{alpha}-Chemokine receptor blockade reduces high mobility group box 1 protein-induced lung inflammation and injury and improves survival in sepsis

Xinchun Lin,1,2 Huan Yang,3 Tohru Sakuragi,1,2,5 Maowen Hu,1 Lin L. Mantell,1 Shinichiro Hayashi,5 Yousef Al-Abed,3 Kevin J. Tracey,3 Luis Ulloa,4 and Edmund J. Miller1,2

1Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center; Departments of 2Surgical Immunology and 3Biomedical Sciences, and 4Center for Immunology and Inflammation, Institute for Medical Research at North Shore-LIJ, Manhasset, New York; and Departments of 5Thoracic and Cardiovascular Surgery, and Pulmonary Medicine, Saga University, Saga, Japan

Submitted 28 February 2005 ; accepted in final form 25 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
High mobility group box 1 (HMGB1) protein, a late mediator of lethality in sepsis, can induce acute inflammatory lung injury. Here, we identify the critical role of {alpha}-chemokine receptors in the HMGB1-induced inflammatory injury and show that {alpha}-chemokine receptor inhibition increases survival in sepsis, in a clinically relevant time frame. Intratracheal instillation of recombinant HMGB1 induces a neutrophilic leukocytosis, preceded by alveolar accumulation of the {alpha}-chemokine macrophage inflammatory protein-2 and accompanied by injury and increased inflammatory potential within the air spaces. To investigate the role of {alpha}-chemokine receptors in the injury, we instilled recombinant HMGB1 (0.5 µg) directly into the lungs and administered a subcutaneous {alpha}-chemokine receptor inhibitor, Antileukinate (200 µg). {alpha}-Chemokine receptor blockade reduced HMGB1-induced inflammatory injury (neutrophils: 2.9 ± 3.2 vs. 8.1 ± 2.4 x 104 cells; total protein: 120 ± 48 vs. 311 ± 129 µg/ml; reactive nitrogen species: 2.3 ± 0.3 vs. 3.5 ± 1.3 µM; and macrophage migration inhibitory factor: 6.4 ± 4.2 vs. 37.4 ± 15.9 ng/ml) within the bronchoalveolar lavage fluid, indicating that HMGB1-induced inflammation and injury are {alpha}-chemokine mediated. Because HMGB1 can mediate late septic lethality, we administered Antileukinate to septic mice and observed increased survival (from 58% in controls to 89%) even when the inhibitor treatment was initiated 24 h after the induction of sepsis. These data demonstrate that {alpha}-chemokine receptor inhibition can reduce HMGB1-induced lung injury and lethality in established sepsis and may provide a novel treatment in this devastating disease.

acute lung injury; {alpha}-chemokines; polymorphonuclear neutrophils


SEVERE SEPSIS ANNUALLY AFFECTS ~750,000 individuals in the United States alone (3). The mortality rate from severe sepsis remains high, with patients often succumbing to multiple organ failure. Of particular importance are the lungs, in which acute injury can develop within hours of the onset of sepsis (13). The acute lung injury is associated with a marked increase in polymorphonuclear neutrophils (neutrophils) in the lung (28), which may constitute 80% or more of the total cells obtained by bronchoalveolar lavage (BAL), compared with the 2–3% found in normal subjects (24). The rapid neutrophil influx into the lung in response to systemic bacterial toxins is driven, at least in part, by an accumulation within the lungs of {alpha}-chemokines having the ELR tripeptide motif (8, 24, 25, 34). These {alpha}-chemokines bind to specific receptors on neutrophils and function as potent chemoattractants and activators (10). The increased accumulation of neutrophils within the lung is associated with severe injury to the pulmonary alveolar-capillary membrane that leads to flooding of the alveolar spaces. The alveolar flooding disrupts normal gas exchange and severe hypoxemia results. High mobility group box 1 (HMGB1) has been shown to be a late-acting mediator in lethal systemic inflammation (39, 44). HMGB1 release can be induced in macrophages and monocytes by lipopolysaccharide (LPS) (42) and is regulated, at least in part, by {alpha}7-nicotinic acetylcholine receptors (43). Extracellular HMGB1 can trigger acute lung injury (1, 14) and a lethal inflammatory process (31) by significantly increasing the release of inflammatory cytokines such as TNF, IL-1{beta}, IL-6, IL-8, and macrophage inflammatory protein (MIP)-1{beta} (2).

