Tumour necrosis factor-α (TNF), interleukin-1β (IL-1) and interleukin-6 (IL-6) constitute important cytokines in the inflammatory response to trauma and infection [1-9]. The infusion of TNF in animals may induce stress hormone responses and lethal shock . Groups of patients selected for documented bacteraemia or signs of sepsis have been found to have increased circulating concentrations of TNF, IL-1, and IL-6. [4,11-14]. Both TNF and IL-1 participate in the endothelial activation [15-17]. Procoagulant activities have been attributed to TNF [18,19] and IL-1 . IL-1 may also suppress cell-surface anticoagulant activity . The cytokine network also comprises proteins such as interleukin-1 receptor antagonist (IL-1 ra) [22,23] and soluble TNF receptors I and II (sTNF-R I and sTNF-R II)  which may attenuate the effects of their respective cytokine. A soluble receptor has also been characterized for IL-6 .
Leucocyte adhesion and extravasation are supported by a number of different adhesion molecules present on the leucocytes and endothelial cells [26-28]. Cytokines have multiple roles in the mobilization of leucocytes to inflammatory sites and may up-regulate the expression of the endothelial ligands E-selectin, vascular cellular adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), which promote leukocte extravasation. In recent years, soluble (s) forms of the adhesion molecules have been identified [29,30]. They have been shown to be markers of endothelial activation or damage in the experimental setting .
Fibrin, soluble (SF) represents an intermediate state in the development of cross-linked polymerized fibrin and has been related to a complicated clinical history in critically ill patients . Antithrombin (AT) is a coagulation inhibitor. Fibrin D-dimer is a specific degradation product of cross-linked fibrin. The presence of D-dimer shows that fibrin has been formed and degraded by fibrinolytic activity.
The aim of the study was to investigate the effect of local surgical trauma on the local and the systemic cytokine response in elective hip arthroplasty. The local surgical tissue trauma in hip arthroplasty is rather extensive and the sample collection of the local tissue fluid contaminated with blood can easily be managed in the clinical setting, Moreover, standard methods of anaesthesia, mostly epidural or spinal block, are used. We also hypothesized a relation between TNF and IL-1 on the one hand and haemostatic variables on the other.
Ten patients, seven females and three males, aged 74 (57-78), median (range), years undergoing hip arthroplasty with either spinal (n=5) or epidural block (n=5) were studied with regard to concentrations of TNF, IL-1, IL-6, sTNF-R I, sTNF-R II, IL-1ra, sIL-6-R, soluble E-selectin (sE-selectin), soluble vascular adhesion molecule-1 (sVCAM-1), soluble intracellular adhesion molecule-1 (sICAM-1), AT, SF, and fibrin D-dimer in wound drainage blood and in blood from the systemic circulation for up to 24 h after skin incision. The study protocol is described in Table 1.
All patients were healthy. All patients used paracetamol and dextropropoxifen on demand. No patient was on corticosteroids, immunomodulatory drugs or anti-inflammatory drugs.
Clinical and demographic data are given in Table 2. The hip arthroplasty (cemented) was performed according to Charnley (Ohmed Medical Inc., Leeds, UK). Epidural anaesthesia was administered with bupivacaine, 5 mg mL−1 combined with adrenaline, 5 μg mL−1 (Marcain® adrenaline, Astra, Stockholm, Sweden), spinal anaesthesia with hyperbaric bupivacaine, 5 mg mL−1 (Marcain® adrenaline, Astra, Stockholm, Sweden). All patients received 500 mL of dextran 70, Macrodex® (Pharmacia-Upjohn, Uppsala, Sweden) intra-operatively for thromboprophylaxis. The local anaesthetic (bupivacaine) was administered via the epidural catheter post-operatively for pain relief.
Transfusions of red blood cell concentrates (RBCs) were given when the haemoglobin level was less than 110 g L−1. Four patients received transfusion of RBCs (Table 2). Autotransfusion was not used.
No complications or infections developed post-operatively.
The study was approved by the Local Ethics Committee, Huddinge University Hospital, Karolinska Institute, informed consent was obtained from each patient.
Experimental protocol and sampling
The study protocol is described in Table 1. Venous samples of blood taken from the systemic circulation were obtained via venipuncture. Samples from the local injury site were obtained by aspiration from the wound drainage system (Bellovac, Astra Tech, Mölndal, Sweden) using a sterile cannula. The samples were handled as described earlier .
