We show that the acute inflammatory mediator HMGB1 is significantly elevated in the sputum of humans with CF, a disease dominated by chronic neutrophilic inflammation of the airways, and in a murine model of the disease. At concentrations equivalent to those observed in airway secretions, HMGB1 induced neutrophil chemotactic activity through a CXCR-dependent pathway, and a neutralizing antibody against HMGB1 caused significant reduction in chemotaxis induced by human CF sputum and BALF from Scnn1b
-Tg mice. Intratracheal instillation of HMGB1 in mice induced significant airway neutrophilia and was accompanied by lung matrix degradation, as evidenced by the production of PGP, a bioactive collagen degradation product also shown to be elevated in Scnn1b
-Tg mice BALF and human CF serum. Elevated PGP was previously reported in CF sputum (35
). Although quantification of HMGB1 in serum and airway secretions has been reported to be difficult due to its potential to bind albumin and other extracellular proteins (36
), we were able to quantify HMGB1 in abundance in biologic samples using both immunoblotting (compared with protein standard) and ELISA methods.
Previous reports of HMGB1 activity have focused on its importance as a mediator of acute inflammation of the lung, including its role in sepsis, hemorrhagic shock, ARDS, and ventilator-associated injury (1–3, 9, 14). Our study is the first to suggest that HMGB1 also has a significant role in neutrophilic inflammation in the CF lung and complements findings reported by Kokkola and colleagues (16
), which showed a substantial presence of HMGB1 in the synovial fluid of a rheumatoid arthritis rat model, a disease also mediated by long-standing neutrophilic inflammation. Notably, we demonstrate that HMGB1 acts as a direct neutrophil chemoattractant, and our research supports observations reported by Orlova and colleagues (38
) who showed that HMGB1 induced neutrophil chemotaxis dependent on Mac-1 (CD116/CD18) and RAGE through activation of the NF-κB pathway. Our findings extend these results and establish that neutrophil chemotaxis is also induced directly by HMGB1 in a mechanism dependent on CXCR2. Given that mouse neutrophils, like human PMNs, expressed CXCR2, but did not exhibit chemotaxis in response to purified HMGB1 (), suggests that HMGB1 likely contributes to inflammation in Scnn1b
-Tg mice predominantly through indirect mechanisms, such as the release of TNF-α and KC (through activation of inflammation by Toll-like receptor [TLR]-2, TLR-4, and RAGE ) or by augmenting other proinflammatory cytokines, as suggested by Sha and colleagues (39
). In contrast, in humans, HMGB1 exhibits both direct (CXCR2-dependent) and indirect proinflammatory effects.
Our study is the first report of inhibition of HMGB1-mediated chemotaxis by antibody inhibition in biologic specimens that also supports the potential for in vivo
blockade of chemoattractant activity. HMGB1-dependent chemotactic activity shown in these studies suggests that exaggerated levels, possibly promoted by poor mucociliary clearance of necrotic cells (40
), have deleterious effects in the CF airway that may be self-sustaining. Direct activation of PMNs by HMGB1 would be anticipated to further contribute to airway destruction and progression of bronchiectasis. These findings may have implications in other proinflammatory airway diseases where neutrophils are prominent, including chronic obstructive pulmonary disease (41
) or ischemic/reperfusion injury of the transplanted lung (42
Beyond the intrinsic chemotactic properties of HMGB1, these data show that modest levels of HMGB1 in the lung can lead to collagen matrix degradation and production of bioactive proinflammatory mediators, thus linking influx and activation of PMNs by HMGB1 using a readily obtained, noninvasive measure of lung injury. The finding that HMGB1 is also detected in the Scnn1b
-Tg model of chronic bronchitis, at levels anticipated to activate neutrophils (33
) and sufficient to induce PMN chemotaxis, provides a mechanism by which HMGB1 could contribute to neutrophil migration and activation, lung matrix degradation, and liberation of PGP. We speculate that neutrophil influx by HMGB1 through CXCR2 and activation mediated through the NF-κB pathway may contribute to the release of bioactive enzymes, including neutrophil elastase and matrix metalloproteinase 8 and 9, each previously reported to be present in PMNs highly active in the CF lung (25
). Collagen degradation induced by activation of innate immunity in the inflamed airway results in liberation of PGP, a collagen fragment reported by our laboratory to have proinflammatory effects through both CXCR1 and CXCR2 receptors present in neutrophils (22
). Combined with HMGB1, release of this peptide fragment provides a positive feedback mechanism that contributes to persistent neutrophilic inflammation in the CF lung.
