Circulating C-reactive protein levels as determined by immunoturbidimetry (Olympus, Hamburg, Germany) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for laparoscopic vs open surgery; dagger, P<.05 for preoperative (Preop) vs postoperative (Postop) values.
Circulating interleukin 6 levels as determined by enzyme-linked immunosorbent assays (Biosource, Nivelles, Belgium) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for laparoscopic vs open surgery; dagger, P<.05 for preoperative (Preop) vs postoperative (Postop) values.
B-lymphocyte cell counts as determined by flow cytometry (CD19+) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for preoperative (Preop) vs postoperative (Postop) values.
T-lymphocyte cell counts as determined by flow cytometry (CD3+) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for preoperative (Preop) vs postoperative (Postop) values.
Helper T–cell counts as determined by flow cytometry (CD3+ and CD4+) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for preoperative (Preop) vs postoperative (Postop) values.
Cytotoxic (suppressor) T–cell counts as determined by flow cytometry (CD3+ and CD8+) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for preoperative (Preop) vs postoperative (Postop) values.
Natural killer cell counts as determined by flow cytometry (CD3− and CD16/56+) in patients with colorectal disease treated with open and laparoscopic surgery. Asterisk indicates P<.05 for laparoscopic vs open surgery; dagger, P<.05 for preoperative (Preop) vs postoperative (Postop) values.
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Wichmann MW, Hüttl TP, Winter H, et al. Immunological Effects of Laparoscopic vs Open Colorectal Surgery: A Prospective Clinical Study. Arch Surg. 2005;140(7):692–697. doi:10.1001/archsurg.140.7.692
Laparoscopy has become a popular approach for the surgical treatment of benign and even malignant colorectal diseases. Several authors have reported better preserved immunity in patients undergoing laparoscopic compared with conventional colorectal surgery. The present study addresses the hypothesis that specific and nonspecific immunity are differently affected by laparoscopic and conventional colorectal surgery.
Nonrandomized control trial.
Seventy prospectively enrolled patients with colorectal diseases undergoing laparoscopic (n = 35) or open (n = 35) surgery.
Main Outcome Measures
Blood samples were taken prior to surgery as well as on days 1, 3, and 5 after surgery. Cell numbers of lymphocyte subpopulations as well as natural killer cells were determined by flow cytometry, and interleukin 6 and C-reactive protein serum levels were measured.
Significant differences between study and control patients (P<.05) were detected regarding circulating interleukin 6 and C-reactive protein levels with a reduced proinflammatory response to surgery in patients after laparoscopic surgery. Furthermore, postoperative natural killer cell counts were significantly higher in patients after laparoscopic surgery. The levels of B lymphocytes and T lymphocytes and helper T–cell counts and cytotoxic (suppressor) T–cell counts did not show significant differences after open or laparoscopic surgery.
Our findings indicate a less pronounced proinflammatory response to surgical trauma in patients after minimally invasive surgery. The nonspecific immune response appears to be less affected by laparoscopic surgery when compared with open surgery while the specific cell-mediated immunity is equally affected. These findings are important because a divergent effect on specific and nonspecific immunity of laparoscopic surgery for colorectal disease has not been reported before.
The development of minimally invasive surgery has allowed major changes in the surgical treatment of various benign and malignant diseases, especially because it limits surgical trauma.1 During recent years, the laparoscopic approach has developed as an interesting therapeutic alternative for the resection of various colorectal diseases. This procedure has been shown to be feasible in most patients with benign disease and can be performed without an increase of perioperative morbidity and mortality rates.2-7 Because the surgical trauma is limited, the laparoscopic approach usually allows for a rapid return to preoperative activity levels with significantly shorter hospitalization. Recent reports also indicate reduced rates of postoperative ileus, wound infection, and cardiorespiratory complications after laparoscopic surgery when compared with the open approach.1,8,9
Recently, the Clinical Outcomes of Surgical Therapy Study Group10 has reported that laparoscopic-assisted colectomy and open colectomy for colon cancer provide comparable long-term results in a multi-institutional setting. These findings of a large multi-institutional study support the data published by Lacy et al,11 who were able to show that laparoscopic surgery was superior to open surgery in colon cancer therapy in a single-center study when comparing morbidity, hospital stay, tumor recurrence, and cancer-related survival.
