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Ann Thorac Surg 1998;66:1145-1150
© 1998 The Society of Thoracic Surgeons
a Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, USA
Accepted for publication April 18, 1998.
Address reprint requests to Dr McBride, Department of Anesthesiology, Royal Victoria Hospital, Belfast, BT12 6BA, Northern Ireland, United Kingdom
Abstract
Background. A homeostatic balance of proinflammatory and antiinflammatory cytokines is thought to be important for the maintenance of health. Cytokine baseline levels and response patterns to cardiac and nonmalignant abdominal operations have been investigated. The purpose of this study was to investigate the cytokine patterns at operation for thoracic cancer; the hypothesis tested was that cytokine baseline levels and response patterns would be unique for patients with malignant disease undergoing thoracic operation.
Methods. Ten patients undergoing pulmonary tumor resections were studied. Blood samples were collected at six perioperative time points.
Results. The cytokine response of these patients differed from patients undergoing cardiac operations: baseline tumor necrosis factor-
(39.1 pg/mL) and interleukin-10 (76.76 pg/mL) were elevated without significant changes. Interleukin-1 receptor antagonist became elevated postoperatively (871.6 pg/mL) compared with baseline (332.8 pg/mL) (p < 0.01). The level of tumor necrosis factor soluble receptor-2 was elevated at baseline (4,823.3 pg/mL) and remained elevated postoperatively (7,293.4 pg/mL) (p < 0.01).
Conclusions. Our hypothesis was supported; a separate pattern of proinflammatory and antiinflammatory cytokine levels and responses to thoracic operation was determined. This pattern may be indicative of tumor burden or detrimental to tumor surveillance; it merits further evaluation.
Baseline plasma cytokine levels as measured in healthy volunteers before general cardiac operations are within normal ranges (illustrated at the bottom of Figs 1 through 6 ); they are defined by almost undetectable levels of proinflammatory (tumor necrosis factor- [TNF], interleukin-8 [IL-8], interleukin-1ß [IL-1ß]), and the antiinflammatory cytokine interleukin-10 (IL-10). In healthy persons before undergoing abdominal and cardiac procedures, the other antiinflammatory cytokines, interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor soluble receptors (TNFsr-1 and TNFsr-2) are detectable in plasma but within defined ranges (see Figs 1 through 6). It has been shown that maintenance of this homeostatic balance of proinflammatory and antiinflammatory cytokines within the plasma is important for maintenance of health, as well as the prevention of, and recovery from certain disease states [1–3]. Increasingly, a beneficial physiologic role for a balanced operative inflammatory response is being understood [4]. Wound healing and resistance to infection may be beneficial effects of a perioperative proinflammatory response held in homeostatic balance by a concomitant antiinflammatory response [5, 6]. After uncomplicated cardiac and general operations, there are transient increases in proinflammatory cytokines accompanied by transient increases in antiinflammatory cytokines. The magnitude and time course of these changes are characteristic of the type of operation. For example, it is well known that during and after cardiopulmonary bypass (CPB) there is a significant increase in the plasma proinflammatory cytokines: IL-1ß, TNF, and IL-8 [7, 8]. This proinflammatory response is balanced by a phased plasma antiinflammatory cytokine response characterized by an immediate increase in IL-10 followed by IL-1ra and 24 hours later by the TNFsr-1 and TNFsr-2 [7, 8]. Others have confirmed an IL-10 response after operation using CPB and suggested that the magnitude of this response after CPB may be modulated by previous administration of methylprednisolone [9]. A similar perioperative proinflammatory response, accompanied by an antiinflammatory response has also been shown at nonmalignant abdominal operations [10]. It is not yet known to what extent these changes in cytokine balance accompany noncardiac thoracic operations, which does not involve CPB. In addition, it is not known to what extent the presence of malignancy alters baseline cytokine production. This study investigates the hypothesis that the perioperative proinflammatory and antiinflammatory cytokine baseline levels and patterns of response differ from those determined for cardiac and nonmalignant abdominal operations.
