ORIGINAL ARTICLE
Intraalveolar TNF-α in Combined Burn and Inhalation Injury Compared With Intraalveolar Interleukin-6 Jan-Philipp Stromps, MD,* Paul Fuchs, PhD,*† Erhan Demir, MD,*† Gerrit Grieb, MD,* Kai Reuber, MD,* Norbert Pallua, PhD*
The objective of this study was to evaluate the role of intraalveolar tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a combination of skin burn and smoke inhalation injuries because this combined trauma is associated with an increased morbidity and mortality compared with either of these traumas alone. We used a standardized small animal model (rats n = 84) to investigate the early intraalveolar excretion of TNFα during the first one, three, and six hours after a singular skin burn injury, singular smoke inhalation injury, and a combination involving both the traumas. The data were compared with the data from control rats that only received preparation and mechanical ventilation. The TNF-α serum levels and intraalveolar IL-6 concentrations were also measured. One hour after trauma, there was a significant difference in the TNF-α concentration between the controls and both the singular traumas (control vs burn P < .0444 and control vs smoke P < .005) and between the inhalation injury and the combined trauma (smoke vs burn + smoke P < .0084). After three and six hours, no significant differences among the groups were observed. Compared with the controls, both the singular skin burn and smoke inhalation injuries led to increased intraalveolar TNF-α excretion, whereas the combined trauma showed the least intraalveolar TNF-α levels at three and six hours post-trauma. These findings differed from the serum TNFα levels. Compared with the IL-6 levels, we observed a negative correlation within the intraalveolar cytokine concentrations after one hour (r = −.809), three hours (r = −.627), and six hours (r = −.746). This study confirms the importance of the intraalveolar cytokine reaction in the early posttraumatic stage after a combined burn and inhalation injury. The differences between the combined and singular traumas indicate that TNFα plays a role in the immunologic hyporesponsiveness of the lung and therefore in the systemic pathophysiological pathway, that often leads to patient mortality. In addition, an inverse correlation between TNF-α and IL-6, both classical markers of inflammation, in the intraalveolar space was observed. (J Burn Care Res 2015;36:e55–e61)
Although burn therapy and management have continuously developed over recent decades, the combination of comprehensive skin burn and smoke inhalation injuries is still associated with an increased morbidity and mortality compared with either of these traumas alone.1–3 The immunologic response From the *Department of Plastic Surgery, Hand Surgery, Burn Center University Hospital RWTH Aachen, Pauwelsstrasse, Germany; and †Department of Plastic Surgery, Hand Surgery, Burn Center University of Witten/Herdecke, Cologne-Merheim Medical Center (CMMC), Germany. Address Correspondence to Jan-Philipp Stromps, MD, Department of Plastic Surgery, Hand Surgery, Burn Center University Hospital RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. Copyright © 2015 by the American Burn Association 1559-047X/2015 DOI: 10.1097/BCR.0000000000000108
following these traumas remains incompletely understood. However, many studies have confirmed the importance of the early intraalveolar cytokine reactions and the proposed consequent immune hyporesponsiveness.4–6 Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are known to be important initiators of the inflammatory cascade in the lungs after inhalation injury and are also involved in burn injuries.7,8 In the lungs, this cascade often leads to endothelial leakage, pulmonary edema, surfactant dysfunction, and hyaline membrane formation. The role of TNFα in these processes has recently been reevaluated, because it has also been reported of having protective functions in the lungs.9–12 During the early response to trauma, TNF-α is primarily expressed by e55
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alveolar macrophages in the early stage13 and is later expressed by polymorphonuclear leukocytes.14 Apart from these local cytokine dependent reactions the systemic effects of a combined burn and inhalation injury can lead to acute respiratory distress syndrome and multi organ dysfunction syndrome. Previous studies have shown that the mortality after combined burn and inhalation injury is occasionally correlated with the serum concentrations of TNF-α and IL-6.15,16 In this context, previous studies have shown that the intraalveolar excretion of those cytokines occurs earlier than can be measured in serum samples.4 Therefore, examining the bronchoalveolar lavage fluid (BALF) is important to gain a better understanding of the early post-trauma stage and to help develop new therapeutic strategies.
