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IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock
Cytokine 45 (2009) 26–31
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock Philipp Kobbe a,b,*, Burkhard Stoffels c, Joachim Schmidt c, Takeshi Tsukamoto a, Dmitry W. Gutkin d, Anthony J. Bauer c, Hans-Christoph Pape a a
Department of Orthopaedic Surgery, University of Pittsburgh, Kaufmann Medical Building, Suite 1010, 3471 Fifth Avenue, Pittsburgh, PA 15213, USA Department of Trauma Surgery, University Hospital Essen, Essen, Germany c Department of Gastroenterology, University of Pittsburgh, Pittsburgh, USA d Department of Pathology, VAMC Oakland, Pittsburgh, USA b
a r t i c l e
i n f o
Article history: Received 2 May 2008 Received in revised form 5 August 2008 Accepted 10 October 2008
Keywords: Hemorrhagic shock Interleukin-10 Systemic inflammation Acute lung injury Acute liver injury
a b s t r a c t In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3 h followed by resuscitation. Animals were either sacrificed 3 or 24 h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the LuminexTM multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3 h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Systemic inflammation with consecutive organ dysfunction continues to be a major problem after hemorrhagic shock, trauma, severe burns, and sepsis. Several components of the immune system have been implicated in this process including the systemic release of inflammatory cytokines and organ infiltration of polymorphonucleated neutrophils (PMNs); therefore modulation of this systemic inflammatory response appears to be a tempting strategy [1]. IL-10 is known to be an immuno-regulatory cytokine which inhibits the synthesis of pro-inflammatory cytokines [2,3] and the recruitment of PMNs [4]. However, whether the release of IL-10 following severe injury is beneficial or not is controversial. Some studies suggest that increased IL-10 levels are an indicator of poor prognosis with higher risk for multiple organ failure, infection, and death [5–8], whereas other studies indicate beneficial ef-
* Corresponding author. Address: Department of Orthopaedic Surgery, University of Pittsburgh, Kaufmann Medical Building, Suite 1010, 3471 Fifth Avenue, Pittsburgh, PA 15213, USA. Fax: +1 412 687 3724. E-mail address: [email protected] (P. Kobbe). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.10.004
fects of early systemic IL-10 release following injury [9–11]. In view of these diverse findings, we hypothesized that an endogenous IL-10 deficiency will augment acute lung and liver injury following hemorrhagic shock.
2. Materials and methods 2.1. Animal care 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 University of Pittsburgh. Male IL-10 KO (IL-10tm1Cgn) and C57/BL6 mice, the background strain for IL-10 KO mice, 8–10 weeks old and weighing 20–30 g, were used in the experiment (Jackson Laboratories, Bar Harbor, ME). The animals were maintained in the University of Pittsburgh Animal Research Center with a 12:12-h light–dark cycle and free access to standard laboratory feed and water. Animals were anesthetized with inhaled isoflurane (Abbott Labs, Chicago, IL).
P. Kobbe et al. / Cytokine 45 (2009) 26–31
2.2. Groups C57/BL6 and IL-10 KO mice were divided into four subgroups, respectively. In the Control-group, animals were euthanized after the induction of anesthesia to obtain physiological baseline levels. Animals in the Sham group were subjected to femoral artery catheterization, 3 h of anesthesia and 3 h of recovery before euthanasia. Animals of the Shock 3/3 group were subjected to femoral artery catheterization, 3 h of anesthesia with hemorrhagic shock and 3 h of recovery before euthanasia. Animals of the Shock 3/24 group were subjected to femoral artery catheterization, 3 h of anesthesia with hemorrhagic shock, and 24 h of recovery before euthanasia.
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dure 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, KC, and MCP-1 levels were assessed with the LuminexTM multiplexing platform (MiraiBio, Alameda, CA) using the BioSource 20-plex mouse cytokine bead set (BioSource-Invitrogen, San Diego, CA) as per manufacturer’s specifications. Serum ALT levels were measured using the Vitros 950 Chemistry System (Johnson & Johnson, Riaritan, NJ). 2.5. Pulmonary and hepatic myeloperoxidase (MPO) activity
A sterile technique was used to perform a left groin exploration, and the left femoral artery was cannulated with tapered polyethylene-10 tubing. Volume controlled hemorrhagic shock was performed by withdrawing 0.035 ml blood per g bodyweight over a period of 15 min in a syringe with 0.07 ml of heparin (1000 USP units/ml) [12]. Hemorrhagic shock was maintained for 3 h and was followed by resuscitation with shed blood plus an equal volume of 0.9% saline.
