The Effects of Epidural Anaesthesia and Analgesia on Natural Killer Cell Cytotoxicity and Cytokine Response in Patients with Epithelial Ovarian Cancer Undergoing Radical Resection

Abstract

Objective:

Epidural anaesthesia appears to promote antitumourigenic activity in patients with malignant disease who are undergoing surgery. This study investigated immune function in women with epithelial ovarian cancer undergoing radical resection with either general anaesthesia alone or in combination with epidural anaesthesia.

Methods:

Patients (n = 61) were randomized to receive either combined general/epidural anaesthesia (study group) or general anaesthesia alone (control group). Natural killer cell cytotoxicity (NKCC) and serum concentrations of four cytokines (interleukin [IL]-1β, -8 and -10 and interferon [IFN]-γ) were measured before anaesthesia (T_pre) and 4h after skinincision (T_{4 h}) in both groups.

Results:

In both groups, concentrations of protumourigenic cytokines (IL-1β and IL-8) were significantly higher at T_{4 h} than at T_pre, while concentrations of antitumourigenic cytokines (IL-10 and IFN-γ) and NKCC were significantly lower at T_{4 h}. The study group had significantly higher NKCC, IL-10 and IFN-γ levels and lower IL-1β and IL-8 levels at T_{T h} compared with the control group.

Conclusion:

Combined general/epidural anaesthesia appeared to promote antitumourigenic NKCC and cytokine responses.

Introduction

Epithelial ovarian cancer (EOC) is the most common cause of gynaecological malignancy-related mortality in women. ¹ Although surgery is the first-line treatment for EOC, > 70% of patients usually have a poor prognosis for disease recurrence even after radical resection of the tumour mass. ² This is because micrometastases and scattered tumour cells may have already existed at the time of surgery, and even the best surgical technique is associated with the release of tumour cells into the blood and lymphatic streams. ³ Whether this minimal residual disease results in local or metastatic recurrence has been shown to depend largely on the body's immune status. ⁴

Many factors have been confirmed to contribute to perioperative immuno -suppression. First, surgery per se depresses cell-mediated immunity, ⁵ releases growth factors that promote the growth of local and distant malignant tissue, ⁶ increases circulating concentrations of proangiogenic factors such as vascular endothelial growth factor, ⁷ reduces concentrations of tumour-related antiangiogenic factors, ⁸ and releases tumour cells into the circulation. ⁹ Secondly, anaesthesia per se impairs neutrophil, macrophage, natural killer (NK) cell, T cell, and dendritic cell functions.^10,11 Thirdly, opioid analgesics such as morphine and fentanyl, which are given to control surgical pain, might inhibit both cellular and humoral immune function in humans.^10,12 Fourthly, pain per se enhances the tumour-promoting effects of surgery and suppresses cell-mediated immunity. ⁴

Fortunately, previous investigations have demonstrated that regional anaesthesia, including epidural anaesthesia and spinal block, can attenuate or prevent these adverse effects.^4,13 Furthermore, the use of regional anaesthesia has been associated with decreased recurrence rates and prolonged overall survival in comparison with general anaesthesia in patients with melanoma, ¹⁴ prostate, ¹¹ breast ¹⁵ or localized colon cancer. ¹⁶ However, the precise mechanisms of how regional anaesthesia and analgesia help to maintain the body's immune activity and inhibit tumour progression remain unclear.

Both NK cells and cytokines are of key importance in antitumour immunity. The NK cells can detect and destroy circulating tumour cells and play major roles against tumour progression and metastasis. ¹⁷ It is now evident that intratumoural numbers of NK cells have prognostic significance in a variety of neoplasms.^18–20 Some cytokines have an antitumourigenic function, and stimulate and promote the immune system's antitumour capability, whereas others have been confirmed as protumourigenic and, thus, promote tumour growth by inhibiting an effective immune response. ²¹ The present prospective study, therefore, set out to measure NK cell cytotoxicity (NKCC) and the serum concentrations of four cytokines (interleukin [IL]-1β, -8 and -10 and interferon [IFN]-γ), all of which have a corroborated association with the cell-mediated immune response, in patients with primary EOC undergoing radical resection. The study tested, for what we believe is the first time, the hypothesis that patients with EOC who receive combined general/epidural anaesthesia exhibit greater preservation of NK cell function, elevated serum concentrations of antitumourigenic cytokines and reduced levels of protumourigenic cytokines, compared with patients receiving general anaesthesia alone.

