Hypercapnic Conditions After Experimental Blunt Chest Trauma Increase Efferocytosis of Alveolar Macrophages and Reduce Local Inflammation.

Blunt chest trauma induces severe local and systemic inflammatory alterations and an accumulation of apoptotic polymorphonuclear granulocytes (aPMN) in the lungs, frequently followed by bacterial infection. Alveolar macrophages (AM) represent one of the main actors for their clearance. However, little is known regarding regulatory and influencing factors of AM efferocytic and phagocytic activities. In this context, we investigated the influence of impaired gas exchange on AM activity.Male rats underwent blunt chest trauma or sham procedure and aPMN or Escherichia coli (E. coli) were instilled. Subsequently, the efferocytic and phagocytic activities were assessed by analyzing AM obtained from bronchoalveolar lavage fluids at three time points. To determine whether efferocytic and phagocytic activities of AM are affected by shifting gas concentrations, AM were subjected in vitro to hypoxic and hypercapnic conditions.Trauma significantly upregulated the capacity of AM to ingest E. coli starting 24 h after trauma, whereas the aPMN uptake rate remained virtually unchanged. In vitro, AM reacted to hypercapnic conditions by enhanced efferocytosis associated with increased release of anti-inflammatory cytokines. Additionally, phagocytosis and the pro-inflammatory reaction of AM after trauma appeared to be impaired. In contrast, hypoxic conditions displayed no regulatory effect on AM.In conclusion, blunt chest trauma enhances phagocytic activity of AM. On the other hand, hypercapnic conditions in the lungs may significantly contribute to the clearance of aPMN. The application of CO2 in clinical settings must be properly assessed, with the benefits of CO2 balanced against the detrimental effects of impaired bacterial clearance.

Role of alveolar macrophages in the regulation of local and systemic inflammation after lung contusion.

BACKGROUND: Blunt chest trauma is an injury that enhances the morbidity and mortality rate, particularly in the context of polytrauma. Our previous studies showed local and systemic inflammatory alterations after blunt chest trauma in mice. This study was designed to determine whether alveolar macrophages (AMΦ) have an alleviative role in this posttraumatic inflammation. METHODS: AMΦ of male C3H/HeN mice were depleted by instillation of clodronate liposomes into the lung before blunt chest trauma induced by a single blast wave. In bronchoalveolar lavage, lung homogenates, plasma, and cell culture supernatants of Kupffer cells, peripheral blood mononuclear cells, splenic macrophages, and splenocytes isolated 2 hours or 24 hours after chest trauma mediator concentrations were determined by multiplex assay or enzyme-linked immunosorbent assay. RESULTS: In bronchoalveolar lavage, AMΦ depletion led to increased monocyte chemoattractant protein 1 and regulated and normal T cell expressed and secreted (RANTES) concentrations as well as an attenuated increase of interleukin 6 concentrations after chest trauma. Bronchoalveolar lavage keratinocyte-derived chemokine concentrations increased in nontraumatized but AMΦ-depleted animals with no further change after chest trauma. Cytokine concentrations in lung homogenates were altered in the same way as in bronchoalveolar lavage early after trauma. In the plasma of AMΦ-depleted animals, interleukin 6 concentrations were slightly decreased after chest trauma. Depletion of AMΦ abrogated the trauma-induced decrease of Kupffer cell chemokine release. Cytokine concentrations of blood monocytes, splenic macrophages, and splenocyte supernatants were not influenced by AMΦ depletion. CONCLUSION: These depletion experiments show that AMΦ ameliorate the inflammatory response after blunt chest trauma. Taken together, this study gives relevant insights into the regulative role of AMΦ during the local and systemic inflammation after lung contusion.

Inhaled hydrogen sulfide induces suspended animation, but does not alter the inflammatory response after blunt chest trauma.

