Several studies report immunomodulatory effects of endogenous IL-10 after trauma. The present study investigates the effect of inhalative IL-10 administration on systemic and pulmonary inflammation in hemorrhagic shock.
Male C57/BL6 mice (8 animals per group) were subjected to pressure-controlled hemorrhagic shock for 1.5 hrs followed by resuscitation and inhalative administration of either 50
Hemorrhagic shock initiates a systemic inflammatory response which is thought to be responsible for the development of ARDS and MOF [
This research protocol complied with the regulations regarding the care and use of experimental animals published by the NIH and was approved by the Institutional Animal Use and Care Committee of the RWTH Aachen University. Male C57/BL6 mice (Charles Rivers Laboratories, Germany), 6–10 weeks old and weighing 20–30 g, were used in the experiments. The animals were maintained in the Animal Research Center of the RWTH Aachen University with a 12:12 h light-dark cycle and free access to standard laboratory feed and water. Animals were anesthetized with inhaled isoflurane (Abbott Laboratories, Wiesbaden, Germany), 70 mg/kg i.p. pentobarbital (Merial GmbH, Hallbergmoos, Germany), and 0.5 mg/kg buprenorphine (Reckitt-Benckiser, Bergheim, Germany).
C57/BL6 mice were divided into four groups. In the Control group (
Animals were subjected to anesthesia as described above. A sterile technique was used to perform a left groin exploration, and the left femoral artery was cannulated with tapered polyethylene-10 tubing. The catheter was connected to a digital blood pressure monitor (TSE Systems, Bad Homburg, Germany) and the mean arterial pressure (MAP) was recorded. Pressure-controlled hemorrhagic shock was performed by withdrawing blood over a period of 15 min in a syringe with 0.07 mL of heparin (1000 USP units/mL) until a MAP of 35 mmHg was reached. Hemorrhagic shock was maintained for 1.5 hrs followed by resuscitation with shed blood and an equal volume of 0.9% saline. The catheter was removed, the artery ligated, and the skin incision closed. After a recovery phase of 4.5 hrs the animals were sacrificed.
The inhalative administration of PBS or IL-10 was performed using a MicroSprayer Aeroliser (Penn-Century, Philadelphia, Pa, USA) connected to a high-pressure syringe (FMJ-250, Penn-Century, Philadelphia, Pa, USA). The endotracheal intubation was carried out as described by Bivas-Benita et al. [
Following thoracotomy, cardiac blood was withdrawn under deep anesthesia as part of the procedure of exsanguination for euthanasia. Plasma samples were allowed to clot at 4°C and then were centrifuged at 7000 rpm for 7 min in order to separate the serum from cellular blood components. Serum was stored at −20°C until thawed for further evaluation. Serum IL-6, IL-10, MCP-1, and KC levels were quantified with ELISA kits (R&D System Inc., Minneapolis, Minn, USA) as per manufacturer’s specifications.
To minimize background MPO activity by remaining nonadherent intravascular polymorphonuclear cells, a needle was inserted into the beating right ventricle, after withdrawal of cardiac blood, and the circulation was perfused with 1.5 mL of PBS. The left lung was harvested and immediately snapped-frozen in liquid nitrogen and stored at −80° Celsius. To determine tissue MPO activity, the samples were thawed and homogenized in a lysis buffer according to the manufacturer’s protocol. The MPO activity was measured using an MPO-ELISA kit (Hycultec GmbH, Beutelsbach, Germany) and normalized to the protein concentration of the sample (BCA Protein Assay Kit, Pierce, Rockford, Ill, USA).