Enzyme immunoassays (Quantikine™, R&D Systems Inc., Minneapolis, MN, USA) were used to analyse the concentrations of cytokines, soluble TNF and IL-6 receptors, IL-1ra, and soluble adhesion molecules. In the analysis of IL-1, IL-6, and TNF in the systemic circulation (low concentrations of pro-inflammatory cytokines were expected), high-sensitivity kits able to detect lower levels but with more restricted working ranges were compared with normal-sensitivity kits. For wound drainage blood (high concentrations of pro-inflammatory cytokines were expected), normal sensitivity kits were used but if no cytokine was detected the sample was reanalysed using the high sensitivity kit. The microtiter plates were read at 450 nm on a spectrophotometer (Microplate autoreader EL 311, Bio Tek Instruments Inc., Winooski, VT, USA). A standard curve was plotted to obtain the concentrations of the samples. Assays were carried out with the appropriate positive and negative controls included. This method does not distinguish between bound and unbound cytokines.
The detection levels (pg mL−1) (intra- and interassay coefficients of variation) for TNF, IL-1, and IL-6 using the high-sensitivity kit were 0.17 (6.1, 10.0), 0.059 (3.0, 6.7), and 0.094 (5.3, 7.0), respectively. The corresponding detection levels for TNF, IL-1, IL-6, soluble TNF receptor I and II, IL-1ra, sIL-6-R, sE-Selectin, sVCAM-1 and sICAM-1 using normal sensitivity kits were 4.4, 1.0, 0.7, 25, 5, 22, 3.5, 7000, 2000 and 100 000, respectively. The intra- and interassay coefficients of variation were less than 10%.
Concentrations of plasma antithrombin (AT)  and fibrin, soluble (SF)  were analysed by amidolytic methods, using chromogenic substrates with kits from Chromogenix (Mölindal, Sweden). Plasma D-dimer  was analysed by enzyme-linked immunosorbent assay (ELISA) with kits from Biopool (Umeå, Sweden). Reference values for AT, SF and D-dimer were 0.80-1.20 IU mL−1, <25 nmol L−1 and <80 μg L−1 and the intra- and interassay coefficients of variation were less than 10% for each assay. The detection limits for AT, SF and D-dimer were 0.05 IU mL−1, 10 nmol L−1 and 10 μg L1, respectively.
Values are presented as means ± SEM with 95% confidence intervals in parenthesis. As a result of multiple comparisons possible differences between samples from wound drainage blood and the systemic circulation, respectively, and over time were analysed by analysis of variance (ANOVA) with repeated measures. The Scheffe F-test was used as a post-hoc test. Adjustments for degrees of freedoms (d.f.) were made. Correlations were calculated applying the principle of product moment correlation. Regression lines were plotted using the method of least squares. The level of significance (two-tailed) was set at 5%.
Cytokines and cytokine inhibitors/neutralizing molecules
In general a considerable interindividual variation in concentrations of cytokines and cytokine inhibitors/neutralizing molecules was noted.
Wound drainage blood(Fig. 1). TNF was detected in 18 of the 50 investigated samples using normal sensitivity kits. The other samples were analysed using the high-sensitivity kit (low detection level). Altogether, TNF was detected in 43 samples. The highest concentrations were found in samples obtained 3 h after connection of drainage, but these concentrations were not significantly increased compared with concentrations in samples obtained at 1 h after connection of drainage (P=0.29).
Compared with 1 h after connection of drainage, the concentrations of sTNF-R I were increased at 12 (P<0.05) and 24 h (P<0.05) and sTNF-R II concentrations were increased at 6, 12 and 24 h (P<0.05 for all comparisons).
Using normal sensitivity kits, IL-1 was not detected in any patient at 1 h after start of drainage, in three patients at 3 h, and in seven patients at 6 h after start of drainage. IL-1 increased during the course of the sampling period and approached the level of significance (P=0.05) at 24 h when compared with values at 1 h. An increase was seen in IL-1ra at 12 and 24 h, respectively (P<0.05 for both comparisons). The IL-6 concentrations were increased at 6 (P<0.05), 12 (P<0.01), and 24 h (P<0.01), respectively, compared with values obtained 1 h after connection to drainage. Concentrations of sIL-6-R did not change during the study period.
Blood drawn from the systemic circulation(Fig. 2). The TNF concentrations were lower at the time of connection of drainage (end of surgery) than before premedication (P<0.05). Soluble TNF receptors did not change. IL-1 and IL-1ra concentrations did not change over the study period. The IL-6 concentrations were higher at 6 (P<0.05) and 24 h (P<0.05), respectively, compared with the concentrations before premedication. Concentrations of sIL-6-R decreased at the end of surgery and at 1 and 24 h after connection of drainage compared with concentrations before premedication (P<0.05 for all comparisons).