Experimental and clinical data, therefore, suggest that laparoscopic surgery is also suitable for the treatment of malignant disease. It appears that laparoscopic resection of colorectal cancer is associated with clinically relevant benefits during the first weeks after surgery and that it can be performed with the same intention of radical treatment as conventional resection.10-12 A recent study, however, reported that only minimal, short-term quality-of-life benefits could be observed with laparoscopic-assisted colectomy when compared with open colectomy for colon cancer.13 Nonetheless, a finding that the laparoscopic approach to colorectal cancer results in less immunosuppression may have implications for the long-term prognosis of patients with cancer.7
Despite the promising clinical results, only limited information is available regarding the perioperative immunological effects of laparoscopic surgery when compared with conventional open large-bowel and rectal surgery. This issue is of major clinical interest because the reduced surgical trauma should result in reduced postoperative immune dysfunction in patients undergoing laparoscopic surgery, thus contributing to clinical and oncologic advantages for these patients.
Until now, it has been reported that the degree of postoperative inflammation is reduced after laparoscopic surgery.14,15 Other groups also observed significantly better preservation of lymphocyte subpopulations, neutrophil function, and cell-mediated immunity after laparoscopic vs open colorectal surgery.16-19 Furthermore, it has been observed that cell-mediated immunity, as assessed by delayed-type hypersensitivity testing in humans, is better preserved after laparoscopic vs open colorectal resection.20 This lesser degree of operative stress was also confirmed by experimental animal studies by Kuntz et al.21
This prospective clinical study was performed to evaluate perioperative immune parameters in 70 patients with various colorectal diseases. We herein analyze the effects of laparoscopic and open surgery on proinflammatory cytokine levels (interleukin 6 [IL-6]) and C-reactive protein (CRP) levels. Furthermore, we measured lymphocyte subpopulations, leukocyte and granulocyte counts, and circulating natural killer (NK) cells before surgery and on days 1, 3, and 5 after surgery. This study, therefore, allows assessment of the effects of laparoscopic and conventional colorectal surgery on specific and nonspecific immune responses after major abdominal surgery.
Patients with known immunological dysfunction (advanced liver disease, HIV [human immunodeficiency virus] infection, hepatitis C virus infection), drug addiction, and cardiac or pulmonary insufficiency were excluded from the study. Patients recruited for laparoscopic resection were not considered for analysis when converted to the open procedure (n = 2 during the study period).
During the study period, we asked all patients who had surgery on one minimally invasive surgical ward (that of M.M.H.) to participate in our clinical case-control study. Control patients were recruited on another surgical ward (that of T.P.H.). Patient selection was based on their admission to different surgical wards in the same institution. All patients recruited for our clinical case-control study were informed that additional blood was taken during the perioperative period for immunological evaluation, and written consent was obtained.
Minimally invasive colorectal surgery was performed as a laparoscopic-assisted procedure with removal of the resected specimen via a horizontal minilaparotomy (5 cm) just above the mons pubis. Laparoscopic surgery was done using a 4-trocar technique with 1 trocar (10 mm) inserted via a paraumbilical incision (camera port). Two additional (5- or 10-mm) trocars were inserted in the right and left lower abdomen, and 1 trocar (12 mm) was inserted in the midline just above the mons pubis (the site of the minilaparotomy). After removal of the resected specimen and preparation of the stapler anastomosis, we closed the minilaparotomy and reintroduced pneumoperitoneum.
Conventional colorectal surgery was performed via a vertical midline incision ranging from 5 to 10 cm above the umbilicus to the mons pubis. After we removed the resected specimen, we performed a stapler anastomosis.
