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β-Adrenergic blockade during systemic inflammation: Impact on cellular immune functions and survival in a murine model of sepsis
Resuscitation (2007) 72, 286—294
EXPERIMENTAL PAPER
-Adrenergic blockade during systemic inflammation: Impact on cellular immune functions and survival in a murine model of sepsis夽 Daniel Schmitz a, Klaus Wilsenack a, Sven Lendemanns a, Manfred Schedlowski b, Reiner Oberbeck a,∗ a b
Department Trauma Surgery, University Hospital, University Duisburg-Essen, Essen, Germany Institute of Medical Psychology, University Hospital, University Duisburg-Essen, Essen, Germany
Received 5 April 2006 ; received in revised form 22 June 2006; accepted 3 July 2006 KEYWORDS Immune system; Apoptosis; Cytokines; Propranolol; Septic shock; Catecholamines; -Adrenergic receptor; -Adrenergic blockade
Summary Aim of the study: Adrenergic immuno-modulation mediated by -adrenergic receptors has been demonstrated. Pharmacological blockade of -adrenergic receptors is a therapeutic intervention frequently used in critically ill patients. The effect of -adrenergic blockade on cellular immune functions in a critical illness, such as polymicrobial sepsis, has not been investigated. Methods: Male NMRI-mice were subjected to sham operation or to sepsis (caecal ligation and puncture, CLP) following administration of either the non-selective -adrenergic antagonist propranolol (0.5 mg/kg s.c. every 12 h in 1 ml vehicle) or saline 0.9% (1 ml s.c. every 12 h). Mice were kept in metabolic cages and were sacrificed 48 h after induction of sepsis. Survival rate, clinical situation (body weight and temperature, fluid and food intake, urine output), and immunological variables (splenocyte proliferation, apoptosis, and IFN-␥ and IL-6 release) were determined. Results: Administration of propranolol in septic mice increased the splenocyte apoptosis rate, reduced the proliferative capacity of splenocytes, and modulated cellular cytokine release (IL-6, IFN-␥). This was paralleled by a higher loss of body weight and temperature, and a decreased urine output. Furthermore, treatment with propranolol increased the sepsis-induced lethality from 47% up to 68%, respectively. Conclusion: -Adrenergic blockade was accompanied by alterations of cellular immune functions, a deterioration in the clinical situation and a reduced survival in a
夽 A Spanish translated version of the summary of this article appears as Appendix in the final online version at 10.1016/j.resuscitation.2006.07.001. ∗ Corresponding author at: Klinik f. Unfallchirurgie, Universit¨ atsklinikum Essen, Hufelandstr. 55, 45147 Essen, Germany. Tel.: +49 201 723 1301; fax: +49 201 723 5936. E-mail address: [email protected] (R. Oberbeck).
0300-9572/$ — see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.resuscitation.2006.07.001
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murine model of sepsis. These data demonstrate the potential immuno-modulatory effects of -adrenergic antagonists. © 2006 Elsevier Ireland Ltd. All rights reserved.
Introduction Pharmacological blockade of -adrenergic receptors is a frequent therapeutic intervention in critically ill patients.1—3 Examples are the long-term treatment with -adrenergic antagonists due to chronic cardiovascular disease and the use of -adrenergic blocking agents in the treatment of acute cardiac arrhythmias and hypertension.1 Furthermore, a pharmacologic blockade of adrenergic receptors is used to prevent the catecholamine-mediated hypermetabolism in critically ill patients suffering from severe trauma or burn injury. In this group of patients a reduction in cardiac work, resting energy expenditure, and muscle-protein catabolism was recorded after pharmacological -adrenergic blockade.2—4 In addition to these effects it has been demonstrated that the adrenergic system modulated cellular immune functions through activation of -adrenergic receptors.5—9 The potential immunological side effects of a -adrenergic blockade have not been investigated although infectious complications are the leading cause of death in critically ill patients after trauma or burn injury.10—12 In line with this, several reports demonstrate an adrenergic modulation of cellular immune functions that is at least partly mediated via activation or inhibition of -adrenergic receptors.5—9 Accordingly, it was reported that non-selective -adrenergic blockade following administration of the non-selective -adrenergic antagonist propranolol in healthy human volunteers induced a reduction in the numbers of circulating CD8+lymphocytes and natural killer (NK)-cells. Furthermore, this effect was accompanied by inhibition of the antibody-dependent cellular cytotoxicity and the cytotoxic activity of NK-cells.5,6,13 Moreover, administration of the non-selective -adrenergic antagonist propranolol or of the 1 -selective adrenergic antagonist metoprolol in a clinically relevant murine haemorrhagic shock model revealed that administration of either -adrenergic antagonist abolished the haemorrhage-induced increase of circulating CD8+-lymphoyctes and NK-cells and modulated splenocyte apoptosis.14 However, the effect of a -adrenergic blockade on cellular immune functions during systemic inflammation and its impact on the clinical cause of this disease remains to be established.
We therefore investigated the effect of a non-selective -adrenergic blockade on the clinical course and cellular immune functions during polymicrobial sepsis in mice.
Materials and methods Animals and animal care Male NMRI-mice (Charles River Laboratories, Wilmington, MA), 8—9 weeks old with a body weight between 30 and 34 g were used in this study. Mice were kept with a 12 h dark/light cycle and received water and food ad libitum. The experiments were performed in adherence to the guidelines for the Care and Use of Laboratory Animals of the National Institute of Health. The experimental protocol was approved by the local legislative committee.