To minimize background MPO activity by remaining nonadherent intravascular polymorphonucleated cells, a needle was inserted into the beating right ventricle following cardiac blood withdrawal and the circulation was perfused with 1.5 ml of PBS. The left lung and left liver lobe were harvested and immediately snap frozen in liquid nitrogen and stored at 90 °C. To determine tissue MPO activity, the samples were thawed and homogenized in a lysis buffer exactly as directed by the manufacturer. The MPO activity was measured using a MPO-ELISA kit (Cell Sciences, Canton, MA) and normalized to the protein concentration of the sample (BCA Protein Assay Kit, Pierce, Rockford, IL).
2.4. Blood collection for serum cytokines and serum ALT levels
2.6. Liver tissue IL-6 concentration
At the planned time of sacrifice, cardiac blood was withdrawn through a thoracotomy under deep anesthesia as part of the proce-
To determine liver tissue IL-6 levels, the right liver lobe was harvested and immediately snap frozen in liquid nitrogen and stored at
2.3. Femoral artery cannulation and induction of hemorrhagic shock
Fig. 1. Comparison of serum IL-6 (A), KC (B), and MCP-1 (C) levels in C57/BL6 and IL-10 KO mice following hemorrhagic shock 3 h (Shock 3/3) and 24 h (Shock 3/24) after resuscitation. Results are expressed as means ± SEM of 4–6 animals per group (*p < 0.05 C57/BL6 vs. IL-10 KO mice).
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P. Kobbe et al. / Cytokine 45 (2009) 26–31
90° C. Liver tissue samples were thawed and homogenized in a lysis buffer exactly as directed by the manufacturer. Tissue IL-6 levels were quantified with ELISA kits (R&D Systems, Minneapolis, MN) and were normalized to the sample’s protein concentration (BCA Protein Assay Kit, Pierce, Rockford, IL).
Rank Sum Test. Comparisons between C57/BL6 and IL-10 KO mice were assessed using the Mann-Whitney Rank Sum Test. The null hypothesis was rejected for p < 0.05 (a = 0.05). Data were analyzed using StatView Version 5.0 (SAS Institute, Cary, NC). 3. Results
2.7. Pulmonary histology 3.1. Serum cytokine levels For the detection of intra-alveolar edema and the formation of hyaline membranes the right lung was harvested and immediately fixed in buffered formalin. Paraffin-embedded blocks were cut at 5 lm thickness and stained with H&E (Hematoxylin and Eosin) and PAS (Periodic Acid Schiff). 2.8. Statistical analysis Results are expressed as the means ± SEM of 4–6 animals per group. Subgroup comparisons within one group were assessed using the Kruskal–Wallis Test followed by the Mann–Whitney
Serum IL-6 levels were significantly increased in C57/BL6 and IL10 KO mice in the Shock 3/3 group compared to Control and Sham animals. In both strains, serum IL-6 levels waned with time but remained significantly elevated in the Shock 3/24 group as compared to Control and Sham animals. In comparison between C57/BL6 and IL-10 KO mice in the Shock 3/3 group, IL-10 deficient animals showed increased IL-6 levels, however, this did not reach statistical significance (Fig. 1A). Serum IL-10 levels were significantly elevated in C57/BL6 mice in the Shock 3/3 and Shock 3/24 group as compared to Control and Sham animals. Predictably, no IL-10 was detectable in the serum of any IL-10 KO mice. Serum KC levels were significantly increased in C57/BL6 and IL-10 KO mice in the Shock 3/3 group as compared to the Control, Sham, and Shock 3/24 group. Furthermore, KC serum concentrations were significantly increased in the Sham and Shock 3/24 group as compared to the Control-group. In comparison between C57/BL6 and IL-10 KO mice in the Shock 3/3 group, IL10 deficient animals showed increased serum KC levels; however, this did not reach statistical significance (Fig. 1B). Serum MCP-1 levels were not significantly elevated in C57/BL6 animals following hemorrhagic shock. In contrast, IL-10 KO mice of the Shock 3/3 and Shock 3/24 showed significantly increased serum MCP-1 concentrations as compared to Control and Sham animals. Comparing IL-10 KO with C57/BL6 mice, MCP-1 levels were significantly higher in IL-10 KO mice of the Shock 3/3 and Shock 3/24 group (Fig. 1C). 3.2. Lung injury
Fig. 2. Pulmonary myeloperoxidase (MPO) activity in C57/BL6 and IL-10 KO mice following hemorrhagic shock 3 h (Shock 3/3) and 24 h (Shock 3/24) after resuscitation. Results are expressed as means ± SEM of 4–6 animals per group (*p < 0.05 C57/BL6 vs. IL-10 KO mice).