Blood samples were taken on the day before surgery as well as on days 1, 3, and 5 after surgery.
Peripheral venous blood samples were collected in EDTA collection tubes (Kabe, Nümbrecht-Elsenroth, Germany). The monoclonal antibodies used for immunophenotyping were purchased from Becton Dickinson (San Jose, Calif). The samples were prepared by labeling 50 μL of whole blood with 10 μL of monoclonal antibody for 10 minutes in the dark using the antibody combinations indicated in the Table. The monoclonal antibodies were conjugated to the fluorochrome fluorescein or phycoerythrin. The blood samples subsequently underwent a hypotonic lysis of red blood cells for 10 minutes (FACS Lysing Solution; Becton Dickinson) and were washed with phosphate buffered saline. The samples were made into pellets (300 g, room temperature, 5 minutes) and then resuspended in 150 μL of phosphate buffered saline solution. The fluorescence was measured using a FACScan (Becton Dickinson) within 60 minutes after processing the samples.
Fluorescence-activated cell sorter analysis was performed on a FACScan flow cytometer after calibration with CaliBRITE beads (Becton Dickinson) using the AutoCOMP software package (Becton Dickinson). A minimum of 10 000 cells were measured for each determination. For 2-parameter evaluation dot plots and quadrant statistics, we used the SimulSET software package (Becton Dickinson). The lymphocyte populations were automatically gated, including at least 98% of all lymphocytes measured within each sample.
Peripheral venous blood samples were collected in serum collection tubes (Kabe) and were subsequently centrifuged at 300 g for 15 minutes at 4°C. Serum aliquots were subsequently stored at −80°C until assayed for IL-6.
Circulating serum IL-6 levels were determined using enzyme-linked immunosorbent assays (Biosource, Nivelles, Belgium) as described by the manufacturer. The concentration of IL-6 present in the samples was determined at 450 nm on a Bio-Tek plate reader (EL-311; Bio-Tek Instruments Inc, Winooski, Vt). C-reactive protein was measured with the immunoturbidimetric method (Olympus, Hamburg, Germany).22
Data were analyzed using 1-way analysis of variance, analysis of variance on ranks, the Newman-Keuls test, and the Dunn test. Differences were considered statistically significant at P<.05. Data are presented as mean ± SEM.
Among the study patients (laparoscopic colorectal surgery), the mean ± SEM patient age was 64.2 ± 1.6 years, and most study patients were men (71%). The majority of study patients underwent surgery for colorectal cancer (76%). The mean ± SEM length of surgery was 188 ± 28 minutes. During the early postoperative period (until discharge from the hospital), complications (anastomotic leakage, surgical site infection, hemorrhage) occurred in 23% of the study patients. One patient died from myocardial infarction during the postoperative period (mortality rate, 5%).
Among the control patients (open colorectal surgery), the mean ± SEM patient age was 61.5 ± 1.6 years, and most control patients were men (57%). Again, most control patients underwent surgery for colorectal cancer (77%). The mean ± SEM length of surgery was significantly shorter when compared with patients undergoing laparoscopic surgery, 104 ± 13 minutes (P<.05). During the early postoperative period, complications were observed in 20% of the control patients.
After both laparoscopic and open colorectal surgery, we observed a significant increase of circulating CRP levels (Figure 1). This increase was significantly higher in patients after conventional surgery when compared with patients after minimally invasive surgery.
After conventional and laparoscopic surgery, we observed a significant increase in serum IL-6 levels (Figure 2), which were significantly higher in patients after open surgery during the early postoperative period (days 1 and 3) and were comparable in both patient groups on day 5 after surgery.
After laparoscopic as well as conventional surgery, we saw a rapid but insignificant drop of circulating B lymphocytes (CD19+), which was significantly different from preoperative values only in patients after laparoscopic surgery on postoperative day 5 (Figure 3).
We observed a significant depression of circulating T-lymphocyte (CD3+) cells, which lasted until day 5 after surgery and did not differ between both patient groups (Figure 4).