For the detection of pulmonary inflammation and lung injury the right lung was harvested and immediately fixed in buffered formalin. Paraffin-embedded blocks were cut at 5
All results in this paper are expressed as the mean ± SD of eight animals per group. Data were transformed by the BoxCox transformation (JMP 5.0.1 for Windows). In normally distributed variables group comparisons were assessed using ANOVA followed by Tukey’s HSD test. Nonnormally distributed parameters were tested using the Kruskal-Wallis-Test. The null hypothesis was rejected for
Serum IL-6 levels were significantly higher in animals subjected to hemorrhagic shock as compared to Control and Sham animals. However, the serum IL-6 concentration was indistinguishable between Shock and Shock + IL-10 animals, thus inhalative IL-10 did not significantly alter systemic IL-6 levels (Figure
Comparison of serum IL-6 (a), IL-10 (b), KC (c), and MCP-1 (d) levels in C57/BL6 mice following hemorrhagic shock with (Shock + IL-10) or without (Shock) inhalative administration of IL-10. Results are expressed as means ± SD of 8 animals per group (
Serum IL-10 concentrations showed an increase following hemorrhagic shock; however this did not reach statistical significance. Inhalative IL-10 was not associated with a significant increase in systemic IL-10 levels (Figure
Serum KC levels were significantly increased in Shock and Shock + IL-10 animals as compared with Control and Sham mice. There was no significant difference in serum KC levels between Shock and Shock + IL-10 mice (Figure
Serum MCP-1 levels were significantly higher following hemorrhagic shock as compared to Control and Sham animals. Furthermore, the MCP-1 concentration was significantly reduced in animals with inhalative IL-10 administration, thus inhalative IL-10 alters systemic MCP-1 levels (Figure
Pulmonary MPO activity was significantly higher in the Shock and Shock + IL-10 group as compared with Control and Sham animals. Inhalative IL-10 significantly reduced the pulmonary MPO activity as compared to Shock animals (Figure
Pulmonary myeloperoxidase (MPO) activity in C57/BL6 mice following hemorrhagic shock with (Shock + IL-10) or without (Shock) inhalative administration of IL-10. Results are expressed as means ± SD of 8 animals per group (
This effect was confirmed by histology which showed a reduced pulmonary infiltration with inflammatory cells (Figure
Representative H&E (Hematoxylin and Eosin) lung histology (20x) of the Control (a), Shock (b), and Shock + IL-10 (c) group 4.5 hrs after resuscitation. Inhalative IL-10 reduces the pulmonary influx of inflammatory cells.
The immunoregulatory potential of IL-10 is well recognized and a potential role of IL-10 as a therapeutic agent is increasingly investigated in various animal models [
Trauma hemorrhage is well known to lead to pulmonary inflammation and leukocyte infiltration, that is at least in part mediated by KC [
Interestingly, we observed a significant reduction of systemic MCP-1 following inhalative IL-10 application. MCP-1 is a major attractant for macrophages and monocytes and is upregulated following trauma hemorrhage [
We have previously observed these specific modulations of chemokine release in a model of IL-10 KO mice [
The influx of inflammatory cells in the lung following hemorrhagic shock has been well described and is thought to be a major contributor to the development of remote organ dysfunction following trauma [
Several features and limitations of our study merit further comment. We did not investigate later time points and one may criticize that inhalative IL-10 administration may delay but not prevent pulmonary inflammation following hemorrhagic shock. Further, the unchanged systemic concentrations of IL-6, KC, and IL-10 do not rule out that other important mediators in the posttraumatic inflammatory cascade may be altered by inhalative IL-10 and thereby the susceptibility to infection may again be increased.
In conclusion, this study demonstrates that inhalative IL-10 reduces pulmonary inflammation following hemorrhagic shock without major alterations in the systemic inflammatory response. This may be a valuable therapeutic strategy because inhalative IL-10 administration may solve the dilemma of the two-edged sword: end organ protection is possible without altering the systemic inflammatory response and the susceptibility to infection and sepsis. Nonetheless, further studies are required to elucidate the complex immunomodulatory action of IL-10 under the condition of hemorrhagic shock.
Adult Respiratory Distress Syndrome
Interleukin-6
Interleukin-10
Keratinocyte-derived Cytokine
Macrophage/Monocyte Chemotactic Protein-1
Multiple Organ Failure
Myeloperoxidase
Phosphate Buffered Saline.
This work has been supported by the Else Kröner-Fresenius-Stiftung.