Pulmonary myeloperoxidase (MPO) activity of C57/BL6 and IL10 KO mice was significantly increased in the Shock 3/3 and Shock 3/24 groups as compared with Control and Sham animals. Further,
Fig. 3. H&E (Hematoxylin and Eosin) and PAS (Periodic Acid Schiff) lung histology of shocked C57/BL6 and IL-10 KO mice 24 h after resuscitation. C57/BL6 mice show no histological changes (A) in H&E stain, whereas IL-10 KO mice show interstitial and intra-alveolar edema (?) in H&E stain (B) and focal hyaline membrane formation (?) in PAS stain (C).
P. Kobbe et al. / Cytokine 45 (2009) 26–31
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Fig. 4. Hepatic myeloperoxidase (MPO) activity (A), liver tissue IL-6 concentrations (B), and serum ALT levels (C) in C57/BL6 and IL-10 KO mice following hemorrhagic shock 3 h (Shock 3/3) and 24 h (Shock 3/24) after resuscitation. Results are expressed as means ± SEM of 4–6 animals per group.
IL-10 KO mice of the Shock 3/3 group showed significantly higher pulmonary MPO activity as compared with Shock 3/24 animals. In comparison of both strains, pulmonary MPO activity was significantly higher in IL-10 KO mice of the Shock 3/3 group as compared with C57/BL6 animals (Fig. 2). In addition, only IL-10 deficient mice showed early histological signs of diffuse alveolar damage after 24 h (Shock 3/24 group), including interstitial and intra-alveolar edema and focal hyaline membrane formation (Fig. 3). 3.3. Liver injury Hepatic myeloperoxidase (MPO) activity of C57/BL6 and IL-10 KO mice was significantly increased in the Shock 3/3 and Shock 3/24 group as compared with Control and Sham animals. Liver tissue IL-6 concentrations were significantly increased in C57/BL6 and IL-10 KO mice in the Shock 3/3 group as compared to Control, Sham, and Shock 3/24 animals. Serum ALT levels were significantly increased in C57/BL6 and IL-10 KO mice in the Sham, Shock 3/3, and Shock 3/24 group as compared to Control animals. Furthermore, only C57/BL6 mice of the Shock 3/3 group showed significantly higher serum ALT levels than Sham animals. There were no significant statistical differences between C57/BL6 and IL-10 KO mice in any measured liver injury parameter (Fig. 4). 4. Discussion IL-10 is considered as an immuno-regulatory cytokine which has been shown to modulate the inflammatory response following
injury [2–4,13]. The interaction between IL-10, pro-inflammatory cytokines, and the development of acute lung and liver injury under the condition of hemorrhagic shock is unclear. Our data indicate that IL-10 deficiency in hemorrhagic shock is associated with increased pro-inflammatory cytokine and chemokine serum levels. Although IL-6 and KC were not significantly elevated, there was a significant increase in MCP-1 after 3 and 24 h after resuscitation. This immuno-modulatory effect of IL-10 is in accordance with other studies investigating the role of IL-10 under conditions of stress [14–16]. However, the degree and mechanistic effect of this immuno-modulation appears to be dependent on the type of injury. Karakozis et al. observed a trend towards IL-6 suppression in animals treated with exogenous IL-10 in hemorrhagic shock [14], whereas Welborn et al. reported no differences in serum TNF-a and IL-6 levels following visceral ischemia-reperfusion between IL-10 deficient and competent mice [15]. Yokoyama et al. showed a significant increase of TNF-a and IL-6 production by Kupffer cells following IL-10 inhibition after trauma-hemorrhage [16]. MCP-1 is a major attractant for macrophages and monocytes and is up regulated under stress conditions such as trauma-hemorrhage [17]. Knowledge of the effect of IL-10 on MPC-1 production is limited and refers mainly to models of endotoxemia [18–21]. Herein, IL-10 induced MCP-1 synthesis in resting monocytes, but inhibited MCP-1 production in LPS-stimulated monocytes. Assuming that hemorrhagic shock is a stimulus for monocytes, this is in accordance with our data. In our model, MCP-1 was significantly elevated in IL-10 deficient animals following hemorrhagic shock.