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With institutional review board committee approval and written, informed consent, we studied 10 patients undergoing thoracotomy for pulmonary tumor resection. All patients received standard anesthetic and surgical regimens. All patients had non–small cell lung cancer, stage I or II. A thoracic epidural catheter was placed preoperatively in 6 of the 10 patients. Anesthesia was induced using thiopental (5 mg/kg), fentanyl (1 µg/kg), and pancuronium (0.1 mg/kg). After tracheal intubation (left double lumen endotracheal tube), anesthesia was maintained using oxygen, nitrous oxide, and isoflurane. Postoperative analgesia was by epidural infusion (bupivacaine 0.125% with fentanyl 5 µg/mL) in 6 patients and by morphine patient-controlled analgesia pump in the other 4 patients. Whole blood samples were taken at the following times: 0 = 30 minutes before induction of anesthesia, 1 = 10 minutes after induction of anesthesia, 2 = 10 minutes after collapse of the nondependent lung, 3 = 10 minutes after reinflation of the nondependent lung, 4 and 5 = 2 and 24 hours after reinflation of the nondependent lung. Blood samples were immediately centrifuged in an Ependorf centrifuge for 2 minutes and plasma separated for immediate freezing in polypropylene tubes at -80°C. Stored plasma was later analyzed for cytokines using standard commercial enzyme-linked immunosorbent assays at the cytokine core laboratory (Hasday, Baltimore, MD). The proinflammatory cytokines that were analyzed included TNF, IL-8, and IL-1ß. The antiinflammatory cytokines that were analyzed included IL-10, IL-1ra, TNFsr-1, and TNFsr-2.
The specific assays were performed as follows: Human cytokines were measured by sandwich enzyme-linked immunosorbent assay using commercial reagents (TNF [Endogen, Cambridge, MA], coefficient of variation [CV] 16.5; IL-8 [Medgenix, Milton Keynes, UK], CV 9.2; IL-1ß [Endogen], CV 14.6; IL-10 [Endogen], CV 16.2; IL-1ra [R&D Systems, Abingdon, Oxon, UK] CV 7.1; TNFsr-1 [Medgenix] CV 6.8; TNFsr-2 [Medgenix] CV 16.5). Polystyrene plates (Maxisorb; Nunc, Denmark) were coated with capture mouse mAb against the cytokine in phosphate-buffered saline solution overnight at 25°C. The plates were washed four times with 50 mmol/L Tris, 0.2% Tween-20, pH 7.0 to 7.5. Plates were then blocked for 90 minutes at 25°C in assay buffer (phosphate-buffered saline soplution containing 4% [wt/vol], bovine serum albumin [Sigma, Dorset, UK], and 0.01% Thimerosal, pH 7.2 to 7.4). The wells were washed four times and 50 µL of assay buffer was added to each along with 50 µL of sample or recombinant standard prepared in assay buffer and incubated at 37°C for 2 hours. The wells were washed four times and 100 µL of biotinylated detection antibody in assay buffer was added and incubated for 1 hour at 25°C. The wells were washed four times and strepavidinperoxidase (Dako, Ely, Cambridge, UK) in assay buffer was added and incubated at 25°C for 30 minutes. After four final washes, 100 µL of commercially prepared substrate (TMB; Dako) was added and incubated at 25°C for approximately 30 minutes. The reaction was stopped with 100 µL of 2 N HCl and the A450 (minus A630 background) was read on a microplate reader (Dynatech, Guernsey, UK). A curve was fit to the standards using a computer program (Deltagraph for Macintosh; Deltapoint) and cytokine concentration in each sample was calculated from the standard curve equation. Human IL-1ra was measured using a commercial enzyme-linked immunosorbent assay kit (R&D Systems).
Controls
The data gathered from these patients were compared with baseline data from patients without malignancy about to undergo coronary artery bypass grafting (CABG) with CPB.
Statistical analysis
The data are presented as a mean with a standard error of the mean in parentheses. Within-group samples were compared with preanesthetic baseline (sample 0) using Friedman’s and Wilcoxon signed rank analysis.
Results
The mean age of the patients investigated was 69.2 years and the mean weight was 76.3 kg. There were 9 men and 1 woman. Table 1 and Figures 1 to 5 display the results across six sampling times.
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, IL-1ß, and IL-8 demonstrate unique baseline levels and responses to thoracic cancer operation. The baseline TNF level was above the normal range (0 to 15 pg/mL) and remained elevated throughout the study period (see Fig 1). The level of interleukin-8 remained within the normal range (0 to 10 pg/mL) at baseline, during, and after operation (see Fig 2). Interleukin-1ß levels remained within the normal range at baseline and during and after the operation; therefore, no figure was drawn. The antiinflammatory cytokines IL-10, IL-1ra, TNFsr-1, and TNFsr-2 also demonstrate unique baseline levels and responses to thoracic cancer operation. The baseline IL-10 level was significantly elevated and remained elevated without significant change throughout the study period (see Fig 3). The baseline IL-1ra level was within the normal range at baseline but became significantly elevated 2 hours after lung reexpansion. The magnitude of this response was six times less than that observed 2 hours after CPB [8] (see Fig 4). The baseline TNFsr-1 level was three times higher than the baseline TNFsr-1 level reported at uncomplicated cardiac operations [8]. The level of TNFsr-1 was increased 24 hours after thoracic operation, but this increase was not significant compared with baseline. The baseline TNFsr-2 level was twice as high as that reported in uncomplicated cardiac operation [8]. The level of TNFsr-2 became significantly elevated compared with baseline at 24 hours postoperatively. The magnitude of the TNFsr-2 response was approximately three times that reported 24 hours after uncomplicated cardiac operation [8] (see Fig 5).