Drug administration Propranolol (PROP; 0.5 mg/kg) (Dociton, AstraZeneca, Germany) was administered subcutaneously in control mice, in sham-operated mice and in mice subjected to polymicrobial sepsis by CLP. Mice in the septic and the sham-operated groups received PROP at baseline, and 12, 24, 36 and 48 h after laparotomy or CLP with the baseline dosage being given 20 min prior to laparotomy or CLP. Mice in the control group received a single dose of PROP to determine the baseline effects of the -adrenergic antagonist. Referring to the literature the dose of propranolol used in our experiment is the lowest dose that is be effective in blocking -adrenergic activity.15—17
Experimental groups Mice were randomly assigned to one of the following experimental groups. Groups 1—2 (n = 8/group) served as untreated controls (UNT). Group 1 received 0.9% saline (UNT/saline, 0.2 ml) and group 2 received a single dose of propranolol (UNT/PROP; 0.5 mg/kg s.c.). Groups 3—4 underwent a sham operation (laparotomy: 1 cm midline incision without caecal ligation and puncture, SHAM, n = 16/group) without (SHAM/saline, 0.2 ml s.c.) or with administration of propranolol (SHAM/PROP; 0.5 mg/kg/12 h s.c.). Groups 5—6 were subjected
288 Table 1
D. Schmitz et al. Experimental groups
UNT/saline UNT/PROP SHAM/saline SHAM/PROP CLP/saline CLP/PROP
Untreated mice receiving saline 0.9% Untreated mice receiving propranolol (0.5 mg/kg) Sham-operated mice (laparotomy) receiving saline 0.9% Sham-operated mice (laparotomy) receiving propranolol (0.5 mg/kg) Septic mice (caecal ligation and puncture) receiving saline 0.9% Septic mice (caecal ligation and puncture) receiving propranolol (0.5 mg/kg)
to a polymicrobial sepsis induced by a caecal ligation and puncture (CLP, n = 32/group). Group 5 received a subcutaneous injection of 0.2 ml saline (CLP/saline) and Group 6 received propranolol (CLP/PROP, 0.5 mg/kg/12 h s.c.) (Table 1).
Polymicrobial sepsis model Polymicrobial sepsis was induced using the model of caecal ligation and puncture (CLP) described by Baker et al.18 This model leads to a polymicrobial sepsis with positive blood cultures for gram positive and gram negative bacteria as early as 1 h following CLP, and a reproducible mortality rate of 50% after 48 h, depending on the number and size of the puncture holes.18 Briefly, following a light ether anaesthesia a midline laparotomy was performed. After isolation and ligation, the caecum was punctured twice with a 22-gauge needle, and a small amount of the bowel contents was extruded through the puncture holes. After returning the bowel to the abdomen, the midline incision was closed. All animals received 1 ml Ringer subcutaneously at the end of the operation. Sham animals underwent a laparotomy without caecal ligation and puncture. The animals were then housed in metabolic cages in order to monitor the body weight, heart rate, body temperature, fluid intake, urine production and food consumption. Blind observers recorded these clinical findings. Food and water were allowed ad libitum. All mice were subjected to the same fluid-resuscitation regimen with subcutaneous administration of 1 ml Ringer solution twice a day (08:00 a.m. and 08:00 p.m.). Forty-eight hours after induction of sepsis, or following sham operation, blood was drawn by cardiac puncture and immediately thereafter the spleens were removed aseptically and placed in petri dishes containing ice-cooled phosphate-buffered saline solution (PBS, pH 7.4). All mice were sacrificed at
the same time of day (8 a.m.) to avoid fluctuations due to circadian rhythm. Determination of clinical data and immunologic variables were performed by investigators blinded with regard to the surgical procedure and treatment of experimental groups.
Clinical data Clinical data were obtained using metabolic cages. Body temperature, body weight, food and fluid intake, urine output and faces production were recorded every 12 h in control mice and after induction of either sham-operation or CLP.
Preparation of splenocyte culture and assessment of proliferative capacity After blood withdrawal animals were sacrificed by cervical dislocation and the spleen was removed aseptically. Immediately after removal of the spleen the organ was placed in ice-cooled petri dishes containing ice-cooled phosphate-buffered saline solution (PBS; 4 ◦ C, pH 7.4). Then the organ was punctured with a 20-gauge needle connected to a 20 ml syringe containing ice-cooled PBS solution (4 ◦ C, pH 7.4). A cell suspension was produced by gently flushing the organ with the ice-cooled PBS solution. The suspension was freed from debris by centrifugation at 300 × g for 15 min. Thereafter, erythrocytes were lysed hypotonically, and the remaining cells were washed two times with ice-cooled PBS (4 ◦ C, pH 7.4) by repeated centrifugation (300 × g, 15 min). The ability of the splenocytes to produce lymphokines in response to a mitogenic challenge was assessed in one portion of the cell suspension by incubation for 48 h (at 37 ◦ C, 5% CO2 , and 90% humidity) in the presence of 2.5 g/ml concanavalin A (Sigma—Aldrich Chemicals, Beisenh¨ ofen, Germany). After this incubation period, the cell suspension was centrifuged at 300 × g for 10 min, and the supernatants were harvested, divided into aliquots and stored at −70 ◦ C until assayed for IL-2 and IL-6 concentrations. The second portion of the cell suspension was used to determine the splenocyte proliferative capacity in response to mitogenic stimulation. The splenocyte cell suspension was placed in a 96-well microtiter plate in aliquots of 100 l. The cells ability to proliferate in response to mitogenic stimulation with 0 g/ml (negative control) or 2.5 g/ml Con A was determined after 48 h incubation at 37 ◦ C, 5% CO2 and 90% humidity. The extent of proliferation was determined by 3 H-thymidine incorporation as described previously.19
Beta-adrenergic blockade during systemic inflammation
Assessment of cytokine release The capacity of mixed splenocyte cultures to produce IFN-␥ and IL-6 was assessed by determination of the concentration of these cytokines in the collected culture supernatant 48 h after incubation with 2.5 g/ml Con A using commercial ELISAassays (Biosource International, Nivelles, Belgium; Endogen Inc., Woburn, MA, USA). The sensitivity of the IFN-␥ ELISA was <4 pg/ml. The inter-assay coefficient of variance was <10% and the intra-assay coefficient of variance <4.5%. The sensitivity of the IL-6 ELISA was <3 pg/ml, the inter-assay coefficient of variance was <10% and the intra-assay coefficient of variance was <10%.
