Abstract
The present study was designed to investigate whether serum of animals subjected to hypoxaemic resuscitation from haemorrhagic shock may be a weak stimulant for monocytes or not. Twenty rabbits were subjected to haemorrhagic shock after blood exsanguination; resuscitation was performed by infusion of the shed blood in eight rabbits under normoxaemic conditions (NormoxRes) and in 12 under hypoxaemic conditions (HypoxRes); seven rabbits were subjected to sham operation. Malondialdehyde (MDA) and tumour necrosis factor (TNF)-α were estimated in serum at serial time intervals; the serum was applied for stimulation of U937 monocytes with or without the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Expression of triggering receptor expressed on myeloid cells-1 (TREM-1) on U937 was also assessed by flow cytometric analysis. Death supervened in four animals of the NormoxRes (50%) and in one animal of the HypoxRes group (8·33%, P: 0·032). Serum levels of TNF-α and MDA were higher in NormoxRes compared to HypoxRes animals. Expression of TREM-1 on U937 monocytes was similar after stimulation with serum sampled from both groups. Concentrations of interleukin (IL)-1β, IL-6 and IL-8 of monocyte supernatants were higher after stimulation with serum of NormoxRes than HypoxRes rabbits. Production of cytokines after stimulation with serum was decreased significantly after addition of SB203580. It is concluded that stimulation of monocytes may contribute to the generation of the systemic inflammatory response during reperfusion after ischaemia. Lower stimulation of the p38 MAPK-mediated production of IL-1β, IL-6 and IL-8 by monocytes may be implicated as an explanation for the benefits shown for the host when resuscitation is performed under hypoxaemic conditions.
Keywords: cytokines, hypoxaemic resuscitation, malondialdehyde, monocytes
Introduction
Reperfusion following a period of ischaemia leads to stimulation of the inflammatory response of the host. Theories of pathogenesis implicate oxygen free radial formation following reperfusion as a mainstay for the production of proinflammatory mediators [1]. Former studies of our group have shown that after occlusion of the splanchnic circulation, reperfusion mediated through hypoxaemic conditions may decrease the degree of inflammation in the heart and the intestine in the animal porcine model [2, 3]. Similar results were taken after cerebral ischaemia and hypoxaemic reperfusion in pigs [4, 5]. Results from a recent study in a model of haemorrhagic shock in rats concluded that the benefit of the host is connected to a decrease of proinflammatory mediators when resuscitation is performed under hypoxaemic conditions [6].
The present study was designed to provide a mechanism for the attenuation of the inflammatory response following hypoxaemic resuscitation in a model of haemorrhagic shock in rabbits. The underlying concept was that monocytes are usually the reservoir of proinflammatory mediators in acute inflammatory reactions. As a consequence, monocytes of a human cell line were exposed to serum samples drawn from animals at consecutive time intervals of resuscitation in order to understand whether serum contains factors able to induce direct stimulation of monocytes.
Materials and methods
Animals
The study was approved by the Veterinary Directorate of the Prefecture of Athens according to Greek legislation in conformity with the 160/1991 Council Directive of the European Union. A total of 27 adult male New Zealand white rabbits of 3·1–3·4 kg body weight were used, fasted overnight with access to water ad libitum. They were premedicated with ketamine (35 mg/kg) and xylazine (5–10 mg/kg) intramuscularly.
Study design
A marginal ear vein was cannulated, tracheostomy was performed and mechanical ventilation was instituted on a volume mode using a Siemens 900 respirator. Using a tidal volume of 8 ml/kg the frequency was adjusted to maintain PaCO2 at 33–37 mmHg. A mixture of air and oxygen was administered to keep PaO2 at 95–105 mmHg. Anaesthesia was maintained using the aforementioned doses of ketamine and xylazine given intramuscularly every 90 min.
Left and right carotid arteries and left internal jugular vein were catheterized for blood pressure monitoring, blood withdrawal for shock induction and reinfusion of shed blood for shock resuscitation, respectively. The central body temperature was kept between 38 and 39°C with the aid of an electric blanket.