Comment
Cytokines are glycosylated and nonglycosylated polypeptides, which act as the soluble messengers of the immune system. Among their wide range of biological actions they may have proinflammatory or antiinflammatory activity. Proinflammatory cytokines, such as TNF, IL-1ß, and IL-8, may be associated with a stimulus such as exposure to operation and CPB [7, 10]. Antiinflammatory cytokines include IL-10, IL-1ra, TNFsr-1, and TNFsr-2; it has been suggested that the antiinflammatory cytokines may have the effect of counterbalancing or controlling the proinflammatory response.
Patients have low to undetectable levels of the proinflammatory cytokines before CABG. Similarly, the baseline plasma concentration of the antiinflammatory cytokine IL-10 is nearly undetectable, whereas the baseline concentrations of the other antiinflammatory cytokines IL-1ra, TNFsr-1, and TNFsr-2 lie within well-defined normal ranges [7, 8] (see Figs 1 to 5). After the stimulus of CPB, there is a proinflammatory cytokine response characterized by an initial increase and decrease of the proinflammatory cytokines followed by a later increase and decrease of the antiinflammatory cytokines. This phenomenon has been described as the phased antiinflammatory cytokine response [7, 8]. The demonstration of these transient peaks in proinflammatory and antiinflammatory cytokines at uncomplicated cardiac operation is reflected in earlier pharmacokinetic studies showing relatively short half-lives for proinflammatory and antiinflammatory cytokines [11, 12].
A separate cytokine response was determined for patients undergoing nonmalignant abdominal operation [10]. The proinflammatory cytokine TNF did not change from low baseline levels throughout the perioperative period. However, a different proinflammatory cytokine, interleukin-6 (IL-6), peaked by 4 hours postoperatively and remained elevated above baseline at 24 hours postoperatively. Similarly, the antiinflammatory cytokines IL-10 and IL-1ra started at low baseline levels, peaked at 4 hours postoperatively, and remained elevated at 24 hours postoperatively. This pattern of response was similar for an intravenous or inhalational anesthesia technique.
Thus, varying cytokine responses may be seen with varying types of operation, extent of injury, and intensity of stimuli (such as exposure to CPB). However, a common pattern emerges. In both uncomplicated abdominal and cardiac operation, the pattern of response was similar: baseline cytokine levels were within normal limits and with operation, there was a significant proinflammatory cytokine response accompanied by an antiinflammatory cytokine response. There is now evidence that a wide range of factors including perioperative drugs, age, and disease states [4] may affect such a balanced perioperative cytokine response pattern. For example, the possibility that malignancy may alter such a balanced response is suggested by the finding that patients with lung cancer had abnormally elevated preoperative (baseline) IL-8 [13]. There is also evidence that certain lung tumors produce TNF [14] and IL-10 [15]. These studies support the hypothesis investigated in this study, that lung cancer patients demonstrate a unique baseline and perioperative cytokine pattern as compared with the balanced proinflammatory and antiinflammatory cytokine response seen with uncomplicated cardiac and nonmalignant abdominal operation [10].
This study determined baseline elevations in both proinflammatory (TNF) and antiinflammatory (IL-10, TNFsr-1, TNFsr-2) cytokines in patients with malignant lung cancer undergoing thoracic operation. Postoperatively, the only cytokines that increased significantly from this baseline were IL-1ra and TNFsr-2, both antiinflammatory cytokines. Thus, a selective postoperative increase in these antiinflammatory cytokines occurred in the absence of any further increase in proinflammatory cytokine levels.