Patients and methods

Patients

All consecutive adult patients (age range 18 – 70 years) with an American Society of Anesthesiologists (ASA) physical status of 1 – 3 (0, normal, healthy; 5, moribund, not expected to survive without operation) and scheduled to receive radical resection for EOC in the Department of Anaesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, China, between January 2011 and February 2012, were eligible for enrolment in this prospective randomized trial. Exclusion criteria were: emergency surgery; severe cardiac insufficiency; use of analgesic medication the week before planned surgery; acute medical illness within 16 weeks of the present study; history of substance abuse or cognitive dysfunction; ongoing therapies with immune-regulatory drugs; any contraindication to epidural catheter placement. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University. All the women provided written, informed consent prior to participation in the study.

Randomization and Anaesthesia Protocol

Patients were randomly assigned at study entry to receive combined general/epidural anaesthesia (study group) or general anaesthesia alone (control group), according to a computer-generated randomization schedule.

All patients were premedicated with 0.5 mg atropine intramuscularly 1 h before surgery. In the control group, general anaesthesia was induced with 0.1 mg/kg midazolam, 2 – 4 μg/kg fentanyl, 2 mg/kg propofol and 0.5 – 0.7 mg/kg rocuronium by bolus intravenous injection. Anaesthesia was maintained by continuous intravenous infusion of fentanyl at 2 – 4 μg/kg per h, propofol at 4 – 8 mg/kg per h and atracurium at 0.1 – 0.15 mg/kg per h.

General anaesthesia in the study group was induced using the same method as in the control group, but was maintained by intravenous infusion of fentanyl at 2 μg/kg per h, propofol at 2 – 4 mg/kg per h and atracurium at 0.1 mg/kg per h. The insertion point for the epidural catheter in the study group was at the T10 – T11 intervertebral space. A test dose of 5 ml of 2% lidocaine was given to exclude subarachnoid misplacement of the epidural catheter. These patients then received a mixture of 1% ropivacaine with 2% lidocaine (to produce final concentrations of 0.375% ropivacaine and 1% lidocaine) via the epidural catheter. The loading dose was 8 – 10 ml, followed by an additional infusion of 4 – 5 ml every 1.5 h.

NKCC Assay

Venous blood samples were obtained at two time points: before anaesthesia (T_pre) and 4 h after skin incision (T_{4 h}). For the NKCC assay, heparinized, sterile blood samples were mixed 1 : 3 with McCoy's Modified Medium (Gibco BRL, Grand Island, New York, NY, USA). Culture flasks containing blood and cell culture medium were stored at 20 – 25 °C for further analysis. Immediately before the assay, peripheral blood mononuclear cells (PBMCs) were isolated from stored blood samples by centrifugation on a Histopaque^®-1077 (Sigma-Aldrich, St Louis, MO, USA) gradient for 15 min at 1100 g , at room temperature. The interphase layer was collected with a pipette and washed three times with 40 mM phosphate-buffered saline, pH 7.4 (Gibco BRL). NKCC was determined by measuring the amount of radioactivity released from ⁵¹Cr-labelled (New England Nuclear Corp., Boston, MD, USA) K-562 target cells using a Beckman LS-6800 counter (Beckman Coulter, Brea, CA, USA). A sample (100 μl) of the K-562 cell suspension containing 1 × 10⁵ cells/ml was incubated in triplicate with the same volume of PBMCs at a 1 : 40 ratio in microtitre plates, for 4 h at 37°C in 5% carbon dioxide. Spontaneous release of radioactivity was determined by incubation of the K-562 target cells with medium only. Maximum release of radioactivity was determined by the addition of detergent (2% sodium dodecyl sulphate) to the cells. Percentage cytotoxicity was calculated as: 100 × (experimental counts per minute [cpm] - spontaneous cpm) / (maximum release cpm - spontaneous cpm).^22,23

Cytokine Measurement

For cytokine measurements, venous blood samples were left to clot at room temperature and then stored overnight in a refrigerator to contract the clot. After removing the clot, the remaining material was then centrifuged at 4000 g and the resulting serum was stored at –80 °C until analysis. Serum concentrations of IL-1β, IL-8, IL-10 and IFN-γ were measured by enzyme-linked immunosorbent assays (ELISA) (Bio-Plex^® Human Cytokine Assay; Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Minimum detectable concentrations were 0.06 pg/ml for IL-1β, 0.11 pg/ml for IL-8, 0.15 pg/ml for IL-10 and 0.05 pg/ml for IFN-γ. Intra- and interassay coefficients of variation for all ELISAs were < 7% and < 11%, respectively. All samples were measured in duplicate and the mean was used for statistical analyses.