A stabilization period of 30 min followed the termination of the experimental set-up. Next, shock was induced by withdrawing blood from the left carotid in aliquots of 1·5 ml per min in order to reduce the mean arterial pressure (MAP) to 40 mmHg over the next 30 min. From that point ‘haemorrhagic shock’ began and MAP was maintained at 40 mmHg over the next 60 min by withdrawing or reinjecting blood as required. Syringes with the shed blood were put on a horizontal rotator at 37°C at 100 g.
After the 60 min of shock, animals were allocated randomly to two groups: those receiving normoxaemic (PaO2 = 95–105 mmHg, Normox-Res group, n = 8) and those receiving hypoxaemic (PaO2 = 35–40 mmHg, Hypox-Res group, n = 12) shock resuscitation following a chart of randomization. Another seven animals served as a sham group undergoing all experimental procedures with the exception of shock. During resuscitation the Normox-Res group was ventilated with FiO2 = 0·21–0·28 to achieve a PaO2 at the referred level, while in the Hypox-Res group the PaO2 was lowered to 35–40 mmHg by reducing FiO2 to 0·08–0·10. Shock resuscitation began by infusing the shed blood via a pump over the same period as the induction of shock, i.e. 30 min. From 30 to 60 min of resuscitation Ringer lactate was administered, if needed, to maintain MAP at the baseline level. Concurrently, FiO2 was gradually increased by 0·02 every 10 min in the Hypox-Res group to achieve normoxaemia by the 60 min of resuscitation. From 60 to 120 min of resuscitation no further fluid was administered in order to avoid pulmonary over hydration and oedema.
In all animals, 1 ml of blood was collected after venipuncture of their ear vein. Sampling was performed at the beginning of the induction of shock, at the end of shock and at 30, 60, 90 and 120 min after the start of resuscitation. Animals were then followed-up for another 6 h, i.e. for a total of 8 h from the start of resuscitation and survival was recorded. The animals were then killed by the intravenous administration of sodium thiopental to avoid suffering.
Laboratory techniques
The serum levels of tumour necrosis factor (TNF)-α were measured by rabbit enzyme-linked immunosorbent assay (ELISA). The reagents were provided by the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, UK).
Lipid peroxidation in serum was estimated by the concentration of MDA, as already described [7]. Briefly, a 0·1 ml aliquot of each sample was mixed with 0·9 ml of trichloroacetic acid 20% (Merck, Darmstadt, Germany) and centrifuged at 12 000 g and 4°C for 10 min. The supernatant was removed and incubated with 2 ml of thiobarbituric acid 0·2% (Merck) for 60 min at 90°C. After centrifugation, 10 μl of the supernatant was injected into a high-performance liquid chromatography system (HPLC; Agilent 1100 Series, Waldbronn, Germany) with the following characteristics of elution: Zorbax Eclipse XDB-C18 (4·6 × 150 mm, 5 μm) column under 37°C; mobile phase consisting of a 50 mM K3PO4 (pH: 6·8) buffer and methanol 99% at a 60 : 40 ratio with a flow rate of 1 ml/min; fluorometric detection with signals of excitation at 515 nm and emission at 535 nm. The retention time of MDA was 3·5 min and was estimated as μmol/ml by a standard curve created with 1, 1, 3, 3-tetramethoxy-propane (Merck). All determinations were performed in duplicate.
In order to estimate the ability of serum samples to stimulate human monocytes, cells of the U937 human monocytic cell line were exposed to each one of drawn samples. Confluent cells were washed thoroughly with Hank's solution and distributed in a 96-well plate at a density of 1 × 105 cells per well at a final volume of 200 μl. Culture media in cells consisted of RMPI-1640 supplemented with 2 mM of glutamine. In each well serum samples were added at a 1 : 1 ratio. The plate was incubated at 37°C in a 5% CO2 atmosphere. After 2 h the plate was centrifuged for 8 min at 1400 g at room temperature; the supernatant was collected for estimation of cytokines, whereas attached monocytes were resuspended in 200 μl of phosphate-buffered saline (PBS), pH 7·2. They were then incubated with phycoerythrin (PE)-conjugated monoclonal antibodies anti-IgG1 or anti-triggering receptor expressed on myeloid cells-1 (TREM-1), emission 520 nm (R&D, Minneapolis, USA) for 40 min at 4°C. Expression of TREM-1 receptor on the cell membrane of U937 was assessed after analysis with the EPICS XL/MSL flow cytometer (Beckman Coulter Co., Miami, FL, USA), using unstained cells and cells stained with IgG-PE as negative controls. Results were given as the mean fluorescence intensity (MFI) of TREM-1 on U937 monocytes.