Wound drainage blood vs. blood from systemic circulation(Figs 1 and 2). Concentrations of cytokines and inhibitors were higher in wound drainage blood than in the systemic circulation except for sIL-6-R, which were lower. For TNF, significant differences were found at 3 h (P<0.05) and 6 h (P<0.05), for sTNF-R I at 1 (P<0.01), 6 (P<0.01) and 24 h (P<0.01), respectively, for IL-1 at 1 (P<0.001), 6 (P<0.05) and 24 h (P<0.05), for IL-1ra at 1 (P<0.05), 6 (P<0.001) and 24 h (P<0.001), respectively, for IL-6 at 1 (P<0.01), 6 (P<0.001) and 24 h (P<0.001), and for sIL-6-R at 6 h (P<0.05).
Adhesion molecules (Fig. 3)
The concentrations of soluble adhesion molecules in wound drainage blood and in blood taken from the systemic circulation did not change significantly during the study period. Differences in concentrations between wound drainage blood and that in the systemic circulation were not noted.
The systemic concentrations of AT were decreased and concentrations of SF and fibrin D-dimer were increased at the end of surgery and 1, 6, and 24 h after the end of surgery in comparison with pre-operative values (Table 3).
Concentrations of AT in wound drainage blood did not change during the study period but they were lower than normal reference values (Fig. 4). At 1 h after connection of the drainage, the AT concentration was 0.38±0.02 (0.34-0.43) IU mL−1 and the lowest value was found at 12 h after adaptation of the drainage, 0.35±0.02 [0.30-0.39] IU mL−1.
AT concentrations in all samples from wound drainage blood were higher than the systemic concentrations (P<0.001 for all comparisons) (Fig. 4). The most pronounced differences were found at 24 h, 0.37±0.02 (0.33-0.40) IU mL−1 vs. 0.77±0.42 (0.67-0.86) IU mL−1 (P<0.001, Scheffe F-test 56.28).
In wound drainage blood, SF concentrations were >200 nmol L−1 in all but four samples and the concentrations of SF in these samples ranged from 78 to 171 nmol L−1.
Wound drainage fibrin D-dimer concentrations analysed in two patients ranged from 481 600-1 583 000 μg L−1.
Relations between concentrations of cytokines and coagulation/fibrinolysis variables
Concentrations of cytokines, cytokine inhibitors/neutralizing molecules and soluble adhesion molecules were not related to coagulation/fibrinolysis parameters, neither in wound drainage blood nor in blood from the systemic circulation.
Spinal vs. epidural anaesthesia
Studied parameters did not differ between spinal and epidural anaesthesia. However, the number of patients in each group was too small to make a reliable statistical comparison.
In the present study characteristics of wound drainage blood were used to describe the local trauma response. It cannot be ruled out that the samples could have contained red blood cells able to interfere with the method used to measure concentrations of cytokines, cytokine inhibitors, and soluble adhesion molecules . False negative TNF results may also be obtained because of the interaction with haemoglobin .
Immunoassay methods detecting both bound and free cytokines were used in the present study. The clinical implications of the ratios between cytokines and possible neutralizing molecules or inhibitors have not been elucidated. If circulating receptor-bound cytokine represents a potential reservoir for active factors, then measurements of both free and bound cytokines may be the most relevant approach.
Four of the patients received blood transfusion and this may have influenced the results but not the primary objective of the study, that is local trauma response vs. systemic trauma response.
Finally, the detection of cytokines in wound drainage blood may have been influenced by contact with foreign material. The highest concentrations of IL-1 in wound drainage blood were seen at 24 h. It cannot be excluded that this delayed response could be caused by activation by foreign material as well as by the period of time that the sample remained in the drainage tubings. However, in a previous study , we found TNF and IL-1, but no IL-6, in samples from wound drainage blood obtained at the end of surgery and prior to passing of the wound blood through the drainage, suggesting that cytokine activation actually occurs at the local trauma site.