Because {alpha}-chemokines play an important role in acute lung injury (24), we developed an {alpha}-chemokine receptor inhibitor. This hexapeptide, which we termed Antileukinate, blocks both {alpha}-chemokine receptors 1 and 2 (10), thereby inhibiting neutrophil chemotaxis and activation. We have also determined that this inhibitor protects against acute lung inflammation and injury in several animal models (11, 17, 20, 25). In this study, we have used Antileukinate to more clearly define the role of {alpha}-chemokines in the HMGB1-induced injury and lethality and show that the acute injury initiated by HMGB1 is secondary to {alpha}-chemokine receptor activation. In addition, {alpha}-chemokine receptor blockade can both reduce HMGB1-induced lung injury and improve survival in sepsis.


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Synthesis and use of Antileukinate. The hexapeptide receptor inhibitor Ac-RRWWCR-NH2 (Antileukinate) was synthesized as previously described (10). The peptide was dissolved in sterile saline (0.9% wt/vol) immediately before use and administered subcutaneously.

All animal experiments were approved by the Institutional Animal Care and Use Committee at the Medical Research Institute at North Shore-LIJ.

Induction of polymicrobial bacterial peritonitis. Peritonitis was induced in female BALB/c mice (Taconic, Germantown, NY) by cecal ligation and puncture (CLP). Mice (20–25 g) were anesthetized [ketamine (75–80 mg/kg) + medetomidine (1.0–1.2 mg/kg) in 200 µl ip], and a 1- to 2-cm incision was made on the lower left abdomen. The cecum was exposed and ligated below the ileocecal valve. The cecum was punctured once with a 23-gauge needle, and stool (~1 mm) extruded from the hole. After replacing the cecum, we closed the abdomen, and the mice were resuscitated with 0.5 ml of warm saline (0.9% wt/vol). In some experiments, starting 24 h after the induction of sepsis, groups of animals received either Antileukinate (250 µg in 100 µl sterile pyrogen-free saline, 0.9% wt/vol) or saline alone (control) twice daily for 4 days.

BAL. The mice were anesthetized as before. Blood was collected via cardiac puncture. The trachea was directly visualized, and a 20-gauge x 1-in iv catheter was inserted and secured in place. The lungs were gently lavaged (two times each aliquot) with two aliquots of saline (total 2.5 ml), which were pooled. The recovery ratio of the lavage fluid ranged from 70% to 80% and did not differ significantly among the groups. The total cell count (using a Neubauer hemocytometer) and cell differential (performed on hematoxylin and eosin- stained cytocentrifuge preparations, counting at least 400 cells per condition) were assessed.

Analysis of BAL fluid. MIP-2 and macrophage migration inhibitory factor (MIF) concentrations were measured by ELISA (R&D Systems, Minneapolis, MN and Chemicon International, Temecula, CA, respectively). Total protein concentration was measured using Coomassie Plus assay (Pierce Chemical, Rockford, IL). Reactive nitrogen species (RNS), including NO, nitrate, and nitrite, were measured using a Sievers NO analyzer (model 280; Boulder, CO).

Western blot analysis. Proteins within the BAL fluid were separated on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and located with specific rabbit anti-HMGB1 IgG as previously described (42, 43).

MIP-2 reverse transcriptase polymerase chain reaction. After CLP or sham surgery, the lungs were removed and rapidly frozen in liquid nitrogen. Total mRNA was isolated from the tissues using RNeasy (Qiagen, Valencia, CA), and reverse transcriptase polymerase chain reaction was performed using the method and primers outlined by Mancardi et al. (18). The resultant bands were scanned, and the density of each band was expressed as a ratio of {beta}-actin expression in the same samples (22).