Cytokine stimulation was repeated as described above with serum collected at 60, 90 and 120 min in the absence and presence of 3 μM of SB203580 (Sigma, St. Louis, MO, USA). This is a p38 mitogen-associated protein kinase (MAPK) inhibitor. U937 monocytes were pretreated for 1 h with SB203580 before the addition of serum [8, 9].
Cytokine measurements were performed in the supernatant by flow cytometry using cytometric bead array (CBA) technology [10]. A human inflammation CBA kit (BD Biosciences, San Jose, CA, USA) was used to measure quantitatively interleukin (IL)-8, IL-1β, IL-6, IL-10, TNF-α and IL-12p70 levels. The sensitivity of human inflammation CBA is comparable to conventional ELISA [11]. Samples were analysed using a BD fluorescence activated cell sorter (FACSCalibur) flow cytometer (BD Biosciences), according to the manufacturer's instructions. Corresponding detection limits were: 2·5, 2·1, 1·7, 1·8, 1·1 and 1·3 pg/ml, respectively.
To exclude serum cytotoxicity as a cause of changes of cytokine production by U937 monocytes, experiments in the absence and presence of SB203580 were repeated with serum sampled at 120 min of resuscitation from five animals of the Normox-Res and another five animals of the Hypox-Res groups. That time of sampling was selected because changes in cytokine production were found mainly with the usage of these samples. At the end of the 2-h incubation period, plates were centrifuged as above and the cell pellet was diluted with 200 μl of PBS, pH 7·2. Cell viability was more than 95%, as assessed after trypan blue exclusion of dead cells.
To evaluate the cross-reactivity of rabbit monocytes and human U937 monocytes, peripheral blood mononuclear cells (PBMCs) were isolated from three different healthy rabbits after gradient centrifugation of heparinized whole blood over Ficoll Hypaque (Biochrom, Berlin, Germany). After three washings with ice-cold PBS, pH 7·2, PBMCs were distributed in wells of a 96-well plate at a density of 5 × 105/well with or without stimulation by 5 μg/ml of Pam3Cys (EMC Microcollections GmbH, Tübingen, Germany) and 10 ng/ml of lipopolysaccharide (LPS) of Escherichia coli O111:B4 (Sigma). Pam3Cys and LPS are specific ligands of Toll-like receptor (TLR)2 and TLR4, respectively [12]. The final volume per well was 200 μl and the applied medium was RPMI-1640 enriched with 2 mM of glutamine. The whole experiment was performed in duplicate. After 24 h of incubation, wells were centrifuged at 1400 g for 8 min and the supernatants were collected. TNF-α was estimated by the above-described enzyme immunoabsorbent assay. The same procedure was repeated using U937 monocytes at a density of 5 × 105 cells/well. TNF-α was estimated in supernatants by an enzyme immunoabsorbent assay (R&D Inc.) with 6·25 pg/ml as the lowest limit of detection.
Statistical analysis
Results were expressed as means ± standard error (s.e.). Comparisons between groups were performed by analysis of variance (anova) with post-hoc Bonferroni analysis. The effect of SB203580 on cytokine stimulation was assessed by Wilcoxon's rank sum test. Survival rate at 8 h was compared between groups by Pearson's χ2 test. Any P-value below 0·05 was considered statistically significant.
Results
None of the sham-operated rabbits died. Death supervened during follow-up in four animals of the NormoxRes group (50%) and in one animal of the HypoxRes group (8·33%, P between groups 0·032).
Serum concentrations of TNF-α and MDA for sham-operated rabbits and for the NormoxRes and HypoxRes groups of animals are given in Table 1. Both TNF-α and MDA were higher in the NormoxRes group compared to the HypoxRes group at 30, 60, 90 and 120 min.
Table 1.