Methods Animals To evaluate early intraalveolar TNF-α and IL-6 excretion, we used our standardized small animal model, which has been previously described in detail.17 This study was approved by the regulatory animal committee of the administration of the state of North-Rhine-Westphalia, Germany and the RWTH University Aachen Animal Care Department (protocol number 1142). Care and handling of the animals were in accordance with the European Community Guidelines. The studies were performed in adult Sprague-Dawley rats (n = 84; body weight 300 ± 30 g; Janvier Labs, Saint Berthevin Cedex, France). To avoid gender specific effects, only female rats were used. All the animals were caged in pairs at 22°C and approximately 40% relative humidity with a 12/12 light/dark cycle. The rats were fed standard rat chow and water ad libitum. The animals were randomly divided into four main groups (n = 21) that were then further divided into three sub groups (n = 7) according to the measured time post-trauma (1, 3, and 6 hours). An overview of the different experimental groups is shown in Figure 1. A heating lamp was used to maintain body temperature within a normal range during the experiments. The animals were killed at the end of the experiments with an intravenous overdose of pentobarbital (200 mg/kg, Nembutal®; Abbott Labs, Green Oak, IL).
Controls A group of animals served as the controls (control, n = 21) to estimate the impact of the preparation and mechanical ventilation on the studied parameters.
Figure 1. Random division of the 84 animals into the different main groups and subgroups.
The animals were initially anesthetized by inhaling isoflurane (Forene®; Abbott Labs) and anaesthesia was maintained using intraperitoneal ketamine and medetomidine injections (ratio 2:1, 0.3 mg/kg, Ketamin®; Sanofi-CEVA GmbH, Essen, Germany; and Domitor®; Pfizer GmbH, Berlin, Germany). During the microsurgical preparation, the right jugular vein trachea and right iliac artery were exposed. The artery and vein were catheterized (Vasculon® 0.6 × 19 mm, Becton Dickinson, Franklin Lakes, NJ) for drawing blood samples and for killing at the end of the experiments. A tracheotomy was performed by inserting a sterile, custom made, metal cannula into the trachea. The animals were then connected to the ventilator (Servo 900C; Siemens, Erlangen, Germany) and mechanically ventilated using the settings as shown in Table 1. For baseline resuscitation, the controls received 0.5 ml/hour of lactated Ringer’s solution intravenously.
Burn In addition to the treatment that the controls received, 30% of the TBSATBSA of the animals Table 1. Ventilator settings in pressure-controlled mode FiO2 Frequency PEEP Pmax
100% 25 min−1 8 cm H2O 25 cm H2O
Journal of Burn Care & Research Volume 36, Number 2
in the Burn group (n = 21) was shaved according to the calculations performed using the modified Meeh formula.18 After disinfection, the shaved areas received a standardized third degree contact burn using a custom made burn stamp (Technical Department, University Hospital Aachen, Aachen, Germany) at a temperature of 120°C for 15 seconds under low pressure. Post-trauma, fluid resuscitation therapy with lactated Ringer’s solution was performed according to the modified Parkland formula (2 ml/hour for a 300 g rat).19
Inhalation Injury The rats in this group (smoke group, n = 21) received a smoke inhalation injury in addition to the control treatment. For smoke production, we used a standardized smoldering unit (according to DIN 53436; EPA-Aachen GmbH, Aachen, Germany) to combust a mixture of beech wood and polyvinylchloride granules (ratio of 8:1). After cooling, the smoke was administered directly to the animals for one minute through a connection between the smoldering unit and the ventilator. The smoke inhalation injury was confirmed during preliminary experiments, by measuring the hydrochloride, carbon monoxide, and carbon dioxide concentrations of the produced smoke (Accuro® Gasspürpumpe; Dräger Safety, Lübeck, Germany) and by performing a blood gas analysis 15 minutes post-trauma; this analysis revealed increased carbon monoxide-Hemoglobin levels and negative base excess within the “Smoke” and “Burn + Smoke” groups compared with the “Control” group (radiometer ABL510; Radiometer, Bronshoj, Denmark). The arterial carbon monoxide-Hb levels never transcended 20% in any animal and decreased to the normal range one hour post-trauma. The smoke group received fluid resuscitation therapy with lactated Ringer’s solution (0.5 ml/hour) post-trauma.