Assessment of splenocyte apoptosis and total viable cell yield After isolation of splenocytes, the cells were washed twice with ice cold PBS (4 ◦ C, pH 7.4) and then resuspended in binding buffer at 4 ◦ C. Cell concentration was adjusted to 1 × 106 cells/ml. One millilitre of this cell suspension was placed into 1.5 ml microcentrifuge tubes for cell staining. FITC-conjugated recombinant Annexin V (Annexin V-FITC, PharMingen, San Diego, USA) and/or propidium iodide (PI) staining solution was added to 100 l of the cell solution with the cells subsequently incubated for 15 min at room temperature in the dark. Immediately following the incubation period cells were analyzed for Annexin V binding and PI-incorporation by flow cytometry. Determination of the total viable cell count was performed by trypan blue exclusion. Total viable cell yield was performed using a microscope, and was conducted by an investigator experienced with this method. A number of 106 cells/ml was counted.
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Furthermore, two-colour fluorescence analyses of splenocyte subpopulations were performed using FITC-conjugated Annexin V (Annexin V-FITC, PharMingen, San Diego, USA) and the commercially available Abs Anti-CD4 (PE), Anti-CD8 (PE), AntiNK1.1 (PE), F4/80 (PE) (Biozol Diagnostica, Eching, Germany).
Statistical analysis Results are presented as means ± S.E.M. Data were analyzed for significance by one-way ANOVA including post-hoc analysis (Scheff´ e-test and Bonferronicorrection). The Kaplan—Meier and Cox regression test were used for comparing survival. A p-value < 0.05 was considered statistically significant. Statistical analysis were performed by using the software SPSS (version 12.0 for windows).
Results Every CLP-experiment was conducted twice with 16 animals/group. For each septic group similar results were found in both experiments, so the data were pooled. Sepsis induced a lethality rate of 47% 48 h after induction of CLP (p < 0.001, CLP versus SHAM). Administration of propranolol was accompanied by a significant increase of sepsisinduced lethality (68%; p < 0.01, CLP/PROP versus CLP/saline) (Figure 1). Concerning the clinical data no significant differences were found comparing the control and the sham-operated groups (UNT versus SHAM/saline; UNT versus SHAM/PROP; SHAM/saline versus SHAM/PROP). A statistically significant reduction in body temperature, body weight, fluid intake, and
Figure 1 Survival rate of mice subjected to either sham operation (SHAM) or sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of the -adrenergic antagonist propranolol.
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Table 2 Metabolic data (body temperature and weight, fluid and food intake, urine output) of mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of non-selective -adrenoceptor antagonist propranolol
SHAM/saline SHAM/PROP CLP/saline CLP/PROP
body temperature (◦ C)
body weight (g)
fluid intake (ml)
food intake (g)
urine output (ml)
1.17* a (±0.8) 0.9* b (±1) −7.47* b (±6.7) −9.08 (±5.2)
−1.1* a (±0.1) −0.9* b (±0.2) −2.9** b (±0.1) −4.9 (±0.07)
8.25*** a (±0.17) 10.62*** b (±0.29) 4.29 (±0.29) 3.39 (±0.21)
7.62# a (±0.13) 8.85# b (±0.22) 0.76 (±0.15) 0.52 (±0.1)
1.8 (±0.4) 2.2 (±0.6) 1.1* b (±0.6) 1.4 (±0.7)
a: vs. CLP/saline; b: vs. CLP/PROP. * p < 0.05; ** p < 0.005;
*** p < 0.01; # p < 0.001.
food intake was found in saline and propranololtreated septic animals compared with the shamoperated groups. Furthermore, administration of propranolol in septic animals was accompanied by a significant reduction in body temperature, body weight and urine output (Table 2). Monitoring of splenocyte proliferation by determination of H3 -thymidine incorporation following stimulation with Con A revealed a sepsis-induced inhibition of splenocyte proliferative ability 48 h after onset of sepsis (p < 0.0001, SHAM/saline versus CLP/saline). Administration of propranolol in septic mice augmented the sepsis-induced inhibition of splenocyte proliferation significantly (CLP/saline versus CLP/PROP, p < 0.05) (Figure 2). Splenocyte apoptosis, determined by FACScan analysis of splenocyte Annexin V binding capacity, revealed an increased splenocyte apoptosis in
saline-treated septic animals 48 h after induction of sepsis (p < 0.05, SHAM/saline versus CLP/saline). This increase in splenocyte apoptosis was further increased in propranolol-treated septic mice (p < 0.001, CLP/saline versus CLP/PROP) (Figure 3). The results obtained for the total viable cell count corresponded inversely to the results reported for splenocyte Annexin V binding capacity (data not shown). Induction of CLP in saline-treated animals was accompanied by a significant decrease in splenocyte IFN-␥ and IL-6 release (p < 0.05, CLP/saline versus SHAM/saline). Administration of propranolol induced a further inhibition of IFN-␥ release (p < 0.001, CLP/saline versus CLP/PROP) but attenuated the sepsis-induced inhibition of splenocyte IL-6 release (p < 0.05, CLP/saline versus CLP/PROP) (Figure 4).
Figure 2 Splenocyte proliferation recorded in mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of propranolol. Data are presented as percentage of changes compared to untreated animals with means ± S.D.
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Figure 3 Splenocyte apoptosis determined in mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of the -adrenergic antagonist propranolol. Data are presented as percentage of changes compared to untreated animals with means ± S.D.
Figure 4 Determination of IFN-␥ and IL-6 concentrations in the supernatant of cultured splenocytes obtained from mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of the -adrenergic antagonist propranolol. Data are presented as percentage of changes compared to untreated animals with means ± S.D.