Serum concentrations of tumour necrosis factor (TNF)-α and of malondialdehyde (MDA) at serial time intervals of rabbits undergoing normoxaemic (NormoxRes) and hypoxaemic (HypoxRes) resuscitation. Data of sham-operated rabbits are also provided.
| Time (min) | Sham (n = 7) | NormoxRes (n = 8) | HypoxRes (n = 12) |
|---|---|---|---|
| TNF-α (mean ± s.e., pg/ml) | |||
| Before | 17·1 ± 5·8 | 25·5 ± 5·3 | 22·8 ± 8·9 |
| 0 | 31·3 ± 7·1 | 56·1 ± 10·1 | 37·5 ± 7·8 |
| 30 | 48·7 ± 7·8 | 144·3 ± 22·3†‡ | 72·3 ± 13·9‡ |
| 60 | 65·1 ± 10·7 | 265·5 ± 38·1†‡ | 94·1 ± 11·1‡ |
| 90 | 100·0 ± 24·9 | 404·5 ± 38·2†‡ | 141·3 ± 20·7‡ |
| 120 | 160·7 ± 45·2 | 574·7 ± 48·9†‡ | 234·8 ± 31·7‡ |
| MDA (mean ± s.e., nmol/ml) | |||
| Before | 0·53 ± 0·04 | 0·83 ± 0·05 | 0·87 ± 0·08 |
| 0 | 0·86 ± 0·02 | 1·69 ± 0·09 | 0·92 ± 0·09 |
| 30 | 0·92 ± 0·04 | 2·23 ± 0·06†‡ | 0·89 ± 0·13§ |
| 60 | 0·98 ± 0·05 | 2·62 ± 0·08†‡ | 1·05 ± 0·09¶ |
| 90 | 1·07 ± 0·09 | 2·90 ± 0·10†‡ | 1·00 ± 0·12§ |
| 120 | 1·09 ± 0·11 | 3·31 ± 0·84†‡ | 0·95 ± 0·13§ |
P < 0·001 compared to the HypoxRes group
P < 0·001 compared to 0 time within the same group
P n.s. (non-significant) compared to 0 time within the same group
P: 0·012 compared to 0 time within the same group.
Expression of TREM-1 on U937 monocytes is shown in Fig. 1. No differences were found between stimulation with serum sampled at different times of resuscitation from the studied groups. Concentrations of TNF-α and IL-10 in supernatants of cultures of U937 monocytes are shown in Fig. 1. Increases of both TNF-α and IL-10 compared to baseline were found after stimulation with serum sampled 60, 90 and 120 min of resuscitation of either the NormoxRes or the HypoxRes rabbits (P < 0·001). However, no differences were found between the two groups.
Fig. 1.
Expression of triggering receptor expressed on myeloid cells-1 (TREM-1) on U937 monocytes and concentrations of tumour necrosis factor (TNF)-α and interleukin (IL)-10 in supernatants of U937 monocytes after stimulation with sera drawn at the indicated time intervals. Sham (n = 7); normoxaemic resuscitation (Normox-Res, n = 8); hypoxaemic resuscitation (Hypox-Res, n = 12).
Concentrations of IL-1β, IL-6, IL-8 and IL-12p70 in supernatants of cultures of U937 monocytes are shown in Fig. 2. Concentrations of IL-1β were increased compared to baseline after stimulation by sera sampled after the start of resuscitation, reaching statistical significance with samples of 90 and 120 min of resuscitation (P < 0·001). That increase involved both NormoxRes and HypoxRes animals. However, those of HypoxRes at 120 min were lower than those of NormoxRes. Similar kinetics were found for both IL-6 and IL-8; in the latter cases differences between the NormoxRes and the HypoxRes groups were found after stimulation with serum sampled at 90 and 120 min of resuscitation. IL-12p70 was increased compared to baseline after stimulation with serum drawn at 60, 90 and 120 min of resuscitation of either NormoxRes or HypoxRes rabbits (P < 0·001). However, no differences were found between the two groups.
Fig. 2.
Concentrations of interleukin (IL)-1β, IL-6, IL-8 and IL-12p70 in supernatants of U937 monocytes after stimulation with sera drawn at the indicated time intervals. Sham (n = 7); normoxaemic resuscitation (Normox-Res, n = 8); hypoxaemic resuscitation (Hypox-Res, n = 12). aP < 0·001 between Normox-Res and Hypox-Res at the indicated time interval.