Combined Trauma In the combined trauma group (burn + smoke, n = 21) the animals first received a 30% TBSA third degree skin burn as described above; after 15 minutes the animals received the smoke inhalation injury. This group of animals received the same Parkland formula fluid resuscitation as the “Burn” group (2 ml/hour).
Bronchoalveolar Lavage After killing the animals, they were disconnected from the ventilator, and bronchoalveolar lavage (30 ml/kg) was performed over the sterile tracheostoma five
Stromps et al e57
times using calcium chloride saline (1.5 mmol/L). The BALF was centrifuged (400 × g for 10 minutes) to remove the cell debris. The samples were labeled and stored at −70°C for later analysis.
Serum Samples The blood was drawn into heparinzed syringes (Omnifix® 1 ml; B. Braun GmbH, Melsungen, Germany) through the arterial line at one, three, and six hours post-trauma and immediately centrifuged for 10 minutes. The serum was collected, and the pellet containing the cellular elements was discarded. All the serum samples were labeled and stored at −70°C until analysis.
Assays The TNF-α and IL-6 levels in the BALF and serum samples were measured with rat specific enzyme linked immunosorbent assay kits (ELISA-Duo-Set No DY510, No DY506; R&D Systems, Minneapolis, MN). The protocols provided by the manufacturer were strictly followed. The measured cytokine concentrations were normalized to the total protein concentration.
Statistical Analysis The differences among the study groups were analyzed by unpaired t-testing and one way analysis of variance (ANOVA). The significance was defined as P value less than .05. All of the data are expressed as the mean ± the standard error of the mean (SEM). The correlation between cytokines was assessed using the nonparametric Spearman’s rank correlation coefficient. All the analyses were performed using Prism® 5 for Macintosh (GraphPad Software Inc., La Jolla, CA).
Results The intraalveolar TNF-α levels for each group at the different time points (one, three, and six hours posttrauma) are summarized in Figure 2. One hour after trauma, there was a significant difference in the intraalveolar TNF-α levels between the controls and both singular traumas (control 197 ± 31 pg/ml vs burn 362 ± 65 pg/ml, P < .0444; and control vs smoke 367 ± 13 pg/ml, P < .005) and between the inhalation injury and the combined trauma (smoke 367 ± 13 pg/ml vs burn+smoke 221 ± 41 pg/ml, P < .0084). At three and six hours post-trauma, there were no statistically significant differences in the intraalveolar TNF-α levels between the groups.
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serum levels of the combined trauma group (burn + smoke) were below the detection limit of the assay; however, significant differences were observed between the control and smoke groups (112 ± 16 vs. 28 ± 6 pg/ml, P < .0007) and the burn and smoke groups (187 ± 32 vs 28 ± 6 pg/ml, P < .0006). Overall, we observed a slight decrease in the serum TNFα excretion in the trauma groups over time, and peak TNF-α levels were observed in the control animals at three hours(Figures 3 and 4). Figure 2. Intraalveolar tumor necrosis factor-α (TNF-α) levels (pg/ml) ± SEM in the different groups at the indicated hours post-trauma (*P < .05).