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However, the cellular and intra-cellular mechanisms by which IL10 influences MCP-1 production remain unclear and warrant further investigation. In our model Sham animals showed significantly increased KC and ALT serum levels as compared with Control mice. This observation is in accordance with other studies which reported on increased KC and ALT serum levels following minor soft tissue injury (e.g. catheterization) [15,22] and anesthesia [23–25]. Ample existing evidence indicates that polymorphonucleated cells (PMNs) play a key role in the induction of remote organ dysfunction following trauma [26,27]. Our data showed that IL-10 deficiency augmented PMN infiltration in the shocked lung and that this correlated with histological evidence of ARDS 24 h after resuscitation only in IL-10 KO mice. The beneficial pulmonary effect of endogenous IL-10 has also been shown in other animal models [15,27] and is further supported by clinical studies which report that non-survivors of ARDS had significantly lower levels of IL-10 in BAL-fluid as compared to survivors [28]. However, the role of endogenous IL-10 appeared to be organ specific in this model of hemorrhagic shock as the livers of C57/BL6 and IL-10 KO mice had similar serum ALT levels and hepatic tissue concentrations of IL-6, as well as, being equally infiltrated with PMNs. The organ selective protective property of IL-10 observed in our model is in accordance with Welborn et al. who showed that endogenous IL10 in visceral ischemia-reperfusion attenuates the associated pulmonary neutrophil infiltration but has no effect upon hepatic injury [15]. However, other studies showed beneficial effects of IL10 on hepatic injury following endotoxemia [29,30]. These apparent diverse findings may be explained by the hypothesis that the immuno-modulatory effects of IL-10 are dependent on the type of injury. Our data show only a significant difference in pulmonary PMN infiltration between C57/BL6 and IL-10 KO mice 3 h after resuscitation which may explain the results of several studies which reported beneficial effects of exogenous IL-10 early but not later on after trauma [9–11,31]. Endogenous IL-10 may mediate its protective effects on the lung after hemorrhagic shock partly by reducing systemic cytokine levels and thereby reducing PMN infiltration. However, it may be assumed that in regards of the organ selective benefits of IL-10, the local organ microenvironment may also play a pivotal role [32]. Evidence of early cytokine release of hepatic Kupffer cells [33,34] resulted in the definition of the liver as the motor of systemic inflammation and MODS [35]. This is inline with our observation of significantly increased hepatic IL-6 levels 3 h after resuscitation. It may be speculated that the local synthesis of cytokines in the liver in response to hemorrhage may initiate an organ microenvironment in which the beneficial effects of IL10 may not be mediated. However, we are yet not able to explain the organ selective properties of IL-10 in our model of hemorrhagic shock. This observation warrants further investigation, which should elucidate intra- and intercellular signaling mechanism responsible for acute lung and liver injury. Several features and limitations of the study merit further comment. The magnitude of the systemic inflammatory response and the degree of lung injury were fairly modest although they served as major endpoints. However, in preliminary studies during model development, few shocked animals survived for 24 h after resuscitation when the degree of hemorrhagic shock was more severe. Furthermore, we did not investigate later timepoints and whether endogenous IL-10 provides a significant benefit for survival or may just delay but not prevent organ injury following hemorrhagic shock. In summary, our data shows that in a model of hemorrhagic shock endogenous IL-10 is beneficial in terms of early pulmonary but not hepatic inflammatory changes. The role of IL-10 following injury appears to be very complex and dependent on the inciting
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[15] Welborn III MB, Moldawer LL, Seeger JM, Minter RM, Huber TS. Role of endogenous interleukin-10 in local and distant organ injury after visceral ischemia-reperfusion. Shock 2003;20:35–40. [16] Yokoyama Y, Kitchens WC, Toth B, Schwacha MG, Rue III LW, Bland KI, et al. Role of IL-10 in regulating proinflammatory cytokine release by Kupffer cells following trauma-hemorrhage. Am J Physiol Gastrointest Liver Physiol 2004;286:G942–6. [17] Frink M, Lu A, Thobe BM, Hsieh YC, Choudhry MA, Schwacha MG, et al. Monocyte chemoattractant protein-1 influences trauma-hemorrhageinduced distal organ damage via regulation of keratinocyte-derived chemokine production. Am J Physiol Regul Integr Comp Physiol 2007;292:R1110–6. [18] Kucharzik T, Lugering N, Pauels HG, Domschke W, Stoll R. IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells. Clin Exp Immunol 1998;111:152–7. 