All production of cytokines after stimulation with serum drawn at 60, 90 and 120 min was blocked completely after the addition of SB203580 with the exception of IL-8, which was decreased significantly (Table 2).
Table 2.
Modulation of cytokine release by U937 mocoytes after stimulation with serum drawn at 60, 90 and 120 min by rabbits undergoing normoxaemic (NormoxRes) and hypoxaemic (HypoxRes) resuscitation by the p38 inhibitor SB203580.
| NormoxRes | HypoxRes | |||
|---|---|---|---|---|
| (h) | −SB203580 | +SB203580 | −SB203580 | +SB203580 |
| TNF-α (pg/ml, mean ± s.e.) | ||||
| 60 | 18·7 ± 2·9 | < 1·1* | 18·5 ± 1·9 | < 1·1* |
| 90 | 32·3 ± 4·5 | < 1·1* | 25·8 ± 2·7 | < 1·1* |
| 120 | 48·3 ± 7·7 | < 1·1* | 38·3 ± 4·4 | < 1·1* |
| IL-10 (pg/ml, mean ± s.e.) | ||||
| 60 | 18·6 ± 3·9 | < 1·8* | 19·5 ± 2·2 | < 1·8* |
| 90 | 39·2 ± 5·6 | < 1·8* | 33·4 ± 3·8 | < 1·8* |
| 120 | 54·9 ± 11·6 | 1·9 ± 0·8* | 46·1 ± 5·1 | < 1·8* |
| IL-1β (pg/ml, mean ± s.e.) | ||||
| 60 | 134·1 ± 17·5 | < 2·1* | 144·8 ± 10·2 | < 2·1* |
| 90 | 184·8 ± 25·1 | < 2·1* | 171·1 ± 14·7 | < 2·1* |
| 120 | 298·3 ± 57·9 | < 2·1* | 248·9 ± 26·5 | < 2·1* |
| IL-6 (pg/ml, mean ± s.e.) | ||||
| 60 | 78·3 ± 8·7 | < 2·1* | 62·7 ± 7·9 | < 2·1* |
| 90 | 153·8 ± 25·9 | < 2·1* | 97·2 ± 12·8 | < 2·1* |
| 120 | 312·3 ± 58·9 | < 2·1* | 150·6 ± 16·7 | < 2·1* |
| IL-8 (pg/ml, mean ± s.e.) | ||||
| 60 | 1041·3 ± 162·8 | 477·3 ± 46·3* | 832·5 ± 103·7 | 195·6 ± 30·3* |
| 90 | 1726·3 ± 262·5 | 243·2 ± 31·8* | 882·5 ± 92·5 | 597·4 ± 54·1* |
| 120 | 2847·3 ± 369·5 | 742·9 ± 45·7 | 960·7 ± 81·8 | 300·6 ± 27·3* |
| IL-12p70 (pg/ml, mean ± s.e.) | ||||
| 60 | 28·3 ± 4·7 | < 1·3* | 34·6 ± 4·4 | < 1·3* |
| 90 | 37·1 ± 7·8 | < 1·3* | 52·4 ± 4·87 | < 1·3* |
| 120 | 65·2 ± 15·4 | < 1·3* | 68·2 ± 5·9 | < 1·3* |
P < 0·05 compared to the presence of SB203580. IL: interleukin; TNF: tumour necrosis factor.
During resuscitation, animals of the Normox-Res group required a greater volume of Ringer's lactate to achieve MAP baseline levels compared with hypoxaemic animals. This is an advantageous point for hypoxaemic resuscitation.Concentrations of serum factors and therefore stimulation for cytokine production should be inferior in normoxaemic animals because of higher-volume resuscitation due to dilution by the added amount of exogenous fluids, and not the contrary.
Mean TNF-α release by PBMCs isolated from three rabbits was 65, 651 and 1120 pg/ml after stimulation with medium, Pam3Cys and LPS, respectively. Respective release of TNF-α by U937 monocytes was < 6·25, 690 and 220 pg/ml. These results show that rabbit monocytes reacted in a similar fashion to U937 monocytes after stimulation with TLR ligands.