A significant depression of circulating helper T cells (CD3+ and CD4+) was observed (Figure 5). No differences were detected when comparing study and control patients.
We observed a lasting significant depression of circulating cytotoxic (suppressor) T cells (CD3+ and CD8+) (Figure 6). The depression of cell counts was comparable in study and control patients.
We observed an initial significant depression of NK-cell (CD16/56+ and CD3−) counts in both patient groups (Figure 7). In patients who had minimally invasive surgery, this depression was reversed on day 5, and circulating NK cells were significantly higher on days 1 and 5 after surgery, when compared with patients after open colorectal surgery.
Laparoscopic surgery is as safe as open colorectal surgery, and laparoscopic colorectal cancer surgery is feasible but should be performed within clinical studies and in surgical centers that have sufficient experience with laparoscopic colorectal surgery for benign diseases.6,7,23 In addition to the ongoing discussion about the oncologic safety of this approach, it is still not completely clear whether the laparoscopic approach offers significant immunological advantages over the conventional open approach.23,24 Whelan et al20 recently reported a significantly better preservation of delayed-type hypersensitivity responses after laparoscopic vs conventional surgery. These findings indicate better preserved cell-mediated immune responses in patients after laparoscopic colorectal surgery.20 The postoperative immune dysfunction is important for patients undergoing surgery for benign as well as malignant disease because it influences the rate of infectious complications as well as the growth of disseminated tumor cells.25,26 Especially in patients with cancer, better preserved postoperative immunity could result in better long-term oncologic results.
Recently, the Clinical Outcomes of Surgical Therapy Study Group10 demonstrated in a multicenter setting with 872 patients that laparoscopic-assisted colectomy and open colectomy for colon cancer provide comparable long-term results. These authors provided good evidence to suggest that the laparoscopic approach is an acceptable alternative to open surgery for colon cancer. The findings reported by this study group support the results of Lacy et al,11 who even reported better oncologic and clinical results of laparoscopic surgery when compared with open surgery.
We conducted this prospective clinical study to address the issue of potential differences in postoperative immunological alterations after conventional and laparoscopic colorectal surgery. In addition, this work focused on the potential differences in trauma-induced alterations of specific and nonspecific immunity.
As done in other clinical studies, we assessed immune function by measuring circulating immunocompetent cells (ie, helper T cells, cytotoxic T cells, and NK cells) as well as circulating IL-6 and CRP.27-30 It has been reported that the functional capacity of NK cells correlates well with absolute cell numbers.31 Furthermore, absolute numbers of circulating immunocompetent cells have been considered good indicators of the individual patient’s immune function.27-30
Our findings show that minimally invasive surgery results in a less pronounced proinflammatory response to surgical trauma. In addition, we observed that the effector cells of the nonspecific immune response (NK cells) are less affected by laparoscopic surgery. The effector cells of the specific immune response (helper T cells and cytotoxic T cells), however, are equally depressed after conventional open as well as laparoscopic surgery for colorectal diseases.