Our previously published results, showed that the intraalveolar IL-6 levels were reduced when associated with either the burn or inhalation injury alone but increased when the traumas were combined;17 in this study, we found an inverse correlation between the intraalveolar IL-6 and TNF-α levels. This inverse correlation was significant within the combined group after 1 (r = −.809), 3 (r = −.627), and 6 (r = −.746) hours (Figure 3). The serum TNF-α levels for each group at the different times (one, three, and six hours post-trauma) are summarized in Figure 4. After one hour, we found significant differences between some of the groups (control 161 ± 37 pg/ml vs burn 334 ± 47 pg/ml, P
Intraalveolar TNF-α in Combined Burn and Inhalation Injury Compared With Intraalveolar Interleukin-6 Jan-Philipp Stromps, MD,* Paul Fuchs, PhD,*† Erhan Demir, MD,*† Gerrit Grieb, MD,* Kai Reuber, MD,* Norbert Pallua, PhD*
The objective of this study was to evaluate the role of intraalveolar tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a combination of skin burn and smoke inhalation injuries because this combined trauma is associated with an increased morbidity and mortality compared with either of these traumas alone. We used a standardized small animal model (rats n = 84) to investigate the early intraalveolar excretion of TNFα during the first one, three, and six hours after a singular skin burn injury, singular smoke inhalation injury, and a combination involving both the traumas. The data were compared with the data from control rats that only received preparation and mechanical ventilation. The TNF-α serum levels and intraalveolar IL-6 concentrations were also measured. One hour after trauma, there was a significant difference in the TNF-α concentration between the controls and both the singular traumas (control vs burn P < .0444 and control vs smoke P < .005) and between the inhalation injury and the combined trauma (smoke vs burn + smoke P < .0084). After three and six hours, no significant differences among the groups were observed. Compared with the controls, both the singular skin burn and smoke inhalation injuries led to increased intraalveolar TNF-α excretion, whereas the combined trauma showed the least intraalveolar TNF-α levels at three and six hours post-trauma. These findings differed from the serum TNFα levels. Compared with the IL-6 levels, we observed a negative correlation within the intraalveolar cytokine concentrations after one hour (r = −.809), three hours (r = −.627), and six hours (r = −.746). This study confirms the importance of the intraalveolar cytokine reaction in the early posttraumatic stage after a combined burn and inhalation injury. The differences between the combined and singular traumas indicate that TNFα plays a role in the immunologic hyporesponsiveness of the lung and therefore in the systemic pathophysiological pathway, that often leads to patient mortality. In addition, an inverse correlation between TNF-α and IL-6, both classical markers of inflammation, in the intraalveolar space was observed. (J Burn Care Res 2015;36:e55–e61)
Although burn therapy and management have continuously developed over recent decades, the combination of comprehensive skin burn and smoke inhalation injuries is still associated with an increased morbidity and mortality compared with either of these traumas alone.1–3 The immunologic response From the *Department of Plastic Surgery, Hand Surgery, Burn Center University Hospital RWTH Aachen, Pauwelsstrasse, Germany; and †Department of Plastic Surgery, Hand Surgery, Burn Center University of Witten/Herdecke, Cologne-Merheim Medical Center (CMMC), Germany. Address Correspondence to Jan-Philipp Stromps, MD, Department of Plastic Surgery, Hand Surgery, Burn Center University Hospital RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. Copyright © 2015 by the American Burn Association 1559-047X/2015 DOI: 10.1097/BCR.0000000000000108
following these traumas remains incompletely understood. However, many studies have confirmed the importance of the early intraalveolar cytokine reactions and the proposed consequent immune hyporesponsiveness.4–6 Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are known to be important initiators of the inflammatory cascade in the lungs after inhalation injury and are also involved in burn injuries.7,8 In the lungs, this cascade often leads to endothelial leakage, pulmonary edema, surfactant dysfunction, and hyaline membrane formation. The role of TNFα in these processes has recently been reevaluated, because it has also been reported of having protective functions in the lungs.9–12 During the early response to trauma, TNF-α is primarily expressed by e55
Journal of Burn Care & Research March/April 2015
e56 Stromps et al
alveolar macrophages in the early stage13 and is later expressed by polymorphonuclear leukocytes.14 Apart from these local cytokine dependent reactions the systemic effects of a combined burn and inhalation injury can lead to acute respiratory distress syndrome and multi organ dysfunction syndrome. Previous studies have shown that the mortality after combined burn and inhalation injury is occasionally correlated with the serum concentrations of TNF-α and IL-6.15,16 In this context, previous studies have shown that the intraalveolar excretion of those cytokines occurs earlier than can be measured in serum samples.4 Therefore, examining the bronchoalveolar lavage fluid (BALF) is important to gain a better understanding of the early post-trauma stage and to help develop new therapeutic strategies.