Production and purification of recombinant HMGB1. Recombinant rat HMGB1 (rHMGB1) was expressed in Escherichia coli and purified as described previously (42). The protein was dialyzed extensively against phosphate-buffered saline (PBS), passed over a polymixin B column, lyophilized, and redissolved in sterile water before use. rHMGB1 preparations were shown to be free of residual LPS contamination using a colorimetric limulus assay. In some experiments, the protein was digested with trypsin before instillation.

Instillation of rHMGB1. Mice were anesthetized as described above. An incision (2–3 mm) was made, and the trachea was directly visualized. A 25-gauge needle was used to make a 1-mm hole in the trachea, and the HMGB1 was instilled via a 50-µl Hamilton syringe. Each group contained between 5 and 11 animals, and experiments were performed independently at least twice.

Bacterial counts. Bacterial counts in septic animals were performed as previously described (40). In brief, the spleen was removed by a sterile technique and homogenized in PBS (0.05 M, pH 7.4). After serial dilutions with PBS, the homogenate was plated as 0.15-ml aliquots on tryptic soy agar plates (Difco, Detroit, MI), and colony forming units (CFU) were counted after overnight incubation at 37°C. Bacterial load was expressed as CFU/g tissue.

Statistical analysis. The data are expressed as means ± SD and were analyzed for significance by Student's t-test (for two groups) or for multiple comparisons by analysis of variance with a post hoc Dunn's or Bonferroni analysis. A P value of <0.05 was considered statistically significant. Differences in the survival curves were assessed using the Mann-Whitney U-test.


    RESULTS
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HMGB1 accumulates within the alveolar spaces following CLP. HMGB1 accumulated within the lungs of mice following the induction of sepsis by ligation and puncture of the cecum. Although there were detectable amounts of HMGB1 within the BAL fluid in the control mice (those having undergone anesthesia and laparotomy without ligation and puncture of the cecum), there was a steady accumulation of HMGB1 within the alveoli over the 36 h of evaluation following CLP (Fig. 1).



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Fig. 1. High mobility group box 1 (HMGB1) accumulates in the alveoli following sepsis. Lungs from mice (n = 5 per group) 12, 24, or 36 h after the induction of peritonitis or from sham controls were lavaged. Bronchoalveolar lavage (BAL) samples were analyzed for HMGB1 content by Western blot. Culture medium from LPS-stimulated mouse RAW 264.7 macrophage-like cells was used as a positive control. CLP, cecal ligation and puncture.

 
MIP-2 accumulates within the alveolar spaces during sepsis. mRNA encoding MIP-2 was elevated in the lung tissues 6 h after the induction of sepsis and was further increased at 24 h (Fig. 2A). This was associated with an increase in MIP-2 protein within the BAL fluid at 12 h post-CLP. The level remained elevated at 48 h postsurgery (Fig. 2B).



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Fig. 2. Macrophage inflammatory protein (MIP)-2 expression and concentration in the lung increases following sepsis. A: after CLP or sham surgery, the total mRNA in the lungs was isolated and analyzed by RT-PCR. The resultant bands were scanned, and the density of each band was expressed as a ratio of {beta}-actin mRNA in the same samples (n = 3 per group). B: concentration of MIP-2 in BAL collected 12 or 48 h post-CLP (n = 5 per group). *Means are significantly different from control values (P < 0.05).

 
Inflammatory neutrophils within the alveoli following direct instillation of HMGB1. Sepsis is often associated with acute lung inflammation and injury. Therefore, we examined whether HMGB1 alone was sufficient to induce lung inflammation and injury. rHMGB1 was instilled directly into the lungs, and the alveolar spaces were lavaged 18 h postinstillation. Leukocyte accumulation occurred within the alveoli in a dose-dependent manner with respect to rHMGB1 (Fig. 3). Even modest instillations of as little as 0.5 µg of rHMGB1 (~20 pmol) increased the number of lavaged leukocytes to 8- to 10-fold higher than those animals instilled with saline alone. At 18 h after the instillation of HMGB1, a differential cell count revealed that the percentage of neutrophils had increased to 72.6 ± 14.9% compared with the saline-instilled controls that contained 2.3 ± 1.3% neutrophils within the lavage fluid. Furthermore, the neutrophil accumulation induced by 0.5 µg of rHMGB1 was comparable to that induced by 5 µg of LPS. The leukocyte accumulation was time dependent with significant increases occurring at 6 h postinstillation (Fig. 4A) and due predominantly to an influx of neutrophils. The ability to induce accumulation of inflammatory cells was lost when the rHMGB1 was digested with trypsin before instillation (Fig. 4B), indicating a specific effect of the rHMGB1 protein.