Statistical Analyses

Statistical analyses were performed using the SPSS^® statistical package, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. The mean ± SD of the data from the study and control groups were calculated and compared using the unpaired Student's t-test. The T_pre and T_{4 h} data within groups were compared using the paired Student's t-test. All tests were two-tailed and a P-value < 0.05 was considered statistically significant.

Results

A total of 61 consecutive eligible patients participated in this study; 31 women were randomized to the study group (general/epidural anaesthesia) and 30 to the control group (general anaesthesia alone). The two groups were homogeneous for age, weight, ASA physical status, baseline value of percentage NKCC, baseline cytokine concentrations, blood loss and length of operation (Tables 1 and 2). None of the patients in the study group experienced complications due to the epidural catheter.

TABLE 1:

Demographic and intraoperative characteristics of patients with epithelial ovarian cancer who underwent radical resection with either general anaesthesia alone (control group) or general and epidural anaesthesia (study group)

Characteristic	Control group n = 30	Study group n = 31
Age, years	56 ± 8.6	59 ± 7.2
Weight, kg	62 ± 5.3	64 ± 4.9
Length of operation, min	182 ± 31.5	170 ± 26.3
Blood loss, ml	453 ± 43.2	401 ± 51.7
ASA physical status scale (0 – 5) a	2.2 ± 0.3	2.0 ± 0.3

Data presented as mean ± SD.

ASA, American Society of Anesthesiologists.

a

0, normal, healthy; 5, moribund, not expected to survive without operation.

There were no statistically significant between-group differences (P  0.05).

TABLE 2:

Natural killer cell cytotoxicity (NKCC) and serum cytokine concentrations before anaesthesia (T_pre) and 4 h after skin incision (T_{4 h}), in patients with epithelial ovarian cancer who underwent radical resection with either general anaesthesia alone (control group) or general and epidural anaesthesia (study group)

NKCC or cytokine	Control group n = 30	Study group n = 31	Statistical significance a
NKCC, %
Tpre	32.4 ± 3.6	34.7 ± 4.1	NS
T4 h	18.8 ± 2.3	28.4 ± 3.9	P < 0.001
Statistical significance b	P < 0.001	P = 0.024
IL-1β, pg/ml
Tpre	4.7 ± 0.5	4.5 ± 0.6	NS
T4 h	6.1 ± 0.7	5.5 ± 0.6	P = 0.003
Statistical significance b	P < 0.001	P = 0.015
IL-8, pg/ml
Tpre	36.5 ± 4.6	35.7 ± 4.2	NS
T4 h	6.6 ± 7.5	48.9 ± 5.6	P = 0.020
Statistical significance b	P < 0.001	P = 0.008
IL-10, pg/ml
Tpre	1977 ± 215	2019 ± 233	NS
T4h	1498 ± 162	1796 ± 186	P < 0.001
Statistical significance b	P < 0.001	P = 0.005
IFN-γ, pg/ml
Tpre	1.5 ± 0.2	1.4 ± 0.2	NS
T4 h	0.9 ± 0.1	1.1 ± 0.1	P = 0.017
Statistical significance b	P < 0.001	P = 0.006

Data presented as mean ± SD.

a

Unpaired Student's t-test

b

paired Student's t-test.

IL, interleukin; IFN, interferon; NS, not statistically significant (P  0.05).

At T_pre, there was no significant difference in mean percentage NKCC between the two groups (Table 2). At T_{4 h}, mean percentage NKCC was significantly higher in the study group compared with the control group (P < 0.001). Both the control and the study group showed a significant decrease in NKCC at T_{4 h} compared with T_pre (P < 0.001 and P = 0.024, respectively).