The treatment of acute lung injury and septic complications after blunt chest trauma remains a challenge. Inhaled hydrogen sulfide (H2S) may cause a hibernation-like metabolic state, which refers to an attenuated systemic inflammatory response. Therefore, we tested the hypothesis that inhaled H2S-induced suspended animation may attenuate the inflammation after pulmonary contusion. Male Sprague-Dawley rats were subjected to blunt chest trauma (blast wave) or sham procedure and subsequently exposed to a continuous flow of H2S (100 ppm) or control gas for 6 h. Body temperature and activity were measured by an implanted transmitter. At 6, 24, or 48 h after trauma, animals were killed, and the cellular contents of bronchoalveolar lavage (BAL) as well as cytokine concentrations in BAL, plasma, and culture supernatants of blood mononuclear cells, Kupffer cells, splenic macrophages, and splenocytes were determined. Hydrogen sulfide inhalation caused a significant reduction in body temperature and activity. The trauma-induced increase in alveolar macrophage counts was abrogated 48 h after trauma when animals received H2S, whereas the trauma-induced increase in neutrophil counts was unaltered. Furthermore, H2S inhalation partially attenuated the mediator release in BAL and culture supernatants of Kupffer cells as well as splenic cells; it altered plasma cytokine concentrations but did not affect the trauma-induced changes in mononuclear cell culture supernatants. These findings indicate that inhaled H2S induced a reduced metabolic expenditure and partially attenuated inflammation after trauma. Nevertheless, in contrast to hypoxic- or pathogen-induced lung injury, H2S treatment appears to have no protective effect after blunt chest trauma.

Alveolar macrophage phagocytosis is enhanced after blunt chest trauma and alters the posttraumatic mediator release.

Blunt chest trauma is known to induce a pulmonary invasion of short-lived polymorphonuclear neutrophils and apoptosis of alveolar epithelial type 2 (AT2) cells. Apoptotic cells are removed by alveolar macrophages (AMΦ). We hypothesized that chest trauma alters the phagocytic response of AMΦ as well as the mediator release of AMΦ during phagocytosis. To study this, male Sprague-Dawley rats were subjected to blunt chest trauma. Phagocytosis assays were performed in AMΦ isolated 2 or 24 h after trauma with apoptotic cells or opsonized beads. Phagocytosis of apoptotic AT2 cells by unstimulated AMΦ was significantly increased 2 h after trauma. At 24 h, AMΦ from traumatized animals, stimulated with phorbol-12-myristate-13-acetate, ingested significantly more apoptotic polymorphonuclear neutrophils than AMΦ from sham animals. Alveolar macrophages after trauma released significantly higher levels of tumor necrosis factor α, macrophage inflammatory protein 1α, and cytokine-induced neutrophil chemoattractant 1 when they incorporated latex beads, but significantly lower levels of interleukin 1ß and macrophage inflammatory protein 1α when they ingested apoptotic cells. In vivo, phagocytosis of intratracheally instilled latex beads was decreased in traumatized rats. The bronchoalveolar lavage concentrations of the phagocytosis-supporting surfactant proteins A and D after blunt chest trauma were slightly decreased, whereas surfactant protein D mRNA expression in AT2 cells was significantly increased after 2 h. These findings indicate that chest trauma augments the phagocytosis of apoptotic cells by AMΦ. Phagocytosis of opsonized beads enhances and ingestion of apoptotic cells downregulates the immunologic response following lung contusion. Our data emphasize the important role of phagocytosis during posttraumatic inflammation after lung contusion.

Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung contusion in mice.

BACKGROUND: When used as a pretreatment, hydrogen sulfide (H2S) either attenuated or aggravated lung injury. Therefore, we tested the hypothesis whether posttreatment intravenous Na2S (sulfide) may attenuate lung injury. METHODS: After blast wave blunt chest trauma or sham procedure, anesthetized and instrumented mice received continuous intravenous sulfide or vehicle while being kept at 37°C or 32°C core temperature. After 4 hours of pressure-controlled, thoracopulmonary compliance-titrated, lung-protective mechanical ventilation, blood and tissue were harvested for cytokine concentrations, heme oxygenase-1, IκBα, Bcl-Xl, and pBad expression (western blotting), nuclear factor-κB activation (electrophoretic mobility shift assay), and activated caspase-3, cystathionine-ß synthase and cystathionine-γ lyase (immunohistochemistry). RESULTS: Hypothermia caused marked bradycardia and metabolic acidosis unaltered by sulfide. Chest trauma impaired thoracopulmonary compliance and arterial Po2, again without sulfide effect. Cytokine levels showed inconsistent response. Sulfide increased nuclear factor-κB activation during normothermia, but this effect was blunted during hypothermia. While histologic lung injury was variable, both sulfide and hypothermia attenuated the trauma-related increase in heme oxygenase-1 expression and activated caspase-3 staining, which coincided with increased Bad phosphorylation and Bcl-Xl expression. Sulfide and hypothermia also attenuated the trauma-induced cystathionine-ß synthase and cystathionine-γ lyase expression. CONCLUSIONS: Posttreatment sulfide infusion after blunt chest trauma did not affect the impaired lung mechanics and gas exchange but attenuated stress protein expression and apoptotic cell death. This protective effect was amplified by moderate hypothermia. The simultaneous reduction in cystathionine-ß synthase and cystathionine-γ lyase expression supports the role of H2S-generating enzymes as an adaptive response during stress states.