The magnitude of this cytokine response may be compared with that in patients who underwent uncomplicated cardiac and nonmalignant abdominal operations. In contrast to patients undergoing CPB and abdominal procedures, the thoracic cancer patients had baseline TNF and IL-10 levels greater than the maximal response observed after CPB and abdominal operation; no further perioperative increases in TNF and IL-10 were observed in thoracic cancer patients. In thoracic cancer patients, TNFsr-1 and TNFsr-2 levels were also elevated at baseline, being approximately two times higher than TNFsr-1 and TNFsr-2 levels determined in patients after CPB. The level of TNFsr-2 further increased after thoracic operation to levels that were approximately three times higher than those reported after CABG. The other antiinflammatory cytokine measured, IL-1ra, was normal at baseline (as with patients before CABG) but became significantly elevated after thoracic operation to a low level, six times less than the response seen after procedures involving CPB. Levels of IL-1ra were similar at baseline for nonmalignant abdominal operations and the increase with thoracic procedures was similar to the response seen with abdominal operations. The cause for these differences in baseline perioperative cytokine levels possibly relates to a baseline inflammatory response to tumor burden with a balancing antiinflammatory response. The meaning of why concentrations of specific cytokines are elevated at baseline as compared with others remains to be elucidated.
Previous research can be compared with these findings. Tonnesen and colleagues [16] found that TNF was detectable in only 2 of 21 patients with lung cancer. In contrast, all of our patients had supranormal TNF baseline levels. Martinet and associates [17] demonstrated increased TNF activity in plasma and pleural fluid of lung cancer patients. This may have been attributable to production by the tumor itself or secondary to accompanying inflammation. Arias-Diaz and colleagues [14] found significantly increased TNF in bronchoalveolar lavage fluid of patients with lung cancer compared with control and Tsukaguchi and associates [18] showed that monocyte cultures from patients with lung cancer produced significantly greater quantities of IL-1, TNF, and IL-6 than in healthy controls. These studies may indicate that lung tumors may produce proinflammatory cytokines locally [18].
Partanen and colleagues [19] showed that a significant increase in plasma TNF occurred in patients with asbestosis in whom lung cancer later developed, as compared with those in whom cancer did not develop. This work suggested that increases in TNF may provide a method of early diagnosis of cancer related to asbestosis. Alternatively, it has been suggested that the ability of patients to produce TNF may be associated with better prognosis. Shimura and associates [20] showed that the TNFß allele was found at significantly lower frequency in lung cancer patients than in noncancer controls. Furthermore, the TNFß allele was associated with enhanced postoperative survival.
The finding of significantly elevated baseline levels of the antiinflammatory cytokine IL-10 suggests that these cancer patients were already maximally producing IL-10 before operation. Furthermore, these levels did not decrease postoperatively. Smith and colleagues [15] demonstrated increased levels of antigenic IL-10 in tissue homogenates of human bronchogenic carcinomas compared with normal lung tissue. Analysis of several in vitro unstimulated human bronchogenic cell lines demonstrated the ability of tumor cells to constitutively produce IL-10 [15]. Interestingly, mononuclear cells cultured in the conditioned medium from a bronchogenic cell line were able to produce significantly greater TNF after neutralizing antibodies to IL-10 were added to the cultures. This work suggests that the elevated plasma IL-10 in our patients may be attributable, at least in part, to IL-10 production by the tumor itself.
Our patients also had elevated baseline plasma levels of the antiinflammatory cytokines TNFsr-1 and TNFsr-2. This is consistent with Martinet and associates [17] who demonstrated increased TNF inhibitor activity in sera and pleural fluid of patients with lung cancer as compared with noncancer controls. They suggested that this TNF inhibitor activity may enable the tumor to evade TNF-mediated cytotoxic activity. In our study, there was a further significant increase in TNFsr-2 postoperatively. The clinical significance of this postoperative increase in TNFsr-2 is not clear. Future studies should evaluate the hypothesis that such a reduction in TNF-mediated cytotoxicity may not be to the cancer patient’s advantage.
Our study suggests that the presence of lung cancer does not alter baseline plasma IL-1ra, in contrast to the baseline elevations of the other antiinflammatory cytokines IL-10, TNFsr-1, and TNFsr-2. IL-1ra has been found in malignant pleural effusion fluid [21]. Conversely, other investigators have found that IL-1ra is significantly increased after general operations (as we found after thoracic operations) unrelated to CPB or malignant disease, suggesting that this may be a response to the surgical stress [22, 23]. Future work is necessary to determine the correlation between IL-1ra and other markers of the stress response to operation.
This pilot study is limited by several factors. First, the study number was small, as it was intended to define perioperative proinflammatory and antiinflammatory cytokine response pattern to thoracic operation in patients with malignancy. Second, the baseline levels of cytokines were compared to baseline levels in patients about to undergo CABG. It will be necessary to confirm that baseline cytokine levels in patients undergoing thoracic operation for noncancer indications are the same as baseline cytokine levels in patients undergoing CABG. The low baseline levels of proinflammatory and antiinflammatory cytokines found in patients undergoing nonmalignant abdominal operation [10] support the validity of this comparison. This is further suggested by an earlier demonstration that baseline plasma cytokine levels in 5 healthy volunteers (whose blood was being submitted to an isolated in vitro CPB experiment) were similar to baseline levels in patients before cardiac operation [7]. Third, the duration of the study was brief. Correlation of baseline and perioperative cytokine levels with outcome and survival over time should be considered. Fourth, the role of comorbid disease states, which may alter perioperative cytokine responses, needs to be investigated in larger studies.