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Fig. 3. HMGB1 instilled into the lungs induces leukocytosis. Purified recombinant HMGB1 free from measurable LPS (0.5, 1.5, 4.4, 13.3, or 40 µg in 50 µl of sterile pyrogen-free saline, 0.9% wt/vol) was instilled directly into the lungs via the trachea. An equal volume of saline alone or LPS (5 µg) was instilled as negative or positive control, respectively (n = 5 per group). *Significantly different from instillation of saline alone (P < 0.05).

 


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Fig. 4. HMGB1 induces neutrophil accumulation in the lung. Purified recombinant HMGB1 free from measurable LPS (0.5 µg in 50 µl of sterile pyrogen-free saline, 0.9% wt/vol) instilled directly into the lungs induced significant increases in cell accumulation in a time-dependent manner. A: *significantly different from instillation of saline alone (P < 0.05). This increase was predominantly due to an increase in neutrophils. The inflammatory affects of HMGB1 were abolished following trypsin digestion (n = 9 per group). B: there was a significant reduction in cell accumulation following the of instillation of trypsinized HMGB1 [0.5 µg in 50 µl of sterile pyrogen-free saline, 0.9% wt/vol (n = 11)] vs. instillation of an equal concentration of native protein (n = 14). *P < 0.05.

 
Neutrophil chemoattractant MIP-2 accumulates within the alveoli following instillation of rHMGB1. Neutrophil accumulation within the lung during periods of acute inflammation, particularly associated with sepsis, are associated with {alpha}-chemokines (8, 23, 24). Therefore, we examined the role of {alpha}-chemokines in rHMGB1-induced neutrophil accumulation within the lung. After instillation of rHMGB1 into the alveoli, there was a time-dependent increase in the {alpha}-chemokine MIP-2 (Fig. 5). This increase, which was evident 1 h after rHMGB1 instillation and peaked at around 3 h, preceded the neutrophil accumulation. Additionally, 6 h after instillation of rHMGB1 (0.5 µg) into the lungs, there was an accumulation of MIP-2 (26.5 ± 5.1 pg/ml) in the plasma that was significantly higher than the plasma concentration of MIP-2 (11.0 ± 2.7 pg/ml) following instillation of trypsin-degraded rHMGB1 (P ≤ 0.05).



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Fig. 5. HMGB1 induces MIP-2 accumulation in the lung. Purified recombinant human (rh) HMGB1 free from measurable LPS (0.5 µg in 50 µl of sterile pyrogen-free saline, 0.9% wt/vol) instilled directly into the lungs induced significant increases in MIP-2 measured by ELISA (n = 6 per group). The concentration of MIP-2 in the BAL fluid was at a maximum at 3 h postinstillation and returned to control levels after 18 h. *Significantly different from instillation of saline alone (P < 0.05).

 
{alpha}-Chemokine receptor blockade reduces rHMGB1-induced acute lung injury. Because neutrophil accumulation following the instillation of rHMGB1 occurred in association with an increase in {alpha}-chemokine, we evaluated the effect of specific receptor blockade on the inflammatory response and acute lung injury. {alpha}-Chemokine receptor blockade was achieved with the use of Antileukinate, which we have shown previously effectively binds both {alpha}-chemokine receptors 1 and 2 (10) and suppresses acute lung injury in mice (11, 17). Antileukinate administered subcutaneously following intratracheal instillation of rHMGB1 (0.5 µg) significantly decreased, in a dose-dependent manner, the total leukocyte count within the BAL (Fig. 6A) by reducing the neutrophil trafficking to the alveolar spaces (Fig. 6B). Concurrent with the decreased influx of inflammatory cells in the lung, following {alpha}-chemokine receptor blockade, there was a decrease in oxidative stress as assessed by total RNS (Fig. 7A), injury evaluated by increased total protein (Fig. 7B), and inflammatory potential measured by the accumulation of other inflammatory mediators, such as MIF (Fig. 7C), which has also been shown to play a key role in the pathogenesis of sepsis (5, 6). These results indicate that there were decreases both in the injury and the potential for further damage to the epithelium. However, blockade of {alpha}-chemokine receptors had no significant effect on MIP-2 accumulation within the plasma (rHMGB1-treated animals = 26.5 ± 5.1 pg/ml vs. rHMGB1 + Antileukinate animals = 20.5 ± 2.9 pg/ml) of rHMGB1-treated animals.