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[30] Santucci L, Fiorucci S, Chiorean M, Brunori PM, Di Matteo FM, Sidoni A, et al. Interleukin 10 reduces lethality and hepatic injury induced by lipopolysaccharide in galactosamine-sensitized mice. Gastroenterology 1996;111:736–44. [31] Lyons A, Goebel A, Mannick JA, Lederer JA. Protective effects of early interleukin 10 antagonism on injury-induced immune dysfunction. Arch Surg 1999;134:1317–23. [32] Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy–review of a new approach. Pharmacol Rev 2003;55:241–69. [33] Frink M, Hsieh YC, Hsieh CH, Pape HC, Choudhry MA, Schwacha MG, et al. Keratinocyte-derived chemokine plays a critical role in the induction of systemic inflammation and tissue damage after trauma-hemorrhage. Shock 2007;28:576–81. [34] Poeze M, Ramsay G, Buurman WA, Greve JW, Dentener M, Takala J. Increased hepatosplanchnic inflammation precedes the development of organ dysfunction after elective high-risk surgery. Shock 2002;17:451–8. [35] Levy RM, Prince JM, Yang R, Mollen KP, Liao H, Watson GA, et al. Systemic inflammation and remote organ damage following bilateral femur fracture requires Toll-like receptor 4. Am J Physiol Regul Integr Comp Physiol 2006;291:R970–6.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock Philipp Kobbe a,b,*, Burkhard Stoffels c, Joachim Schmidt c, Takeshi Tsukamoto a, Dmitry W. Gutkin d, Anthony J. Bauer c, Hans-Christoph Pape a a
Department of Orthopaedic Surgery, University of Pittsburgh, Kaufmann Medical Building, Suite 1010, 3471 Fifth Avenue, Pittsburgh, PA 15213, USA Department of Trauma Surgery, University Hospital Essen, Essen, Germany c Department of Gastroenterology, University of Pittsburgh, Pittsburgh, USA d Department of Pathology, VAMC Oakland, Pittsburgh, USA b
a r t i c l e
i n f o
Article history: Received 2 May 2008 Received in revised form 5 August 2008 Accepted 10 October 2008
Keywords: Hemorrhagic shock Interleukin-10 Systemic inflammation Acute lung injury Acute liver injury
a b s t r a c t In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3 h followed by resuscitation. Animals were either sacrificed 3 or 24 h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the LuminexTM multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3 h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Systemic inflammation with consecutive organ dysfunction continues to be a major problem after hemorrhagic shock, trauma, severe burns, and sepsis. Several components of the immune system have been implicated in this process including the systemic release of inflammatory cytokines and organ infiltration of polymorphonucleated neutrophils (PMNs); therefore modulation of this systemic inflammatory response appears to be a tempting strategy [1]. IL-10 is known to be an immuno-regulatory cytokine which inhibits the synthesis of pro-inflammatory cytokines [2,3] and the recruitment of PMNs [4]. However, whether the release of IL-10 following severe injury is beneficial or not is controversial. Some studies suggest that increased IL-10 levels are an indicator of poor prognosis with higher risk for multiple organ failure, infection, and death [5–8], whereas other studies indicate beneficial ef-
* Corresponding author. Address: Department of Orthopaedic Surgery, University of Pittsburgh, Kaufmann Medical Building, Suite 1010, 3471 Fifth Avenue, Pittsburgh, PA 15213, USA. Fax: +1 412 687 3724. E-mail address: [email protected] (P. Kobbe). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.10.004
fects of early systemic IL-10 release following injury [9–11]. In view of these diverse findings, we hypothesized that an endogenous IL-10 deficiency will augment acute lung and liver injury following hemorrhagic shock.
2. Materials and methods 2.1. Animal care 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 University of Pittsburgh. Male IL-10 KO (IL-10tm1Cgn) and C57/BL6 mice, the background strain for IL-10 KO mice, 8–10 weeks old and weighing 20–30 g, were used in the experiment (Jackson Laboratories, Bar Harbor, ME). The animals were maintained in the University of Pittsburgh Animal Research Center with a 12:12-h light–dark cycle and free access to standard laboratory feed and water. Animals were anesthetized with inhaled isoflurane (Abbott Labs, Chicago, IL).
P. Kobbe et al. / Cytokine 45 (2009) 26–31
2.2. Groups C57/BL6 and IL-10 KO mice were divided into four subgroups, respectively. In the Control-group, animals were euthanized after the induction of anesthesia to obtain physiological baseline levels. Animals in the Sham group were subjected to femoral artery catheterization, 3 h of anesthesia and 3 h of recovery before euthanasia. Animals of the Shock 3/3 group were subjected to femoral artery catheterization, 3 h of anesthesia with hemorrhagic shock and 3 h of recovery before euthanasia. Animals of the Shock 3/24 group were subjected to femoral artery catheterization, 3 h of anesthesia with hemorrhagic shock, and 24 h of recovery before euthanasia.