Altered expression of Fas receptor on alveolar macrophages and inflammatory effects of soluble Fas ligand following blunt chest trauma.

Blunt chest trauma impairs the outcome of multiply-injured patients. Lung contusion induces inflammatory alterations and Fas-dependent apoptosis of alveolar type 2 epithelial (AT2) cells has been described. The Fas/Fas ligand (FasL) system seems to exhibit a proinflammatory potential. We aimed to elucidate the involvement of the Fas/FasL system in the inflammatory response after lung contusion. Chest trauma was induced in male rats by a pressure wave. Soluble FasL concentrations were determined in bronchoalveolar lavage fluids and alveolar macrophage (AMΦ) supernatants. Alveolar macrophages and AT2 cells were isolated to determine the surface expression (FACS) of Fas/FasL, the mRNA expression (reverse transcriptase-polymerase chain reaction) of Fas, FasL, TNF-α, IL-6, and IL-10 and to measure the release of IL-6 and IL-10 after culture with or without stimulation with FasL. After chest trauma, FasL concentration was increased in bronchoalveolar lavage fluid, and AMΦ supernatants and Fas and FasL protein were downregulated on AMΦs and unchanged on AT2 cells. The mRNA expression of Fas was increased in AMΦs and AT2 cells and that of FasL only in AMΦs isolated after lung contusion. Fas ligand stimulation further enhanced IL-6 and suppressed IL-10 release in AMΦs after trauma.The results indicate that the Fas/FasL system is activated after chest trauma, and FasL is associated with the inflammatory response after lung contusion. The proinflammatory response of AMΦs is enhanced by FasL stimulation. Both AMΦs and AT2 cells seem to contribute to the mediator release after lung contusion. These results confirm the importance of the Fas/FasL system in the inflammatory response after chest trauma.

Inflammatory effects of hypothermia and inhaled H2S during resuscitated, hyperdynamic murine septic shock.

Inhaling hydrogen sulfide (H2S) reduced energy expenditure resulting in hypothermia. Because the inflammatory effects of either hypothermia alone or H2S per se still are a matter of debate, we tested the hypothesis whether inhaled H2S amplifies the hypothermia-related modulation of the inflammatory response. Fifteen hours after cecal ligation and puncture or sham laparotomy, anesthetized and mechanically ventilated normothermic and hypothermic mice (core temperature kept at 38°C and 27°C, respectively) received either 100 ppm H2S or vehicle. In the sham-operated animals, inhaled H2S and hypothermia alone comparably reduced the plasma chemokine and IL-6 levels, but combining hypothermia and inhaled H2S had no additional effect. The lung tissue cytokine and chemokine patterns revealed a similar response. During sepsis, inhaled H2S reduced the blood cytokine concentrations only, without effects on the plasma chemokine or the lung tissue levels. Again, inhaled H2S had no major additional effect during hypothermia. With or without sepsis, inhaled H2S and hypothermia alone comparably reduced the lung tissue heme oxygenase 1 expression, whereas inhaled H2S had no additional effect during hypothermia. Lung tissue nuclear transcription factor κB activation was reduced by combining H2S with hypothermia in the sham-operated animals, whereas it was increased by inhaled H2S during sepsis. Hypothermia amplified this response. Hence, during anesthesia and mechanical ventilation, inhaled H2S exerted anti-inflammatory effects, which were, however, not amplified by adding deliberate hypothermia. Sepsis attenuated these anti-inflammatory effects of inhaled H2S, which were at least in part independent of the nuclear transcription factor κB pathway.

Inflammatory alterations in a novel combination model of blunt chest trauma and hemorrhagic shock.