Discussion Pharmacological blockade of -adrenergic receptors is a frequently used therapeutic intervention in the critically ill.1,2 This includes the treatment of acute cardiovascular events like arrhythmia and hypertension as well as the long term administration of -adrenergic antagonists for the treatment
of chronic diseases.1 Furthermore, pharmacological blockade of -adrenergic receptors is recommended to prevent catecholamine-induced hypermetabolism in patients suffering from severe burn injury and the beneficial effect of pharmacological -adrenergic blockade on muscle protein turn-over in severely burned patients has been demonstrated recently.2,3
292 In addition, it is well established that adrenergic receptors are involved in the modulation of the cellular immune system.5—8,14 Interestingly, the potential immunological side effects of a adrenergic blockade in critically ill patients has not been investigated yet although it is known that the majority of these patients develop infectious complications, in particular sepsis due to compromised activity of the immune system.8,10—12 Apoptosis is a form of programmed cell death that is widely distributed among the cells of the immune system.20,21 Some characteristic features of apoptosis are DNA fragmentation, induction of cytosolic proteases, and changes in the cellular plasma membrane.22 The translocation of phospholipids from the inner to the outer leaflet of the plasma membrane is an important step during the early phase of apoptosis and this can be detected by Annexin V binding.22 In line with previous reports about adrenergic immuno-modulation we observed an increased splenocyte apoptosis rate following the administration of a -adrenergic antagonist in our model of polymicrobial sepsis. Several factors are known to trigger apoptotic cell death and it was noticed that the exposure or withdrawal of hormones and neurotransmitters affected cellular apoptosis.23—25 Accordingly, it was reported that catecholamines are capable of modulating lymphocyte apoptosis and that this process involved -adrenergic receptors.23,25 In extension of these data we observed an increase of splenocyte apoptosis following an inhibition of the -adrenergic receptor subtype in a murine model of sepsis. However, it cannot be concluded from our results if this was a direct receptor-mediated effect on the cellular level or if this was consequence of an indirect mechanism, for example a reduced recognition and removal of apoptotic cells by macrophages or reticular endothelial cells.21,26 Interestingly, we observed an increased cellular apoptosis in saline-treated septic mice. It is well known that alterations of cellular apoptosis can be observed during sepsis and a large number of studies demonstrated a sepsis-induced increased in apoptotic cell death. However, data concerning the effect of systemic inflammation on cellular apoptosis are controversial since a reduced cellular apoptosis was reported by other investigators. We assume that these differences can be attributed to differences in the cell types investigated or may reflect differences in the models used in these studies.27—34 Concerning the proliferative activity of splenocytes we observed that pharmacological blockade of -adrenergic receptors was paralleled by an aug-
D. Schmitz et al. mented inhibition of cellular proliferation. Reviewing the literature, only few data are available about -adrenergic modulation of immune cell proliferation. In line with our results, it has been demonstrated that -adrenergic agonists like norepinephrine or isoproterenol influenced splenocyte proliferation in vivo. However, these data are controversial and it was recorded that the nature of this effect depended on the immunologic compartment investigated, the time of cell activation and the specific lymphocyte subset involved.35,36 Several studies demonstrate that catecholamines modulate cytokine secretion in humans and rodents in vitro and in vivo and this effect was mediated via through activation of -adrenergic receptors.35,37—39 Accordingly, we observed an effect of a -adrenergic blockade on cellular cytokine release and that the pharmacological blockade of -adrenergic receptors modulated the production of the Th1-cytokine IFN-␥ and the Th2-cytokine IL-6 in a different way. IFN-␥ is a pro-inflammatory cytokine and a strong activator of the cellular part of the immune system, especially the activation of macrophages and natural killer (NK)-cells.39 In line, it was demonstrated previously that a -adrenergic blockade inhibits the mobilisation and activation of NK-cells and it can be assumed that the propranolol-induced decrease of IFN-␥ release in our study may be one possible mechanism explaining this effect.6,39 Like IFN-␥, IL-6 is a pro-inflammatory cytokine but in contrast to IFN-␥, IL-6 is a Th2-cytokine and is predominantly an activator of the humoral part of the immune system.39 Interestingly it was reported that a shift from the Th1- to the Th2cytokine response may be a factor that promotes the sepsis-induced immune suppression.39 Our cytokine data support the hypothesis that blockade of -adrenergic receptors may augment this effect. The -adrenergic blockade in our study was paralleled by a modulation of cellular immune functions. Furthermore, the propranolol-treated septic animals displayed a higher loss of body weight and body temperature and a further reduction in urine production 48 h after onset of sepsis. The nature of these effects and its relation to the observed alterations of cellular immune functions cannot be concluded from our results but the observed deterioration in the clinical situation may have contributed to the elevated mortality rate. We did not determine cardiovascular variables in our model of sepsis and the administration of propranolol may have induced unfavorable effects on the cardiovascular system that could have affected the results of the immunological as well as the metabolic data. In contrast, it was demon-
Beta-adrenergic blockade during systemic inflammation strated that the adrenergic modulation of the cellular immune system in healthy human volunteers was independent of an altered cardiac function or mean arterial blood pressure (MAP) and previous data demonstrate that a non-selective as well as a 1 -selective -adrenergic blockade did not affect MAP during systemic inflammation or following haemorrhagic shock in humans and animals.6,14,40—43 In addition to these systemic cardiovascular effects, the administration of propranolol could have affected the local perfusion of the spleen but it has been reported previously that -adrenergic blockade neither increased the vascular resistance of the spleen nor decreased splenic perfusion.44 We therefore assume that propranolol-induced changes of the systemic or local perfusion are unlikely to be responsible for our results but we cannot exclude these effects from our data.
Conclusions We conclude that administration of a non-selective -adrenergic antagonist modulates cellular immune functions in a murine model of sepsis. In view of these data further studies are required to reveal the clinical significance of this effect.
Conflict of interest All the authors certify that there were no financial or personal relationships with other people or organisations that could influence their work inappropriately within 3 years of beginning the work submitted.
Acknowledgement The experiment was supported by an internal grant of the University of Duisburg-Essen, Medical Faculty, Hufelandstr. 55, 45145 Essen, Germany.