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Fig. 6. {alpha}-Chemokine receptor blockade abrogates HMGB1-induced leukocytosis. Antileukinate (in 200 µl of sterile pyrogen-free saline, 0.9% wt/vol) was administered subcutaneously, along the dorsum, as a component of the resuscitation fluid, ~30 min after the intratracheal (it) instillation of purified recombinant HMGB1 (0.5 µg in 50 µl of sterile pyrogen-free saline, 0.9% wt/vol). Antileukinate administration significantly reduced, in a dose-dependent manner, the total numbers of white cells (A) and neutrophils (B) lavaged from the lungs after 6 h (n = 5 per group). PMN, polymorphonuclear neutrophil. *Significantly different from administration of subcutaneous (s.q.) saline (P < 0.05).

 


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Fig. 7. {alpha}-Chemokine receptor inhibitor blocks rHMGB1-induced oxidative stress and injury. After the intratracheal instillation of purified rat rHMGB1 (0.5 µg in 50 µl of sterile pyrogen-free saline, 0.9% wt/vol), the animals received a single, subcutaneous bolus of Antileukinate (200 µg in 200 µl of sterile pyrogen-free saline, 0.9% wt/vol) administered subcutaneously. Antileukinate significantly reduced the concentrations of total reactive nitrogen species (A), total protein (B), and macrophage migration inhibitory factor (MIF, C) in the BAL fluid at 6 h post-rHMGB1 administration (n = 6 per group). *Significantly different from administration of subcutaneous saline (P < 0.05).

 
{alpha}-Chemokine receptor blockade increases survival in sepsis. HMGB1 is a late mediator of septic mortality that, as we have shown above, can cause neutrophil-mediated acute lung injury. Because acute lung injury in sepsis is associated with increased mortality (23), we examined whether {alpha}-chemokine receptor blockade would improve survival during sepsis. Because our data indicate that HMGB1 levels in the lung do not significantly increase earlier than 24 h postinduction of sepsis, we delayed initiation of {alpha}-chemokine receptor blockade until 24 h after the CLP procedure. Figure 8 shows that 2 days following induction of polymicrobial sepsis, there was an increase in the tissue bacterial load and that the number of bacteria had subsided by day 4. Administration of Antileukinate in these animals had no significant effect on these numbers. However, {alpha}-chemokine receptor blockade by administration of Antileukinate, starting 24 h after the induction of sepsis, significantly improved the survival rate of these animals from 58% in controls to 89% (Fig. 9).



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Fig. 8. Bacterial load following induction of polymicrobial sepsis. From 24 h after the induction of sepsis, Antileukinate (250 µg in 100 µl of sterile pyrogen-free saline, 0.9% wt/vol) or saline alone (control) was administered twice daily for 4 days. At days 2, 4, and 7, animals were killed, and the spleens were assessed for viable bacteria. There were no significant differences noted between treated and control groups (n = 5 per group). cfu, Colony forming unit.

 


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Fig. 9. {alpha}-Chemokine receptor inhibitor improves survival in sepsis. From 24 h after the induction of sepsis, Antileukinate (250 µg in 100 µl of sterile pyrogen-free saline, 0.9% wt/vol) or saline alone (control) was administered twice daily for 4 days. Survival in each group was assessed daily, and overall survival was evaluated 7 days after the induction of sepsis. Initial numbers of animals in each group were Antileukinate n = 23; control n = 22. The survival curves were significantly different by Mann-Whitney U-analysis (P < 0.05).