Estimates of HMGB1 levels in human sputum were inversely correlated with the duration of intravenous antibiotics in patients with CF and APE (). Previous studies indicate that appropriate antimicrobial therapy is associated with a reduction in markers of inflammation in sputum and serum (particularly IL-8, IL-6, and total neutrophil counts), although not all studies have been consistent (44
). The inverse correlation with antimicrobial therapy in our study further indicates the importance of HMGB1 during periods of disease exacerbation, and suggests that HMGB1 may serve as a biomarker for the activation of innate immunity. Moreover, HMGB1 was intermediately elevated in outpatients with CF, in whom neutrophilic inflammation is also evident (but to a lesser extent), and was inversely correlated with lung function when both outpatients and inpatients with CF were considered, which supports the notion that extracellular HMGB1 levels are correlated with overall disease activity and are particularly sensitive to the presence of acute lung inflammation. The potential indicators of CF APE are an area of significant interest because they may be useful biomarkers for novel CF therapeutics and assist in the clinical management of subjects with CF (47
). The potential to combine the detection of upstream mediators, such as HMGB1, IL-8, and other proteases indicative of acute inflammation, with downstream events, such as the liberation of PGP, might allow the monitoring of both early and late pathways underlying lung inflammation and improve the characterization of CF APE beyond typical clinical criteria. The potential of HMGB1 to serve as a disease biomarker deserves further attention (such as evaluation of paired samples before and after therapeutic intervention) to confirm our initial observations and assess response to systemic antimicrobial therapy.
Inhibitors of HMGB1 are currently being developed for application in sepsis and other inflammatory diseases. In CF, an intervention that blocks the dysregulated activation of the innate immune system could potentially ameliorate excessive chronic neutrophilic inflammation that contributes to disease progression (18
). Elevated HMGB1 levels in the Scnn1b
-Tg mouse model, without acquisition of P. aeruginosa
, suggests its importance in chronic inflammation that is due to mucus stasis, possibly precipitated by the presence of necrotic cellular debris detectible in the airways of these mice (21
). Inhibition of inflammatory mediators that contribute to the CF inflammatory phenotype before the onset of bacterial colonization may be a more suitable therapeutic target than conventional cytokines and other mediators that become elevated primarily after the onset of chronic infection, when immune suppression could carry additional risks (50
). If inhibition of this pathway could reduce infection-independent inflammation, inhibitors of HMGB1 may have a more favorable toxicity profile, thus avoiding the potential worsening of infection associated with potent inhibition of proinflammatory (and antiinfective) pathways, as reported with LTB4 antagonists (51
Although P. aeruginosa
infection is not required, as evidenced by chemotaxis in both CF sputum and murine BALF sample (), these data do not rule out a modulatory role for bacterial infection on the activity of HMGB1-mediated chemotaxis. Additional studies are needed that examine the role of P. aeruginosa
infection and resultant HMGB1 release, to better define this pathway in CF inflammation. Recent reports by Tian and colleagues (53
) and Rouhiainen and colleagues (54
) indicate that immune activation and cytokine release induced by HMGB1 is significantly enhanced after tight binding to bacterial DNA, prokaryotic lipids, and other bacterial substances that are present in high quantities in infected and colonized CF sputum (55
). Not surprisingly, whereas blocking HMGB1 signaling caused a significant reduction in chemotaxis, our in vitro
observations indicate that this is not the only pathway relevant to neutrophil influx (~75% of neutrophil influx remained unabated) and may indicate the importance of other inflammatory mediators such as IL-8 or PGP in these specimens.
In summary, our study shows that HMGB1 is elevated in subjects with CF and in an animal model of the disease, that it is directly chemotactic for neutrophils through a CXCR-dependent mechanism, and that HMGB1 activity can be blocked by antibody binding in vitro. HMGB1 also induces collagen matrix degradation, resulting in a positive feedback loop that is expected to perpetuate and propagate neutrophilic inflammation through liberation of the proinflammatory polypeptide PGP. HMGB1 appears to be an important inflammatory modulator in airway secretions of subjects with CF and a murine model of CF inflammation. Studies using neutralizing anti-HMGB1 antibodies or other antagonists are warranted to confirm its pathogenic role in sterile airway inflammation in Scnn1b-Tg mice. If confirmed, neutralization of HMGB1 and/or PGP may represent potential therapeutic targets to treat the persistent chronic inflammation that is prominent in CF.