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EXPERIMENTAL PAPER
-Adrenergic blockade during systemic inflammation: Impact on cellular immune functions and survival in a murine model of sepsis夽 Daniel Schmitz a, Klaus Wilsenack a, Sven Lendemanns a, Manfred Schedlowski b, Reiner Oberbeck a,∗ a b
Department Trauma Surgery, University Hospital, University Duisburg-Essen, Essen, Germany Institute of Medical Psychology, University Hospital, University Duisburg-Essen, Essen, Germany
Received 5 April 2006 ; received in revised form 22 June 2006; accepted 3 July 2006 KEYWORDS Immune system; Apoptosis; Cytokines; Propranolol; Septic shock; Catecholamines; -Adrenergic receptor; -Adrenergic blockade
Summary Aim of the study: Adrenergic immuno-modulation mediated by -adrenergic receptors has been demonstrated. Pharmacological blockade of -adrenergic receptors is a therapeutic intervention frequently used in critically ill patients. The effect of -adrenergic blockade on cellular immune functions in a critical illness, such as polymicrobial sepsis, has not been investigated. Methods: Male NMRI-mice were subjected to sham operation or to sepsis (caecal ligation and puncture, CLP) following administration of either the non-selective -adrenergic antagonist propranolol (0.5 mg/kg s.c. every 12 h in 1 ml vehicle) or saline 0.9% (1 ml s.c. every 12 h). Mice were kept in metabolic cages and were sacrificed 48 h after induction of sepsis. Survival rate, clinical situation (body weight and temperature, fluid and food intake, urine output), and immunological variables (splenocyte proliferation, apoptosis, and IFN-␥ and IL-6 release) were determined. Results: Administration of propranolol in septic mice increased the splenocyte apoptosis rate, reduced the proliferative capacity of splenocytes, and modulated cellular cytokine release (IL-6, IFN-␥). This was paralleled by a higher loss of body weight and temperature, and a decreased urine output. Furthermore, treatment with propranolol increased the sepsis-induced lethality from 47% up to 68%, respectively. Conclusion: -Adrenergic blockade was accompanied by alterations of cellular immune functions, a deterioration in the clinical situation and a reduced survival in a
夽 A Spanish translated version of the summary of this article appears as Appendix in the final online version at 10.1016/j.resuscitation.2006.07.001. ∗ Corresponding author at: Klinik f. Unfallchirurgie, Universit¨ atsklinikum Essen, Hufelandstr. 55, 45147 Essen, Germany. Tel.: +49 201 723 1301; fax: +49 201 723 5936. E-mail address: [email protected] (R. Oberbeck).
0300-9572/$ — see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.resuscitation.2006.07.001
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murine model of sepsis. These data demonstrate the potential immuno-modulatory effects of -adrenergic antagonists. © 2006 Elsevier Ireland Ltd. All rights reserved.
Introduction Pharmacological blockade of -adrenergic receptors is a frequent therapeutic intervention in critically ill patients.1—3 Examples are the long-term treatment with -adrenergic antagonists due to chronic cardiovascular disease and the use of -adrenergic blocking agents in the treatment of acute cardiac arrhythmias and hypertension.1 Furthermore, a pharmacologic blockade of adrenergic receptors is used to prevent the catecholamine-mediated hypermetabolism in critically ill patients suffering from severe trauma or burn injury. In this group of patients a reduction in cardiac work, resting energy expenditure, and muscle-protein catabolism was recorded after pharmacological -adrenergic blockade.2—4 In addition to these effects it has been demonstrated that the adrenergic system modulated cellular immune functions through activation of -adrenergic receptors.5—9 The potential immunological side effects of a -adrenergic blockade have not been investigated although infectious complications are the leading cause of death in critically ill patients after trauma or burn injury.10—12 In line with this, several reports demonstrate an adrenergic modulation of cellular immune functions that is at least partly mediated via activation or inhibition of -adrenergic receptors.5—9 Accordingly, it was reported that non-selective -adrenergic blockade following administration of the non-selective -adrenergic antagonist propranolol in healthy human volunteers induced a reduction in the numbers of circulating CD8+lymphocytes and natural killer (NK)-cells. Furthermore, this effect was accompanied by inhibition of the antibody-dependent cellular cytotoxicity and the cytotoxic activity of NK-cells.5,6,13 Moreover, administration of the non-selective -adrenergic antagonist propranolol or of the 1 -selective adrenergic antagonist metoprolol in a clinically relevant murine haemorrhagic shock model revealed that administration of either -adrenergic antagonist abolished the haemorrhage-induced increase of circulating CD8+-lymphoyctes and NK-cells and modulated splenocyte apoptosis.14 However, the effect of a -adrenergic blockade on cellular immune functions during systemic inflammation and its impact on the clinical cause of this disease remains to be established.
We therefore investigated the effect of a non-selective -adrenergic blockade on the clinical course and cellular immune functions during polymicrobial sepsis in mice.
Materials and methods Animals and animal care Male NMRI-mice (Charles River Laboratories, Wilmington, MA), 8—9 weeks old with a body weight between 30 and 34 g were used in this study. Mice were kept with a 12 h dark/light cycle and received water and food ad libitum. The experiments were performed in adherence to the guidelines for the Care and Use of Laboratory Animals of the National Institute of Health. The experimental protocol was approved by the local legislative committee.
