effect against lethal, systemic inflammation (e.g., endotoxemia and sepsis) partly through attenuating inflammatory responses mediated by HMGB1 and other proinflammatory cytokines.


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ACKNOWLEDGEMENTS
 
We thank Xiaotian Chen for assistance with immunoassay and Drs. Leslie Goodwin and Dorothy Guzowski of the North Shore–LIJ Research Institute Molecular Genetics/Core Facility for technical assistance with the confocal microscope. This research was supported by the National Institute of General Medical Sciences (NIGMS; R01GM063075 to H. W.) and in part by the National Institute of Allergy and Infectious Diseases (NIAID; R01AI23859 to S. M. G.).

Received April 19, 2004; revised August 3, 2004; accepted August 4, 2004.


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