BACKGROUND: Chest trauma frequently occurs in severely injured patients and is often associated with hemorrhagic shock. Immune dysfunction contributes to the adverse outcome of multiple injuries. The aims of this study were to establish a combined model of lung contusion and hemorrhage and to evaluate the cardiopulmonary and immunologic response. METHODS: Male mice were subjected to sham procedure, chest trauma, hemorrhage (35 mm Hg±5 mm Hg, 60 minutes), or the combination. Respiratory rate, heart rate, and blood pressure were monitored. Plasma, Kupffer cells, blood monocytes, splenocytes, and splenic macrophages were isolated after 20 hours. Tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, 10, 12, 18, and macrophage inflammatory protein-2 levels in plasma and culture supernatants were determined. RESULTS: Heart rate and blood pressure dropped in all groups, and after chest trauma and the double hit, these values remained reduced until the end of observation. Blood pressure was lower after the double hit than after the single hits. Plasma and Kupffer cell TNF-α concentrations were increased after lung contusion but not further enhanced by subsequent hemorrhage. Peripheral blood mononuclear cell (PBMC) TNF-α and IL-6 release were suppressed after the combined insult. IL-18 concentrations were increased in PBMC supernatants after chest trauma and in splenic macrophage supernatants of all groups. CONCLUSIONS: Although physiologic readouts were selectively altered in response to the single or double hits, the combination did not uniformly augment the changes in inflammation. Our results suggest that the leading insult regarding the immunologic response is lung contusion, supporting the concept that lung contusion represents an important prognostic factor in multiple injuries.

Is the function of alveolar macrophages altered following blunt chest trauma?

PURPOSE: The purpose of this study was to characterize the local pulmonary inflammatory environment and to elucidate alterations of alveolar macrophage (AMØ) functions after blunt chest trauma. METHODS: Wistar rats were subjected to blunt chest trauma. AMØ were isolated, stimulated, and cultured. Bronchoalveolar lavage (BAL) was collected. Cytokines/chemokines were quantified in the BAL and in AMØ supernatants via ELISA. AMØ phagocytic and chemotactic activity and respiratory burst capacity were assessed. RESULTS: Following chest trauma, a significant increase of IL-1ß (at 6 and 24 h) and IL-6 (at 24 h) in BAL was observed, whereas IL-10 and TNF-α concentrations were not altered. MIP-2 and CINC were substantially increased as early as 6 h and PGE2 early at 10 min, whereas BAL MCP-1 was not elevated until 24 h after trauma. MIP-2 release by AMØ isolated form trauma animals was markedly increased as early as 10 min after injury. IL-1ß and IL-10 exhibited a late increase at 24 h. AMØ TNF-α release was increased at 6 h. At 6 or 24 h, AMØ from trauma animals incorporated significantly more opsonized latex beads than their sham controls, and their chemotactic activity was substantially enhanced at 24 h. AMØ oxidative burst capacity remained largely unchanged. CONCLUSIONS: Already very early after chest trauma, inflammatory mediators are present in the intraalveolar compartment. Additionally, AMØ are primed to release cytokines and chemokines. Blunt chest trauma also changes the phagocytic and chemotactic activity of AMØ. These functional changes of AMØ might enable them to better ward off potential pathogens in the course after trauma.

Blunt chest trauma induces mediator-dependent monocyte migration to the lung.

OBJECTIVE: This study was designed to determine whether lung contusion induces an increased pulmonary recruitment of monocytes as a source of alveolar macrophages and which mediators are involved. SETTING AND DESIGN: Prospective animal study. SUBJECTS AND INTERVENTIONS: Male Sprague-Dawley rats were subjected to chest trauma by a single blast wave. MEASUREMENTS: Chemokine concentrations in bronchoalveolar lavage fluids and supernatants of alveolar macrophages, chemokine and chemokine receptor mRNA expressions in monocytes, pulmonary interstitial macrophages, and alveolar macrophages isolated after trauma or sham procedure were evaluated. Immigration of monocytes was determined by staining alveolar macrophages with the fluorescent marker PKH26 before chest trauma. Chemotaxis of naïve monocytes in response to bronchoalveolar lavage or supernatants from alveolar macrophages isolated after trauma or sham procedure and the migratory response of monocytes isolated after trauma/sham to recombinant chemokines were measured. MAIN RESULTS: Chemokine levels in bronchoalveolar lavage and alveolar macrophage supernatants and the percentage of monocytes migrated to the lungs were increased after chest trauma. Lung contusion enhanced the mRNA expression for CCR2 in monocytes and interstitial macrophages and for monocyte chemotactic protein-1 in alveolar macrophages. Migration of naïve monocytes vs. bronchoalveolar lavage or alveolar macrophage supernatants from traumatized animals was increased when compared with samples from shams. Monocytes isolated 2 hrs after trauma showed a reduced migration to CINC-1 or monocyte chemotactic protein-1 compared with sham. CONCLUSIONS: Alveolar macrophages seem to contribute to increased chemokine concentrations in alveoli of animals subjected to blunt chest trauma. Mediators released by alveolar macrophage are potent stimuli for monocyte migration. Monocytes alter their chemokine receptor expression and are recruited to the lungs.

Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils.

Alveolar type 2 (AT-2) cell apoptosis is an important mechanism during lung inflammation, lung injury, and regeneration. Blunt chest trauma has been shown to activate inflammatory cells such as alveolar macrophages (AMs) or neutrophils (polymorphonuclear granulocytes [PMNs]), resulting in an inflammatory response. The present study was performed to determine the capacity of different components/cells of the alveolar compartment (AMs, PMNs, or bronchoalveolar lavage [BAL] fluids) to induce apoptosis in AT-2 cells following blunt chest trauma. To study this, male Sprague-Dawley rats were subjected to either sham procedure or blunt chest trauma induced by a single blast wave. Various time points after injury (6 h to 7 d), the lungs were analyzed by immunohistochemistry, for AT-2 cells, or with antibodies directed against caspase 3, caspase 8, Fas, Fas ligand (FasL), BAX, and BCL-2. Bronchoalveolar lavage concentrations of TNF-alpha, IL-1beta, and soluble FasL were determined by enzyme-linked immunosorbent assay. Furthermore, cultures of AT-2 cells isolated from healthy rats were incubated with supernatants of AMs, PMNs, or BAL fluids obtained from either trauma or sham-operated animals in the presence or absence of oxidative stress. Annexin V staining or TUNEL (terminal deoxynucleotidyl transferase) assay was used to detect apoptotic AT-2 cells. Histological evaluation revealed that the total number of AT-2 cells was significantly reduced at 48 h following trauma. Fas, FasL, active caspase 8, and active caspase 3 were markedly up-regulated in AT-2 cells after chest trauma. BAX and BCL-2 did not show any significant changes between sham and trauma. IL-1beta, but not TNF-alpha, levels were markedly increased at 24 h after the injury, and soluble FasL concentrations were significantly enhanced at 6, 12, 24, and 48 h after the insult. Apoptosis of AT-2 cells incubated with supernatants from cultured AMs, isolated at 48 h following chest trauma was markedly increased when compared with shams. In contrast, no apoptosis was induced in AT-2 cells incubated with supernatants of activated PMNs or BAL fluids of traumatized animals. In summary, blunt chest trauma induced apoptosis in AT-2 cells, possibly involving the extrinsic death receptor pathway. Furthermore, mediators released by AMs appeared to be involved in the induction of AT-2 cell apoptosis.

Cardiopulmonary, histological, and inflammatory alterations after lung contusion in a novel mouse model of blunt chest trauma.

Severe blunt chest trauma remains an important injury with high morbidity and mortality. However, the associated immunological alterations are poorly understood. Existing big animal models require large-scale settings, are often too expensive, and research products for immunological studies are limited. In this study we aimed to establish a new model of blunt, isolated and bilateral chest trauma in mice and to characterize its effects on physiological and inflammatory variables. Male C3H/HeN mice (n = 9-10/group) were anesthetized and a femoral artery was catheterized. The animals were subjected to trauma or sham procedure and monitored for 180 min. Blunt chest trauma was induced by a blast wave focused on the thorax. Trauma intensity was optimized by varying the exposure distance. Blood pressure, heart rate, respiratory rate, arterial blood gases and plasma cytokine levels were measured. Macroscopic and microscopic examinations were performed. In addition, outcome was evaluated in a 10-day survival study. Chest trauma caused a drop (P < 0.05) in blood pressure and heart rate, which partly recovered. Blood gases revealed hypoxemia and hypercarbia (P < 0.05) 180 min after trauma. There was marked damage to the lungs but none to abdominal organs. Histologically, the characteristic signs of a bilateral lung contusion with alveolar and intrabronchial hemorrhage were found. Plasma interleukin-6 and tumor necrosis factor alpha were considerably increased after 180 min. Blunt chest trauma resulted in an early mortality of 10% without subsequent death. On the basis of these findings, this novel mouse model of blunt chest trauma appears suitable for detailed studies on immunological effects of lung contusion.