Drug administration Propranolol (PROP; 0.5 mg/kg) (Dociton, AstraZeneca, Germany) was administered subcutaneously in control mice, in sham-operated mice and in mice subjected to polymicrobial sepsis by CLP. Mice in the septic and the sham-operated groups received PROP at baseline, and 12, 24, 36 and 48 h after laparotomy or CLP with the baseline dosage being given 20 min prior to laparotomy or CLP. Mice in the control group received a single dose of PROP to determine the baseline effects of the -adrenergic antagonist. Referring to the literature the dose of propranolol used in our experiment is the lowest dose that is be effective in blocking -adrenergic activity.15—17
Experimental groups Mice were randomly assigned to one of the following experimental groups. Groups 1—2 (n = 8/group) served as untreated controls (UNT). Group 1 received 0.9% saline (UNT/saline, 0.2 ml) and group 2 received a single dose of propranolol (UNT/PROP; 0.5 mg/kg s.c.). Groups 3—4 underwent a sham operation (laparotomy: 1 cm midline incision without caecal ligation and puncture, SHAM, n = 16/group) without (SHAM/saline, 0.2 ml s.c.) or with administration of propranolol (SHAM/PROP; 0.5 mg/kg/12 h s.c.). Groups 5—6 were subjected
288 Table 1
D. Schmitz et al. Experimental groups
UNT/saline UNT/PROP SHAM/saline SHAM/PROP CLP/saline CLP/PROP
Untreated mice receiving saline 0.9% Untreated mice receiving propranolol (0.5 mg/kg) Sham-operated mice (laparotomy) receiving saline 0.9% Sham-operated mice (laparotomy) receiving propranolol (0.5 mg/kg) Septic mice (caecal ligation and puncture) receiving saline 0.9% Septic mice (caecal ligation and puncture) receiving propranolol (0.5 mg/kg)
to a polymicrobial sepsis induced by a caecal ligation and puncture (CLP, n = 32/group). Group 5 received a subcutaneous injection of 0.2 ml saline (CLP/saline) and Group 6 received propranolol (CLP/PROP, 0.5 mg/kg/12 h s.c.) (Table 1).
Polymicrobial sepsis model Polymicrobial sepsis was induced using the model of caecal ligation and puncture (CLP) described by Baker et al.18 This model leads to a polymicrobial sepsis with positive blood cultures for gram positive and gram negative bacteria as early as 1 h following CLP, and a reproducible mortality rate of 50% after 48 h, depending on the number and size of the puncture holes.18 Briefly, following a light ether anaesthesia a midline laparotomy was performed. After isolation and ligation, the caecum was punctured twice with a 22-gauge needle, and a small amount of the bowel contents was extruded through the puncture holes. After returning the bowel to the abdomen, the midline incision was closed. All animals received 1 ml Ringer subcutaneously at the end of the operation. Sham animals underwent a laparotomy without caecal ligation and puncture. The animals were then housed in metabolic cages in order to monitor the body weight, heart rate, body temperature, fluid intake, urine production and food consumption. Blind observers recorded these clinical findings. Food and water were allowed ad libitum. All mice were subjected to the same fluid-resuscitation regimen with subcutaneous administration of 1 ml Ringer solution twice a day (08:00 a.m. and 08:00 p.m.). Forty-eight hours after induction of sepsis, or following sham operation, blood was drawn by cardiac puncture and immediately thereafter the spleens were removed aseptically and placed in petri dishes containing ice-cooled phosphate-buffered saline solution (PBS, pH 7.4). All mice were sacrificed at
the same time of day (8 a.m.) to avoid fluctuations due to circadian rhythm. Determination of clinical data and immunologic variables were performed by investigators blinded with regard to the surgical procedure and treatment of experimental groups.
Clinical data Clinical data were obtained using metabolic cages. Body temperature, body weight, food and fluid intake, urine output and faces production were recorded every 12 h in control mice and after induction of either sham-operation or CLP.
Preparation of splenocyte culture and assessment of proliferative capacity After blood withdrawal animals were sacrificed by cervical dislocation and the spleen was removed aseptically. Immediately after removal of the spleen the organ was placed in ice-cooled petri dishes containing ice-cooled phosphate-buffered saline solution (PBS; 4 ◦ C, pH 7.4). Then the organ was punctured with a 20-gauge needle connected to a 20 ml syringe containing ice-cooled PBS solution (4 ◦ C, pH 7.4). A cell suspension was produced by gently flushing the organ with the ice-cooled PBS solution. The suspension was freed from debris by centrifugation at 300 × g for 15 min. Thereafter, erythrocytes were lysed hypotonically, and the remaining cells were washed two times with ice-cooled PBS (4 ◦ C, pH 7.4) by repeated centrifugation (300 × g, 15 min). The ability of the splenocytes to produce lymphokines in response to a mitogenic challenge was assessed in one portion of the cell suspension by incubation for 48 h (at 37 ◦ C, 5% CO2 , and 90% humidity) in the presence of 2.5 g/ml concanavalin A (Sigma—Aldrich Chemicals, Beisenh¨ ofen, Germany). After this incubation period, the cell suspension was centrifuged at 300 × g for 10 min, and the supernatants were harvested, divided into aliquots and stored at −70 ◦ C until assayed for IL-2 and IL-6 concentrations. The second portion of the cell suspension was used to determine the splenocyte proliferative capacity in response to mitogenic stimulation. The splenocyte cell suspension was placed in a 96-well microtiter plate in aliquots of 100 l. The cells ability to proliferate in response to mitogenic stimulation with 0 g/ml (negative control) or 2.5 g/ml Con A was determined after 48 h incubation at 37 ◦ C, 5% CO2 and 90% humidity. The extent of proliferation was determined by 3 H-thymidine incorporation as described previously.19
Beta-adrenergic blockade during systemic inflammation
Assessment of cytokine release The capacity of mixed splenocyte cultures to produce IFN-␥ and IL-6 was assessed by determination of the concentration of these cytokines in the collected culture supernatant 48 h after incubation with 2.5 g/ml Con A using commercial ELISAassays (Biosource International, Nivelles, Belgium; Endogen Inc., Woburn, MA, USA). The sensitivity of the IFN-␥ ELISA was <4 pg/ml. The inter-assay coefficient of variance was <10% and the intra-assay coefficient of variance <4.5%. The sensitivity of the IL-6 ELISA was <3 pg/ml, the inter-assay coefficient of variance was <10% and the intra-assay coefficient of variance was <10%.