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dure 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, KC, and MCP-1 levels were assessed with the LuminexTM multiplexing platform (MiraiBio, Alameda, CA) using the BioSource 20-plex mouse cytokine bead set (BioSource-Invitrogen, San Diego, CA) as per manufacturer’s specifications. Serum ALT levels were measured using the Vitros 950 Chemistry System (Johnson & Johnson, Riaritan, NJ). 2.5. Pulmonary and hepatic myeloperoxidase (MPO) activity
A sterile technique was used to perform a left groin exploration, and the left femoral artery was cannulated with tapered polyethylene-10 tubing. Volume controlled hemorrhagic shock was performed by withdrawing 0.035 ml blood per g bodyweight over a period of 15 min in a syringe with 0.07 ml of heparin (1000 USP units/ml) [12]. Hemorrhagic shock was maintained for 3 h and was followed by resuscitation with shed blood plus an equal volume of 0.9% saline.
To minimize background MPO activity by remaining nonadherent intravascular polymorphonucleated cells, a needle was inserted into the beating right ventricle following cardiac blood withdrawal and the circulation was perfused with 1.5 ml of PBS. The left lung and left liver lobe were harvested and immediately snap frozen in liquid nitrogen and stored at 90 °C. To determine tissue MPO activity, the samples were thawed and homogenized in a lysis buffer exactly as directed by the manufacturer. The MPO activity was measured using a MPO-ELISA kit (Cell Sciences, Canton, MA) and normalized to the protein concentration of the sample (BCA Protein Assay Kit, Pierce, Rockford, IL).
2.4. Blood collection for serum cytokines and serum ALT levels
2.6. Liver tissue IL-6 concentration
At the planned time of sacrifice, cardiac blood was withdrawn through a thoracotomy under deep anesthesia as part of the proce-
To determine liver tissue IL-6 levels, the right liver lobe was harvested and immediately snap frozen in liquid nitrogen and stored at
2.3. Femoral artery cannulation and induction of hemorrhagic shock
Fig. 1. Comparison of serum IL-6 (A), KC (B), and MCP-1 (C) levels in C57/BL6 and IL-10 KO mice following hemorrhagic shock 3 h (Shock 3/3) and 24 h (Shock 3/24) after resuscitation. Results are expressed as means ± SEM of 4–6 animals per group (*p < 0.05 C57/BL6 vs. IL-10 KO mice).
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90° C. Liver tissue samples were thawed and homogenized in a lysis buffer exactly as directed by the manufacturer. Tissue IL-6 levels were quantified with ELISA kits (R&D Systems, Minneapolis, MN) and were normalized to the sample’s protein concentration (BCA Protein Assay Kit, Pierce, Rockford, IL).
Rank Sum Test. Comparisons between C57/BL6 and IL-10 KO mice were assessed using the Mann-Whitney Rank Sum Test. The null hypothesis was rejected for p < 0.05 (a = 0.05). Data were analyzed using StatView Version 5.0 (SAS Institute, Cary, NC). 3. Results
2.7. Pulmonary histology 3.1. Serum cytokine levels For the detection of intra-alveolar edema and the formation of hyaline membranes the right lung was harvested and immediately fixed in buffered formalin. Paraffin-embedded blocks were cut at 5 lm thickness and stained with H&E (Hematoxylin and Eosin) and PAS (Periodic Acid Schiff). 2.8. Statistical analysis Results are expressed as the means ± SEM of 4–6 animals per group. Subgroup comparisons within one group were assessed using the Kruskal–Wallis Test followed by the Mann–Whitney
Serum IL-6 levels were significantly increased in C57/BL6 and IL10 KO mice in the Shock 3/3 group compared to Control and Sham animals. In both strains, serum IL-6 levels waned with time but remained significantly elevated in the Shock 3/24 group as compared to Control and Sham animals. In comparison between C57/BL6 and IL-10 KO mice in the Shock 3/3 group, IL-10 deficient animals showed increased IL-6 levels, however, this did not reach statistical significance (Fig. 1A). Serum IL-10 levels were significantly elevated in C57/BL6 mice in the Shock 3/3 and Shock 3/24 group as compared to Control and Sham animals. Predictably, no IL-10 was detectable in the serum of any IL-10 KO mice. Serum KC levels were significantly increased in C57/BL6 and IL-10 KO mice in the Shock 3/3 group as compared to the Control, Sham, and Shock 3/24 group. Furthermore, KC serum concentrations were significantly increased in the Sham and Shock 3/24 group as compared to the Control-group. In comparison between C57/BL6 and IL-10 KO mice in the Shock 3/3 group, IL10 deficient animals showed increased serum KC levels; however, this did not reach statistical significance (Fig. 1B). Serum MCP-1 levels were not significantly elevated in C57/BL6 animals following hemorrhagic shock. In contrast, IL-10 KO mice of the Shock 3/3 and Shock 3/24 showed significantly increased serum MCP-1 concentrations as compared to Control and Sham animals. Comparing IL-10 KO with C57/BL6 mice, MCP-1 levels were significantly higher in IL-10 KO mice of the Shock 3/3 and Shock 3/24 group (Fig. 1C). 3.2. Lung injury