Methods Animals To evaluate early intraalveolar TNF-α and IL-6 excretion, we used our standardized small animal model, which has been previously described in detail.17 This study was approved by the regulatory animal committee of the administration of the state of North-Rhine-Westphalia, Germany and the RWTH University Aachen Animal Care Department (protocol number 1142). Care and handling of the animals were in accordance with the European Community Guidelines. The studies were performed in adult Sprague-Dawley rats (n = 84; body weight 300 ± 30 g; Janvier Labs, Saint Berthevin Cedex, France). To avoid gender specific effects, only female rats were used. All the animals were caged in pairs at 22°C and approximately 40% relative humidity with a 12/12 light/dark cycle. The rats were fed standard rat chow and water ad libitum. The animals were randomly divided into four main groups (n = 21) that were then further divided into three sub groups (n = 7) according to the measured time post-trauma (1, 3, and 6 hours). An overview of the different experimental groups is shown in Figure 1. A heating lamp was used to maintain body temperature within a normal range during the experiments. The animals were killed at the end of the experiments with an intravenous overdose of pentobarbital (200 mg/kg, Nembutal®; Abbott Labs, Green Oak, IL).
Controls A group of animals served as the controls (control, n = 21) to estimate the impact of the preparation and mechanical ventilation on the studied parameters.
Figure 1. Random division of the 84 animals into the different main groups and subgroups.
The animals were initially anesthetized by inhaling isoflurane (Forene®; Abbott Labs) and anaesthesia was maintained using intraperitoneal ketamine and medetomidine injections (ratio 2:1, 0.3 mg/kg, Ketamin®; Sanofi-CEVA GmbH, Essen, Germany; and Domitor®; Pfizer GmbH, Berlin, Germany). During the microsurgical preparation, the right jugular vein trachea and right iliac artery were exposed. The artery and vein were catheterized (Vasculon® 0.6 × 19 mm, Becton Dickinson, Franklin Lakes, NJ) for drawing blood samples and for killing at the end of the experiments. A tracheotomy was performed by inserting a sterile, custom made, metal cannula into the trachea. The animals were then connected to the ventilator (Servo 900C; Siemens, Erlangen, Germany) and mechanically ventilated using the settings as shown in Table 1. For baseline resuscitation, the controls received 0.5 ml/hour of lactated Ringer’s solution intravenously.
Burn In addition to the treatment that the controls received, 30% of the TBSATBSA of the animals Table 1. Ventilator settings in pressure-controlled mode FiO2 Frequency PEEP Pmax
100% 25 min−1 8 cm H2O 25 cm H2O
Journal of Burn Care & Research Volume 36, Number 2
in the Burn group (n = 21) was shaved according to the calculations performed using the modified Meeh formula.18 After disinfection, the shaved areas received a standardized third degree contact burn using a custom made burn stamp (Technical Department, University Hospital Aachen, Aachen, Germany) at a temperature of 120°C for 15 seconds under low pressure. Post-trauma, fluid resuscitation therapy with lactated Ringer’s solution was performed according to the modified Parkland formula (2 ml/hour for a 300 g rat).19
Inhalation Injury The rats in this group (smoke group, n = 21) received a smoke inhalation injury in addition to the control treatment. For smoke production, we used a standardized smoldering unit (according to DIN 53436; EPA-Aachen GmbH, Aachen, Germany) to combust a mixture of beech wood and polyvinylchloride granules (ratio of 8:1). After cooling, the smoke was administered directly to the animals for one minute through a connection between the smoldering unit and the ventilator. The smoke inhalation injury was confirmed during preliminary experiments, by measuring the hydrochloride, carbon monoxide, and carbon dioxide concentrations of the produced smoke (Accuro® Gasspürpumpe; Dräger Safety, Lübeck, Germany) and by performing a blood gas analysis 15 minutes post-trauma; this analysis revealed increased carbon monoxide-Hemoglobin levels and negative base excess within the “Smoke” and “Burn + Smoke” groups compared with the “Control” group (radiometer ABL510; Radiometer, Bronshoj, Denmark). The arterial carbon monoxide-Hb levels never transcended 20% in any animal and decreased to the normal range one hour post-trauma. The smoke group received fluid resuscitation therapy with lactated Ringer’s solution (0.5 ml/hour) post-trauma.