Assessment of splenocyte apoptosis and total viable cell yield After isolation of splenocytes, the cells were washed twice with ice cold PBS (4 ◦ C, pH 7.4) and then resuspended in binding buffer at 4 ◦ C. Cell concentration was adjusted to 1 × 106 cells/ml. One millilitre of this cell suspension was placed into 1.5 ml microcentrifuge tubes for cell staining. FITC-conjugated recombinant Annexin V (Annexin V-FITC, PharMingen, San Diego, USA) and/or propidium iodide (PI) staining solution was added to 100 l of the cell solution with the cells subsequently incubated for 15 min at room temperature in the dark. Immediately following the incubation period cells were analyzed for Annexin V binding and PI-incorporation by flow cytometry. Determination of the total viable cell count was performed by trypan blue exclusion. Total viable cell yield was performed using a microscope, and was conducted by an investigator experienced with this method. A number of 106 cells/ml was counted.
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Furthermore, two-colour fluorescence analyses of splenocyte subpopulations were performed using FITC-conjugated Annexin V (Annexin V-FITC, PharMingen, San Diego, USA) and the commercially available Abs Anti-CD4 (PE), Anti-CD8 (PE), AntiNK1.1 (PE), F4/80 (PE) (Biozol Diagnostica, Eching, Germany).
Statistical analysis Results are presented as means ± S.E.M. Data were analyzed for significance by one-way ANOVA including post-hoc analysis (Scheff´ e-test and Bonferronicorrection). The Kaplan—Meier and Cox regression test were used for comparing survival. A p-value < 0.05 was considered statistically significant. Statistical analysis were performed by using the software SPSS (version 12.0 for windows).
Results Every CLP-experiment was conducted twice with 16 animals/group. For each septic group similar results were found in both experiments, so the data were pooled. Sepsis induced a lethality rate of 47% 48 h after induction of CLP (p < 0.001, CLP versus SHAM). Administration of propranolol was accompanied by a significant increase of sepsisinduced lethality (68%; p < 0.01, CLP/PROP versus CLP/saline) (Figure 1). Concerning the clinical data no significant differences were found comparing the control and the sham-operated groups (UNT versus SHAM/saline; UNT versus SHAM/PROP; SHAM/saline versus SHAM/PROP). A statistically significant reduction in body temperature, body weight, fluid intake, and
Figure 1 Survival rate of mice subjected to either sham operation (SHAM) or sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of the -adrenergic antagonist propranolol.
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Table 2 Metabolic data (body temperature and weight, fluid and food intake, urine output) of mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of non-selective -adrenoceptor antagonist propranolol
SHAM/saline SHAM/PROP CLP/saline CLP/PROP
body temperature (◦ C)
body weight (g)
fluid intake (ml)
food intake (g)
urine output (ml)
1.17* a (±0.8) 0.9* b (±1) −7.47* b (±6.7) −9.08 (±5.2)
−1.1* a (±0.1) −0.9* b (±0.2) −2.9** b (±0.1) −4.9 (±0.07)
8.25*** a (±0.17) 10.62*** b (±0.29) 4.29 (±0.29) 3.39 (±0.21)
7.62# a (±0.13) 8.85# b (±0.22) 0.76 (±0.15) 0.52 (±0.1)
1.8 (±0.4) 2.2 (±0.6) 1.1* b (±0.6) 1.4 (±0.7)
a: vs. CLP/saline; b: vs. CLP/PROP. * p < 0.05; ** p < 0.005;
*** p < 0.01; # p < 0.001.
food intake was found in saline and propranololtreated septic animals compared with the shamoperated groups. Furthermore, administration of propranolol in septic animals was accompanied by a significant reduction in body temperature, body weight and urine output (Table 2). Monitoring of splenocyte proliferation by determination of H3 -thymidine incorporation following stimulation with Con A revealed a sepsis-induced inhibition of splenocyte proliferative ability 48 h after onset of sepsis (p < 0.0001, SHAM/saline versus CLP/saline). Administration of propranolol in septic mice augmented the sepsis-induced inhibition of splenocyte proliferation significantly (CLP/saline versus CLP/PROP, p < 0.05) (Figure 2). Splenocyte apoptosis, determined by FACScan analysis of splenocyte Annexin V binding capacity, revealed an increased splenocyte apoptosis in
saline-treated septic animals 48 h after induction of sepsis (p < 0.05, SHAM/saline versus CLP/saline). This increase in splenocyte apoptosis was further increased in propranolol-treated septic mice (p < 0.001, CLP/saline versus CLP/PROP) (Figure 3). The results obtained for the total viable cell count corresponded inversely to the results reported for splenocyte Annexin V binding capacity (data not shown). Induction of CLP in saline-treated animals was accompanied by a significant decrease in splenocyte IFN-␥ and IL-6 release (p < 0.05, CLP/saline versus SHAM/saline). Administration of propranolol induced a further inhibition of IFN-␥ release (p < 0.001, CLP/saline versus CLP/PROP) but attenuated the sepsis-induced inhibition of splenocyte IL-6 release (p < 0.05, CLP/saline versus CLP/PROP) (Figure 4).
Figure 2 Splenocyte proliferation recorded in mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of propranolol. Data are presented as percentage of changes compared to untreated animals with means ± S.D.
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Figure 3 Splenocyte apoptosis determined in mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of the -adrenergic antagonist propranolol. Data are presented as percentage of changes compared to untreated animals with means ± S.D.
Figure 4 Determination of IFN-␥ and IL-6 concentrations in the supernatant of cultured splenocytes obtained from mice 48 h after sham operation (SHAM) or induction of sepsis (CLP) with (SHAM/PROP; CLP/PROP) or without (SHAM/saline; CLP/saline) administration of the -adrenergic antagonist propranolol. Data are presented as percentage of changes compared to untreated animals with means ± S.D.