Fig. 2. Pulmonary myeloperoxidase (MPO) activity in C57/BL6 and IL-10 KO mice following hemorrhagic shock 3 h (Shock 3/3) and 24 h (Shock 3/24) after resuscitation. Results are expressed as means ± SEM of 4–6 animals per group (*p < 0.05 C57/BL6 vs. IL-10 KO mice).
Pulmonary myeloperoxidase (MPO) activity of C57/BL6 and IL10 KO mice was significantly increased in the Shock 3/3 and Shock 3/24 groups as compared with Control and Sham animals. Further,
Fig. 3. H&E (Hematoxylin and Eosin) and PAS (Periodic Acid Schiff) lung histology of shocked C57/BL6 and IL-10 KO mice 24 h after resuscitation. C57/BL6 mice show no histological changes (A) in H&E stain, whereas IL-10 KO mice show interstitial and intra-alveolar edema (?) in H&E stain (B) and focal hyaline membrane formation (?) in PAS stain (C).
P. Kobbe et al. / Cytokine 45 (2009) 26–31
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Fig. 4. Hepatic myeloperoxidase (MPO) activity (A), liver tissue IL-6 concentrations (B), and serum ALT levels (C) in C57/BL6 and IL-10 KO mice following hemorrhagic shock 3 h (Shock 3/3) and 24 h (Shock 3/24) after resuscitation. Results are expressed as means ± SEM of 4–6 animals per group.
IL-10 KO mice of the Shock 3/3 group showed significantly higher pulmonary MPO activity as compared with Shock 3/24 animals. In comparison of both strains, pulmonary MPO activity was significantly higher in IL-10 KO mice of the Shock 3/3 group as compared with C57/BL6 animals (Fig. 2). In addition, only IL-10 deficient mice showed early histological signs of diffuse alveolar damage after 24 h (Shock 3/24 group), including interstitial and intra-alveolar edema and focal hyaline membrane formation (Fig. 3). 3.3. Liver injury Hepatic myeloperoxidase (MPO) activity of C57/BL6 and IL-10 KO mice was significantly increased in the Shock 3/3 and Shock 3/24 group as compared with Control and Sham animals. Liver tissue IL-6 concentrations were significantly increased in C57/BL6 and IL-10 KO mice in the Shock 3/3 group as compared to Control, Sham, and Shock 3/24 animals. Serum ALT levels were significantly increased in C57/BL6 and IL-10 KO mice in the Sham, Shock 3/3, and Shock 3/24 group as compared to Control animals. Furthermore, only C57/BL6 mice of the Shock 3/3 group showed significantly higher serum ALT levels than Sham animals. There were no significant statistical differences between C57/BL6 and IL-10 KO mice in any measured liver injury parameter (Fig. 4). 4. Discussion IL-10 is considered as an immuno-regulatory cytokine which has been shown to modulate the inflammatory response following
injury [2–4,13]. The interaction between IL-10, pro-inflammatory cytokines, and the development of acute lung and liver injury under the condition of hemorrhagic shock is unclear. Our data indicate that IL-10 deficiency in hemorrhagic shock is associated with increased pro-inflammatory cytokine and chemokine serum levels. Although IL-6 and KC were not significantly elevated, there was a significant increase in MCP-1 after 3 and 24 h after resuscitation. This immuno-modulatory effect of IL-10 is in accordance with other studies investigating the role of IL-10 under conditions of stress [14–16]. However, the degree and mechanistic effect of this immuno-modulation appears to be dependent on the type of injury. Karakozis et al. observed a trend towards IL-6 suppression in animals treated with exogenous IL-10 in hemorrhagic shock [14], whereas Welborn et al. reported no differences in serum TNF-a and IL-6 levels following visceral ischemia-reperfusion between IL-10 deficient and competent mice [15]. Yokoyama et al. showed a significant increase of TNF-a and IL-6 production by Kupffer cells following IL-10 inhibition after trauma-hemorrhage [16]. MCP-1 is a major attractant for macrophages and monocytes and is up regulated under stress conditions such as trauma-hemorrhage [17]. Knowledge of the effect of IL-10 on MPC-1 production is limited and refers mainly to models of endotoxemia [18–21]. Herein, IL-10 induced MCP-1 synthesis in resting monocytes, but inhibited MCP-1 production in LPS-stimulated monocytes. Assuming that hemorrhagic shock is a stimulus for monocytes, this is in accordance with our data. In our model, MCP-1 was significantly elevated in IL-10 deficient animals following hemorrhagic shock.