Many pertinent questions are raised by this study. Further definition of the role of individual cytokines is required, as individual baseline and perioperative cytokine levels were very different from those found in other studies. There is a need to evaluate the possibility of baseline plasma proinflammatory and antiinflammatory cytokine levels being markers for malignancy as suggested by the studies by Martinet, Arias-Diaz, and Partanen and their colleagues [14, 17, 19]. Monitoring postthoracotomy plasma cytokine levels may provide a valuable method for early identification of occurrence, recurrence, or progression of the malignancy. Tissue injury with operation or therapies to alter the cytokine levels of patients with lung cancer may change the balance of proinflammatory and antiinflammatory cytokine responses. Such an effect may change the effective inflammatory response to neoplasm and thus alter tumor surveillance in the perioperative period.
The impact of drugs such as anesthetic agents or immunosuppressive agents on this response is important to determine. Anesthetic technique intraoperatively may influence the perioperative cytokine response; Gilliland and associates [10] found that an inhalational anesthetic technique resulted in lower postoperative levels of IL-10 than an intravenous anesthetic technique. It has also been suggested that opioids and analgesic type can alter cytokine responses [24]. Although in our study general anesthetic technique was uniform for all patients, epidural analgesia was effective in 6 of 10 patients, and subsequently 4 patients required morphine infusions postoperatively. A larger study is required to comment on differences in cytokine concentration between effective and noneffective epidural analgesia. Nonsteroidal antiinflammatory agents, such as ketorolac, may also alter the cytokine response pattern. All of our patients received ketorolac as is standard at our institution. Steroids have also been shown to influence cytokine levels [9]; their use in a situation with a baseline malignancy remains to be determined. Once the varying effects of perioperative immunomodulatory drugs have further been elucidated, one can ponder their use to alter cytokine balance and response patterns in a manner beneficial to the patient.
In summary, this work defines the baseline and perioperative proinflammatory and antiinflammatory cytokine responses during thoracic operation for lung cancer. This pattern is different from the cytokine responses defined for uncomplicated cardiac and nonmalignant abdominal operations. For these lung cancer patients, TNF and IL-10 were elevated preoperatively without significant postoperative changes. However, antiinflammatory cytokines IL-1ra and TNFsr-2 were selectively increased after operation. This suggests a postoperative shift in cytokine balance toward antiinflammatory cytokine activity raising questions about effective perioperative tumor surveillance. Future study could determine whether or not these perioperative cytokine changes in cancer patients correlate with long-term outcome and thus demonstrate whether perioperative immunomodulatory strategies should be considered.
References
This article has been cited by other articles:
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  T. J. Szczesny, R. Slotwinski, A. Stankiewicz, B. Szczygiel, M. Zaleska, and M. Kopacz Interleukin 6 and interleukin 1 receptor antagonist as early markers of complications after lung cancer surgery Eur. J. Cardiothorac. Surg., April 1, 2007; 31(4): 719 - 724. [Abstract] [Full Text] [PDF]
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 K. P. Grichnik and T. A. D'Amico Acute Lung Injury and Acute Respiratory Distress Syndrome After Pulmonary Resection Seminars in Cardiothoracic and Vascular Anesthesia, December 1, 2004; 8(4): 317 - 334. [Abstract] [PDF]
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 T. Kunihara, S. Sasaki, N. Shiiya, T. Miyatake, N. Mafune, and K. Yasuda Proinflammatory cytokines in cerebrospinal fluid in repair of thoracoabdominal aorta Ann. Thorac. Surg., March 1, 2001; 71(3): 801 - 806. [Abstract] [Full Text] [PDF]
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A. P.C. Yim, S. Wan, T. W. Lee, and A. A. Arifi VATS lobectomy reduces cytokine responses compared with conventional surgery Ann. Thorac. Surg., July 1, 2000; 70(1): 243 - 247. [Abstract] [Full Text] [PDF]
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T. Kunihara, S. Sasaki, N. Shiiya, H. Ishikura, Y. Kawarada, A. Matsukawa, and K. Yasuda Lazaroid reduces production of IL-8 and IL-1 receptor antagonist in ischemic spinal cord injury Ann. Thorac. Surg., March 1, 2000; 69(3): 792 - 798. [Abstract] [Full Text] [PDF]
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