Discussion Pharmacological blockade of -adrenergic receptors is a frequently used therapeutic intervention in the critically ill.1,2 This includes the treatment of acute cardiovascular events like arrhythmia and hypertension as well as the long term administration of -adrenergic antagonists for the treatment
of chronic diseases.1 Furthermore, pharmacological blockade of -adrenergic receptors is recommended to prevent catecholamine-induced hypermetabolism in patients suffering from severe burn injury and the beneficial effect of pharmacological -adrenergic blockade on muscle protein turn-over in severely burned patients has been demonstrated recently.2,3
292 In addition, it is well established that adrenergic receptors are involved in the modulation of the cellular immune system.5—8,14 Interestingly, the potential immunological side effects of a adrenergic blockade in critically ill patients has not been investigated yet although it is known that the majority of these patients develop infectious complications, in particular sepsis due to compromised activity of the immune system.8,10—12 Apoptosis is a form of programmed cell death that is widely distributed among the cells of the immune system.20,21 Some characteristic features of apoptosis are DNA fragmentation, induction of cytosolic proteases, and changes in the cellular plasma membrane.22 The translocation of phospholipids from the inner to the outer leaflet of the plasma membrane is an important step during the early phase of apoptosis and this can be detected by Annexin V binding.22 In line with previous reports about adrenergic immuno-modulation we observed an increased splenocyte apoptosis rate following the administration of a -adrenergic antagonist in our model of polymicrobial sepsis. Several factors are known to trigger apoptotic cell death and it was noticed that the exposure or withdrawal of hormones and neurotransmitters affected cellular apoptosis.23—25 Accordingly, it was reported that catecholamines are capable of modulating lymphocyte apoptosis and that this process involved -adrenergic receptors.23,25 In extension of these data we observed an increase of splenocyte apoptosis following an inhibition of the -adrenergic receptor subtype in a murine model of sepsis. However, it cannot be concluded from our results if this was a direct receptor-mediated effect on the cellular level or if this was consequence of an indirect mechanism, for example a reduced recognition and removal of apoptotic cells by macrophages or reticular endothelial cells.21,26 Interestingly, we observed an increased cellular apoptosis in saline-treated septic mice. It is well known that alterations of cellular apoptosis can be observed during sepsis and a large number of studies demonstrated a sepsis-induced increased in apoptotic cell death. However, data concerning the effect of systemic inflammation on cellular apoptosis are controversial since a reduced cellular apoptosis was reported by other investigators. We assume that these differences can be attributed to differences in the cell types investigated or may reflect differences in the models used in these studies.27—34 Concerning the proliferative activity of splenocytes we observed that pharmacological blockade of -adrenergic receptors was paralleled by an aug-
D. Schmitz et al. mented inhibition of cellular proliferation. Reviewing the literature, only few data are available about -adrenergic modulation of immune cell proliferation. In line with our results, it has been demonstrated that -adrenergic agonists like norepinephrine or isoproterenol influenced splenocyte proliferation in vivo. However, these data are controversial and it was recorded that the nature of this effect depended on the immunologic compartment investigated, the time of cell activation and the specific lymphocyte subset involved.35,36 Several studies demonstrate that catecholamines modulate cytokine secretion in humans and rodents in vitro and in vivo and this effect was mediated via through activation of -adrenergic receptors.35,37—39 Accordingly, we observed an effect of a -adrenergic blockade on cellular cytokine release and that the pharmacological blockade of -adrenergic receptors modulated the production of the Th1-cytokine IFN-␥ and the Th2-cytokine IL-6 in a different way. IFN-␥ is a pro-inflammatory cytokine and a strong activator of the cellular part of the immune system, especially the activation of macrophages and natural killer (NK)-cells.39 In line, it was demonstrated previously that a -adrenergic blockade inhibits the mobilisation and activation of NK-cells and it can be assumed that the propranolol-induced decrease of IFN-␥ release in our study may be one possible mechanism explaining this effect.6,39 Like IFN-␥, IL-6 is a pro-inflammatory cytokine but in contrast to IFN-␥, IL-6 is a Th2-cytokine and is predominantly an activator of the humoral part of the immune system.39 Interestingly it was reported that a shift from the Th1- to the Th2cytokine response may be a factor that promotes the sepsis-induced immune suppression.39 Our cytokine data support the hypothesis that blockade of -adrenergic receptors may augment this effect. The -adrenergic blockade in our study was paralleled by a modulation of cellular immune functions. Furthermore, the propranolol-treated septic animals displayed a higher loss of body weight and body temperature and a further reduction in urine production 48 h after onset of sepsis. The nature of these effects and its relation to the observed alterations of cellular immune functions cannot be concluded from our results but the observed deterioration in the clinical situation may have contributed to the elevated mortality rate. We did not determine cardiovascular variables in our model of sepsis and the administration of propranolol may have induced unfavorable effects on the cardiovascular system that could have affected the results of the immunological as well as the metabolic data. In contrast, it was demon-
Beta-adrenergic blockade during systemic inflammation strated that the adrenergic modulation of the cellular immune system in healthy human volunteers was independent of an altered cardiac function or mean arterial blood pressure (MAP) and previous data demonstrate that a non-selective as well as a 1 -selective -adrenergic blockade did not affect MAP during systemic inflammation or following haemorrhagic shock in humans and animals.6,14,40—43 In addition to these systemic cardiovascular effects, the administration of propranolol could have affected the local perfusion of the spleen but it has been reported previously that -adrenergic blockade neither increased the vascular resistance of the spleen nor decreased splenic perfusion.44 We therefore assume that propranolol-induced changes of the systemic or local perfusion are unlikely to be responsible for our results but we cannot exclude these effects from our data.
Conclusions We conclude that administration of a non-selective -adrenergic antagonist modulates cellular immune functions in a murine model of sepsis. In view of these data further studies are required to reveal the clinical significance of this effect.
Conflict of interest All the authors certify that there were no financial or personal relationships with other people or organisations that could influence their work inappropriately within 3 years of beginning the work submitted.
Acknowledgement The experiment was supported by an internal grant of the University of Duisburg-Essen, Medical Faculty, Hufelandstr. 55, 45145 Essen, Germany.
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