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However, the cellular and intra-cellular mechanisms by which IL10 influences MCP-1 production remain unclear and warrant further investigation. In our model Sham animals showed significantly increased KC and ALT serum levels as compared with Control mice. This observation is in accordance with other studies which reported on increased KC and ALT serum levels following minor soft tissue injury (e.g. catheterization) [15,22] and anesthesia [23–25]. Ample existing evidence indicates that polymorphonucleated cells (PMNs) play a key role in the induction of remote organ dysfunction following trauma [26,27]. Our data showed that IL-10 deficiency augmented PMN infiltration in the shocked lung and that this correlated with histological evidence of ARDS 24 h after resuscitation only in IL-10 KO mice. The beneficial pulmonary effect of endogenous IL-10 has also been shown in other animal models [15,27] and is further supported by clinical studies which report that non-survivors of ARDS had significantly lower levels of IL-10 in BAL-fluid as compared to survivors [28]. However, the role of endogenous IL-10 appeared to be organ specific in this model of hemorrhagic shock as the livers of C57/BL6 and IL-10 KO mice had similar serum ALT levels and hepatic tissue concentrations of IL-6, as well as, being equally infiltrated with PMNs. The organ selective protective property of IL-10 observed in our model is in accordance with Welborn et al. who showed that endogenous IL10 in visceral ischemia-reperfusion attenuates the associated pulmonary neutrophil infiltration but has no effect upon hepatic injury [15]. However, other studies showed beneficial effects of IL10 on hepatic injury following endotoxemia [29,30]. These apparent diverse findings may be explained by the hypothesis that the immuno-modulatory effects of IL-10 are dependent on the type of injury. Our data show only a significant difference in pulmonary PMN infiltration between C57/BL6 and IL-10 KO mice 3 h after resuscitation which may explain the results of several studies which reported beneficial effects of exogenous IL-10 early but not later on after trauma [9–11,31]. Endogenous IL-10 may mediate its protective effects on the lung after hemorrhagic shock partly by reducing systemic cytokine levels and thereby reducing PMN infiltration. However, it may be assumed that in regards of the organ selective benefits of IL-10, the local organ microenvironment may also play a pivotal role [32]. Evidence of early cytokine release of hepatic Kupffer cells [33,34] resulted in the definition of the liver as the motor of systemic inflammation and MODS [35]. This is inline with our observation of significantly increased hepatic IL-6 levels 3 h after resuscitation. It may be speculated that the local synthesis of cytokines in the liver in response to hemorrhage may initiate an organ microenvironment in which the beneficial effects of IL10 may not be mediated. However, we are yet not able to explain the organ selective properties of IL-10 in our model of hemorrhagic shock. This observation warrants further investigation, which should elucidate intra- and intercellular signaling mechanism responsible for acute lung and liver injury. Several features and limitations of the study merit further comment. The magnitude of the systemic inflammatory response and the degree of lung injury were fairly modest although they served as major endpoints. However, in preliminary studies during model development, few shocked animals survived for 24 h after resuscitation when the degree of hemorrhagic shock was more severe. Furthermore, we did not investigate later timepoints and whether endogenous IL-10 provides a significant benefit for survival or may just delay but not prevent organ injury following hemorrhagic shock. In summary, our data shows that in a model of hemorrhagic shock endogenous IL-10 is beneficial in terms of early pulmonary but not hepatic inflammatory changes. The role of IL-10 following injury appears to be very complex and dependent on the inciting
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