In both groups, concentrations of IL-1β and IL-8 were significantly higher at T_{4 h} than at T_pre (P < 0.001 for both comparisons), whereas concentrations of IL-10 and IFN-γ were significantly lower at T_{4 h} than at T_pre (P < 0.001 for both comparisons; Table 2). The study group had significantly higher levels of IL-10 and IFN-γ (P < 0.001 and P = 0.017, respectively), and significantly lower levels of IL-1β and IL-8 (P = 0.003 and P = 0.020, respectively) at T_{4 h} compared with the control group.

Discussion

The use of regional anaesthesia and analgesia has consistently been shown to be helpful in maintaining perioperative immune function. ²⁴ Anaesthesia and analgesia attenuate the neuroendocrine response to surgery, reduce the amount of general anaesthesia required during the operation and provide excellent pain relief, essentially obviating the need for postoperative opioids. ²⁵ For patients with EOC, radical surgery may include hysterectomy, bilateral adnexectomy with excision of the pelvic peritoneum, total omentectomy, appendectomy, removal of bulky pelvic and lumboaortic nodes, and even bowel resection, cholecystectomy, splenectomy and diaphragmatic resection in patients diagnosed at an advanced stage. ²⁶ Traditionally, general anaesthesia has been recommended for EOC surgery because the procedure is extensive and surgeons might find more cancerous tissue than expected, lengthening the time needed to complete the operation. As a consequence, no group was given epidural anaesthesia alone in the present study. Measurement of percentage NKCC and serum concentrations of the four cytokines demonstrated that those EOC patients who received the combined general/epidural anaesthesia exhibited less suppression of NK cell activity, higher serum concentrations of IL-10 and IFN-γ, and lower serum concentrations of IL-1β and IL-8 at T_{4 h} compared with those who received general anaesthesia alone.

The NK cells provide important host protection against tumour formation, progression and metastasis. ²⁷ Previous studies have indicated that NK cell activity correlates with tumour development in various types of cancer. ²⁸ Both surgical stress and anaesthetics can inhibit perioperative NK cell activity and suppress immune function. ²⁹ The finding in the present study of higher NKCC in patients with EOC when epidural anaesthesia was combined with general anaesthesia, compared with general anaesthesia alone, is in agreement with some previous studies. For example, Tønnesen and Wahlgreen ³⁰ reported preservation of NK cell activity after epidural use in patients undergoing hysterectomy. Similarly, epidural anaesthesia appeared to preserve perioperative NKCC compared with general anesthesia in a study by Koltun et al. ²² Collectively, these observations suggest that epidural anaesthesia and analgesia may help to attenuate or prevent perioperative suppression of NK cell function and preserve the body's defences against tumour progression.

Cytokines play an important role in immune function, inflammation and general tissue homeostasis. Depending on their concentration and other modulating factors, certain cytokines are protumourigenic, whereas others are antitumourigenic. IL-1β has been shown to promote tumour growth and metastasis, and to induce tumour-mediated immune suppression. ³¹ Evidence also suggests that IL-8 is critical to tumour neovascularity and progression. ³² In contrast, both IL-10 and IFN-γ are well known antitumour cytokines.^33,34 IL-10 exhibits antitumour and antimetastatic activity through enhanced NK cell lysis of tumour cells. ³⁵ IFN-γ has important effects in the tumour microenvironment, including the inhibition of cell proliferation and angiogenesis. ³³ Investigations have revealed that epidural anaesthesia and spinal block may influence cytokine responses and inhibit tumour metastasis.^4,36 The present study showed higher serum concentrations of antitumourigenic IL-10 and IFN-γ, and lower serum concentrations of protumourigenic IL-1β and IL-8 at T_{4 h}, in patients who received combined general/epidural anaesthesia. This may help to explain why epidural anaesthesia and analgesia might protect the body's immune function, and have been associated with decreased recurrence rates and prolonged survival in patients with malignancies. ³⁷

In conclusion, the present prospective randomized trial in 61 women undergoing radical resection for EOC demonstrated significant differences in NKCC and cytokine responses between patients receiving combined general/epidural anaesthesia and general anaesthesia alone. Combined general/epidural anaesthesia appeared to promote antitumourigenic NKCC and cytokine responses. Further studies are required to explore the implications of these observations.