Discussion
The main finding of the present study was that resuscitation under hypoxaemic conditions was accompanied by a significant reduction of mortality in an experimental setting compared with animals that underwent resuscitation under normoxaemic conditions. The study was designed to provide evidence not only about the impact of hypoxaemic resuscitation on survival, but also about the underlying mechanism explaining survival benefit.
Resuscitation following haemorrhagic shock is considered to be accompanied by a series of events leading to proinflammatory phenomena. Although it is conceived as a sequelum of the generation of oxygen free radicals during the period of resuscitation, the role of innate immunity in that process is not elucidated fully. Monocytes produce the main bulk of proinflammatory cytokines in the immediate immune response of the host to an exogenous insult [13].
The present study showed that the sera of rabbits sampled during the period of resuscitation from haemorrhagic shock could stimulate monocytes for the production of proinflammatory and anti-inflammatory cytokines (Figs 1 and 2). The potency of serum for that cellular response increased in parallel to the duration of resuscitation. It might be hypothesized that the sera of animals after 60 min of resuscitation may contain a variety of substances that could act as ligands for receptors located on monocytes. The most important of these receptors are TLRs that recognize phylogenically conserved structures of bacteria [14]. TLRs are also involved in the adrenal response following trauma or shock [15]. A recent study by Thobe et al. showed that haemorrhagic shock followed by resuscitation with Ringer's lactate resulted in hyperactivity of liver Kupffer cells to respond to stimulation of TLRs with the production of proinflammatory cytokines. This was accompanied by elevation of intracellular levels of the p38 MAPK and was inhibited after addition of SB203580, which is a p38 MAPK inhibitor [16]. MAPK p38 had already been implicated as an independent cascade leading to production of proinflammatory cytokines after trauma [17]. The significance of TLR-stimulated pathways for the induction of inflammatory phenomena after haemorrhagic shock resuscitation derives from studies with TLR4-deficient mice. These mice failed to develop liver injury following shock and resuscitation [18].
The results of the present study, showing a marked reduction of cytokines produced after stimulation of U937 monocytes by SB203580 (Table 2), are in accordance with these former findings for the role of the production of cytokines through p38 MAPK in resuscitation under normoxaemic conditions. Our findings render it possible that stimulants of TLRs may circulate after resuscitation and that the application of hypoxaemic conditions makes their effect unlikely.
Former studies of our group have shown less histopathological damage of the host when resuscitation was performed under hypoxaemic than normoxaemic conditions [2–5]. This was supported additionally by the finding of lower concentrations of malondialdehyde, which is produced when oxygen free radicals attack phospholipids embedded in cell membranes [6]. The results of serum MDA in the present study agree with our former studies (Table 1). Another possible explanation may rely on the decreased biosynthesis of IL-1β, of IL-6 and of IL-8 by monocytes observed in the present study (Fig. 2). This hypothesis is in accordance with studies in animals deficient for the IL-1 and the IL-6 genes that could not develop severe renal damage after ischaemia and reperfusion [19, 20]. TNF-α could also be implicated as a cytokine responsible for the attenuation of the inflammatory response following hypoxaemic resuscitation, as its serum concentrations were decreased compared to animals subjected to normoxaemic resuscitation.
The present study attempted to provide evidence about the implication of other cellular receptors in the phenomena of ischaemia and reperfusion and their significance for hypoxaemic resuscitation. TREM-1 is a receptor greatly implicated in the production of TNF-α and IL-8 by neutrophils and monocytes in patients with sepsis [21]. The exact ligand for TREM-1 remains unknown [22]. Stimulation of monocytes in resuscitation post-shock does not seem to be mediated by TREM-1, as sera of animals collected could not increase its expression during resuscitation.
The presented results reveal that hypoxaemic resuscitation was accompanied by survival benefit in an experimental model of haemorrhagic shock. Stimulation of monocytes may contribute to the generation of the systemic inflammatory response during resuscitation after haemorrhagic shock. Lower stimulation of the p38 MAPK-mediated production of IL-1β, IL-6 and IL-8 by monocytes may be implicated as an explanation for the benefits shown for the host when resuscitation is performed under hypoxaemic conditions.
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