 

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Severe sepsis, a lethal systemic inflammatory reaction to infection, kills more than 215,000 people annually in the United States, with over $16.7 billion nationally in costs (1). Severe sepsis frequently precipitates acute lung injury, and even those individuals that survive the septic episode commonly suffer from chronic pulmonary fibrosis, reduced pulmonary function, and a reduced health-related quality of life (7, 12, 27, 32). A better understanding of the mechanisms involved in the injury is essential to a reduction in the morbidity and mortality of acute lung injury associated with sepsis.

Recently, rHMGB1, a lethal mediator of sepsis (44), has been shown to induce acute lung inflammation (1). However, before the current study it remained unclear whether the injury resulted from the direct effects of HMGB1 or from subsequent neutrophil activity. Neutrophils are thought to be a critical component of the pathogenesis of acute lung injury. One of the earliest events in acute lung injury is the sequestration of neutrophils in the lung microvasculature (37). However, sequestration alone is insufficient to cause severe injury to the lung tissue (19). To cause tissue damage, activation of the neutrophils is required. Adherence of the neutrophils to endothelium, epithelium, or to interstitial matrix can trigger the uncontrolled release of proteolytic and oxidative enzymes, as well as reactive oxygen and nitrogen species. This can lead to tissue damage and the release of growth factors, chemokines, and other cytokines that can enhance the inflammatory response (9, 15). Neutrophil adhesion to endothelial cells is mediated via {beta}-integrin molecules on the neutrophil cell surface, with expression and increased avidity of these adhesion molecules being augmented by chemoattractants such as IL-8 (45, 46). Adhesion primes neutrophils, greatly increasing their ability to degranulate and activate the respiratory burst in response to soluble mediators (4, 35), with the amount of oxidant production increasing by up to 1,000-fold over that of nonadherent neutrophils (9). The release of reactive oxygen and nitrogen species from neutrophils can directly injure the lung (33). Tsujimoto et al. (38) have shown the importance of the {alpha}-chemokine MIP-2 in acute lung injury following peritonitis. They demonstrated an early accumulation of the {alpha}-chemokine within the lungs associated with marked interstitial edema and inflammatory infiltration at 6–12 h after the induction of peritonitis. The model was lethal starting at ~24 h, and anti-MIP-2 antibody improved survival at 72 h from 0% in sham-treated animals to 50% in the group receiving anti-MIP-2 antibody. The absolute timing and peak of the MIP-2 generation in our study differ from that of the earlier study. This may be due in part to the use by Tsujimoto et al. of the inhaled anesthetic diethyl ether before the induction of sepsis. Compared with injectable anesthetics, volatile anesthetics have been shown to alter the course of the acute inflammatory response in the lung (26, 29). Our data show that the concentration of MIP-2 within the BAL is significantly increased by 12 h post-CLP. This is consistent with the findings of Walley et al. (41), who found MIP-2 raised in the lung at both 8 and 24 h post-CLP, and consistent with the release of HMGB1 into the alveolar space, which occurs within the first 12 h of sepsis.

From our studies with rHMGB1 instilled directly into the lungs, it might be expected that during sepsis, the release of HMGB1 and subsequent MIP-2 would attract neutrophils to the lungs, and the studies of Tsujimoto et al. (38) showed a marked infiltration of inflammatory cells within the alveolar walls. However, consistent with that study and that of Yin et al. (47), we do not see a large increase of intra-alveolar neutrophils during the first 48 h of sepsis. Although we have shown that in otherwise normal mice, the presence of HMGB1 in the alveolar spaces induces a neutrophilic influx, during sepsis the build up of HMGB1 within the alveoli occurs in the setting of a severe systemic inflammatory response. Although neutrophils are sequestered in the lung, particularly 24 h post-CLP (21, 48), Yin et al. (47) have shown that the neutrophils stay mainly within the pulmonary vasculature and are predominantly immature band cells. Such cells have abnormal surface receptors, cytoskeletal elements, and chemotactic activity that restrict their transendothelial migration. Although sequestration alone is insufficient to cause acute lung injury (19), the potential for injury remains since neutrophils are primed by interaction with the endothelium, greatly increasing their ability to degranulate and activate the respiratory burst in response to soluble mediators (4, 35).