Combined Trauma In the combined trauma group (burn + smoke, n = 21) the animals first received a 30% TBSA third degree skin burn as described above; after 15 minutes the animals received the smoke inhalation injury. This group of animals received the same Parkland formula fluid resuscitation as the “Burn” group (2 ml/hour).
Bronchoalveolar Lavage After killing the animals, they were disconnected from the ventilator, and bronchoalveolar lavage (30 ml/kg) was performed over the sterile tracheostoma five
Stromps et al e57
times using calcium chloride saline (1.5 mmol/L). The BALF was centrifuged (400 × g for 10 minutes) to remove the cell debris. The samples were labeled and stored at −70°C for later analysis.
Serum Samples The blood was drawn into heparinzed syringes (Omnifix® 1 ml; B. Braun GmbH, Melsungen, Germany) through the arterial line at one, three, and six hours post-trauma and immediately centrifuged for 10 minutes. The serum was collected, and the pellet containing the cellular elements was discarded. All the serum samples were labeled and stored at −70°C until analysis.
Assays The TNF-α and IL-6 levels in the BALF and serum samples were measured with rat specific enzyme linked immunosorbent assay kits (ELISA-Duo-Set No DY510, No DY506; R&D Systems, Minneapolis, MN). The protocols provided by the manufacturer were strictly followed. The measured cytokine concentrations were normalized to the total protein concentration.
Statistical Analysis The differences among the study groups were analyzed by unpaired t-testing and one way analysis of variance (ANOVA). The significance was defined as P value less than .05. All of the data are expressed as the mean ± the standard error of the mean (SEM). The correlation between cytokines was assessed using the nonparametric Spearman’s rank correlation coefficient. All the analyses were performed using Prism® 5 for Macintosh (GraphPad Software Inc., La Jolla, CA).
Results The intraalveolar TNF-α levels for each group at the different time points (one, three, and six hours posttrauma) are summarized in Figure 2. One hour after trauma, there was a significant difference in the intraalveolar TNF-α levels between the controls and both singular traumas (control 197 ± 31 pg/ml vs burn 362 ± 65 pg/ml, P < .0444; and control vs smoke 367 ± 13 pg/ml, P < .005) and between the inhalation injury and the combined trauma (smoke 367 ± 13 pg/ml vs burn+smoke 221 ± 41 pg/ml, P < .0084). At three and six hours post-trauma, there were no statistically significant differences in the intraalveolar TNF-α levels between the groups.
Journal of Burn Care & Research March/April 2015
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serum levels of the combined trauma group (burn + smoke) were below the detection limit of the assay; however, significant differences were observed between the control and smoke groups (112 ± 16 vs. 28 ± 6 pg/ml, P < .0007) and the burn and smoke groups (187 ± 32 vs 28 ± 6 pg/ml, P < .0006). Overall, we observed a slight decrease in the serum TNFα excretion in the trauma groups over time, and peak TNF-α levels were observed in the control animals at three hours(Figures 3 and 4). Figure 2. Intraalveolar tumor necrosis factor-α (TNF-α) levels (pg/ml) ± SEM in the different groups at the indicated hours post-trauma (*P < .05).
Our previously published results, showed that the intraalveolar IL-6 levels were reduced when associated with either the burn or inhalation injury alone but increased when the traumas were combined;17 in this study, we found an inverse correlation between the intraalveolar IL-6 and TNF-α levels. This inverse correlation was significant within the combined group after 1 (r = −.809), 3 (r = −.627), and 6 (r = −.746) hours (Figure 3). The serum TNF-α levels for each group at the different times (one, three, and six hours post-trauma) are summarized in Figure 4. After one hour, we found significant differences between some of the groups (control 161 ± 37 pg/ml vs burn 334 ± 47 pg/ml, P