In postoperative immune function, it has been reported that the systemic immune response was better preserved after laparoscopic surgery than after open surgery.19,20,32 Nonetheless, Gupta and Watson32 also showed that laparoscopic surgery was associated with a more severe suppression of intraperitoneal cell-mediated immunity, which may be relevant in the treatment of malignant disease. Braga et al24 recently observed significantly fewer infectious complications and faster recovery after laparoscopic colorectal surgery when compared with open surgery. Moreover, it was reported that the suppressive effect of open surgery on the whole T-cell population was more evident 15 days after operation whereas no suppressive effect was found in the laparoscopic group.24 These authors hypothesized that laparoscopy induced a less pronounced local inflammation than open surgery, which would allow for a faster return of TH1 cells into circulation.24 This hypothesis is in agreement with the lesser degree of proinflammation reported here as well as by other groups.14,15 Nonetheless, our observation of significantly depressed helper T–cell counts after both laparoscopic and conventional colorectal surgery does not support this hypothesis of differently affected TH1 cells and is at odds with the findings reported by Braga et al24 and others who described better preserved cell-specific immunity after laparoscopic surgery.19,20
Our findings of better preserved nonspecific immune functions in patients after laparoscopic colorectal surgery help explain the reported lower rate of infectious complications in these patients.24 Furthermore, it is interesting to note that the NK cell counts were less affected after laparoscopic colorectal surgery, but a more severe significant depression was observed after conventional surgery. This finding is relevant to the discussion of the potential oncologic advantages of laparoscopy because NK cells are important in controlling the growth of metastatic tumor cells, which are disseminated during surgical manipulation.31,33 Moreover, it has been reported that NK cell cytotoxicity increases with the number of NK cells, and low preoperative levels of NK-cell cytotoxicity have been shown to correlate with an increased risk of colorectal cancer recurrence.31 Cristaldi et al34 also observed a less pronounced reduction of NK cells after laparoscopic surgery.
The findings of significant differences between patients after laparoscopic and conventional colorectal surgery regarding the release of CRP and IL-6 suggest a more pronounced proinflammatory response in patients undergoing conventional surgery. This observation confirms findings reported by Leung et al,35 who detected significantly smaller peaks of circulating IL-1β, IL-6, and CRP levels in a group of 34 patients undergoing laparoscopic or conventional resection of rectosigmoid carcinoma. It is not clear whether these differences have immunological relevance because a certain degree of proinflammation is required for the initiation of host defense mechanisms as well as for the activation of repair processes after tissue trauma.36,37 It is nonetheless well known that an overwhelming inflammatory response to surgical trauma may ultimately lead to organ dysfunction.38,39
After laparoscopic as well as conventional colorectal surgery, we observed a significant depression of circulating B and T lymphocytes as well as helper T cells and cytotoxic T cells. This observation is in agreement with the findings of other groups who have previously shown that a significant depression of these cell counts is associated with immune dysfunction and may even promote tumor growth.28,40-42 The findings presented here indicate a comparable significant depression of circulating mediators of the specific immune system (ie, helper T–cell and cytotoxic T–cell counts). This observation is in agreement with the findings of Tang et al,30 who also observed comparable changes of immune responses in patients undergoing laparoscopic or conventional colorectal cancer surgery. Other investigators, however, reported significantly better preservation of lymphocyte subpopulations, neutrophil function, and cell-mediated immunity after laparoscopic vs open colorectal surgery.16-18 Obviously, additional prospective studies are required to elucidate the important question of whether laparoscopy offers significant advantages regarding specific immune function in patients with colorectal diseases. If we consider together all of the studies that have investigated systemic immunological function after laparoscopic and open surgery in both experimental models and in clinical settings, we see their outcomes have consistently demonstrated that laparoscopic approaches are associated with less overall disturbances of the systemic immune function (Gupta and Watson32 provide a review). Nonetheless, our findings do not confirm better preserved specific immune response in patients undergoing laparoscopic colorectal surgery, as previously reported by Whelan et al.20
It is not known whether the observed immunological effects of laparoscopic colorectal surgery are of direct oncologic relevance and contribute to the observed comparable or even better oncologic results of laparoscopic surgery for malignant colorectal disease, which have been reported in the literature.10,11
The long-term effects of immunosuppression in response to treatment and development of metastases still remain obscure. However, we can assume that our observation of better preserved nonspecific immunity in patients after laparoscopic colorectal surgery has beneficial effects on perioperative infectious complication rates—which is true for patients with benign as well as malignant colorectal disease. In addition, our findings are of clinical interest because a divergent effect on specific and nonspecific immunity of laparoscopic surgery for colorectal disease has not been previously reported.
Correspondence: Matthias W. Wichmann, MD, Department of Surgery, Ludwig-Maximilians University, Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany (Matthias.Wichmann@med.uni-muenchen.de).
Accepted for Publication: November 30, 2004.