The studies by Talwar et al. (36) have also shown elevated concentrations of HMGB1 (86 ± 12 ng/ml) in the BAL fluid from patients with the severest form of acute lung injury, acute respiratory distress syndrome. Normal lavage procedures dilute the epithelial lining fluid ~100-fold (24). Therefore, the local concentration of HMGB1 within the alveolar spaces may exceed 8 µg/ml. We found that instillation of rHMGB1 directly into the lungs of normal, healthy mice induced a significant dose-dependent increase in total leukocytes, even at doses as low as 0.5 µg. Because this dose was comparable with that found during acute lung injury, we instilled this amount to assess the effects of {alpha}-chemokine receptor blockade on HMGB1-induced lung injury. Instillation of HMGB1 induced an increase in leukocytes primarily due to a neutrophil influx. The cell influx was specific to the intact rHMGB1 molecule (as it was ablated by trypsin degradation) and preceded by an increase in {alpha}-chemokine MIP-2. We used our specific receptor inhibitor, Antileukinate, to block the {alpha}-chemokine receptors. The blockade of {alpha}-chemokine receptors with Antileukinate reduced the neutrophil influx, total RNS, and injury as assessed by the BAL protein content. The blockade also reduced the accumulation in the air spaces of MIF, another late-acting chemokine important in the pathogenesis of sepsis (16), suggesting a further benefit of the of {alpha}-chemokine receptor blockade strategies. In addition, rHMGB1 instillation into the lung initiated a systemic inflammatory response shown by the increase in plasma MIP-2 concentration; this effect was abolished by trypsin digestion of the HMGB1 but was not reduced by the administration of Antileukinate. It remains to be determined whether the accumulation of the chemokines in the plasma were due to leakage from the air spaces or whether rHMGB1, intratracheally instilled, initiates the release of {alpha}-chemokines from extrapulmonary sources. However, in accordance with HMGB1 being a late mediator of the lethality associated with sepsis, the concentration of HMGB1 in the epithelial lining fluid (sampled in the BAL) increased dramatically between 24 and 36 h after the onset of sepsis. In addition, {alpha}-chemokine receptor blockade did not affect overall bacterial counts in the animals but increased survival during sepsis even when administration was delayed until 24 h after the induction of sepsis. Santos et al. (30) showed that following the induction of polymicrobial sepsis in mice (by CLP), the bacterial burden in the lung is similar to that of other body compartments and, consistent with our data, the CFU peak within the lung 24–48 h postinduction of sepsis and then decrease over the next 48 h. This suggests that the animals are not dying from an overwhelming growth of bacteria but from the inflammatory response set in motion by the original infection.

We conclude that HMGB1 is potentially an important mediator of acute lung injury during severe sepsis and that its effects, which are due predominantly to the recruitment of neutrophils, can be reduced by {alpha}-chemokine receptor inhibition. We have demonstrated that substantial increase in survival can be achieved in "established sepsis" by {alpha}-chemokine receptor blockade. This raises the exciting possibility of the use of anti-{alpha}-chemokine receptor strategies in the treatment of sepsis, since they can be administered in a clinically relevant time frame.


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This work was funded in part by National Institute of General Medical Sciences Grant RO1 GM-65555 (E. J. Miller).


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K. J. Tracey is a coinventor on a patent describing anti-HMGB1 antibodies, which were not used in the work described here. North Shore LIJ licensed this technology to Critical Therapeutics, Inc., which has a sponsored research agreement with Dr. Tracey.

E. J. Miller and S. Hayashi are coinventors on patents describing Antileukinate; these patents are held by The University of Texas Board of Regents and not by the inventors.


    ACKNOWLEDGMENTS
 
We thank Jian-Hua Li, Hong Liao, Renqi Yuan, Mahendar Ochani, Kanta Ochani, John Romashko III, William Franek, and Qiaoting Du for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: E. J. Miller, North Shore-LIJ Research Institute, 350 Community Dr., Manhasset, NY 11030 (e-mail: emiller{at}nshs.edu)

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


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