Cytokines and cytokine inhibitors/neutralizing molecules
The available clinical studies on cytokine activity in trauma have mainly focused on systemic concentrations of cytokines. However, it has been argued that serum/plasma concentrations of cytokines may not accurately reflect the actual cytokine activity at the tissue level. Furthermore, activation patterns of cytokines and their inhibitors have not been delineated previously in elective surgery. The present study high-lights this issue and the results confirm data from both experimental trauma studies [8,38] and humans [33,39,40] suggesting that pro-inflammatory cytokine activity is confined to the local wound site. In the present study, concentrations of cytokines and cytokine inhibtors/cytokine neutralizing molecules were generally, though not always significantly, increased in wound drainage blood as compared with blood taken from the systemic circulation. This is in line with previous data suggesting that cytokines act predominantly as paracrine and autocrine messengers mainly exerting their major effects locally, within organs and tissues .
IL-6 may inhibit the induction of TNF and IL-1 [42,43] and the increase in IL-6 may be part of an anti-inflammatory reaction. The levels of sIL-6-R did not show any obvious relation to the measured IL-6 concentrations. At the present time, the regulation of the in vivo production of sIL-6-R as well as its physiological significance are not well understood. Soluble IL-6-R has been shown to bind to IL-6 in solution and to augment the activity of IL-6 as a result of the binding of the IL-6/sIL-6-R complex to the membrane-bound signal-transducing component gp 130 [44,45].
The induction of endothelial cell adhesion molecules is a critical component in acute inflammatory responses. Cytokines induce the transcription of genes encoding the endothelial cell adhesion molecules E-selectin, ICAM-1, and VCAM-1. The present data do not reveal any firm relations between cytokine concentrations, on the one hand, and concentrations of soluble adhesion molecules, on the other. Increased concentrations of soluble adhesion molecules have been anticipated to reflect activation of the endothelium . In the present study, the concentrations of soluble adhesion molecules did not change, perhaps reflecting the possibility that the moderate degree of trauma studied entailed only a minimal expression of adhesion molecules [17,26-28] compared with the conditions found in major trauma or sepsis.
The interindividual variations in the present study may be associated with differences in activation thresholds to cytokines . Similarly an innate TNF response has been suggested to influence the outcome in meningococcal disease .
The systemic concentrations of sE-selectin have been found to be elevated in the serum of patients with septic shock . Septic shock is associated with endothelial damage and it is conceivable that the cytokine response in such patients elicits a massive expression and subsequent release of soluble adhesion molecules compared with moderate surgery.
The pronounced derangement of coagulation/fibrinolysis variables found in wound drainage blood indicates a prompt response at the local injury site. A systemic response was also noted (Table 3).
Considering haemostatic mechanisms the use of dextran 70 might have influenced the results. Interestingly, dextran 70 may also have an impact on leucocyte adhesion mechanisms . Moreover, the use of regional techniques may have had an impact on concentration levels of the parameters studied.
Bone  has postulated a three-stage development in the systemic inflammatory response syndrome (SIRS), in which stage 1 is a local production of cytokines in response to injury or infection. Stage 2 is the protective release of a small amount of pro-inflammatory cytokines and cytokine inhibitors into the systemic circulation. Stage 3 is the massive systemic reaction in which elevated concentrations of pro-inflammatory cytokines turn destructive by compromising the integrity of the capillary walls and flooding end organs. This may also be influenced by an imbalance between pro-inflammatory cytokines and cytokine inhibitors/neutralizing molecules and the degree of endothelial activation.
Hip arthroplasty for arthrosis is regarded as a routine procedure representing surgery of a moderate magnitude. Provided that the changes in wound drainage blood reflect the conditions at the local trauma site, it may be that the present results reflect a normal local trauma response characterized by initial synthesis and release of TNF and IL-1 at the local trauma site but above all by a subsequent response of cytokine inhibitors/neutralizing molecules in order to counter-balance the initial pro-inflammatory cytokines. Moreover, the unchanged concentrations of soluble adhesion molecules suggest that the endothelial activaton in the wound was minimal and probably reduced the risk of a subsequent massive spread of mediators into the systemic circulation. However, during more extensive surgery or in severely traumatized patients the situation may be quite different and associated with a marked endothelial activation creating the prerequisites for SIRS which may ultimately result in a multiple organ dysfunction syndrome (MODS).
In summary, the local immune and haemostatic trauma response was more marked compared with the systemic one. An initial predominance of pro-inflammatory components was found, subsequently followed by inhibitors in both compartments. The concentrations of soluble adhesion molecules did not change. A relation between TNF and IL-1 on the one hand and coagulation/fibrinolysis parameters was not found.
The authors wish to thank Anna Patoka and Viveka Gustafsson for their excellent technical assistance.
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