Up-regulation of Fas ligand (FasL) mRNA expression in peripheral blood mononuclear cells (PBMC) after major surgery

Abstract

FasL, which is expressed mainly on activated lymphocytes, can induce apoptosis (programmed cell death) of cells which express Fas. Fas/FasL interaction is primarily beneficial in maintaining immunological and physiological homeostasis by eliminating unnecessary cells. Dysregulation of the interaction, however, leads to tissue damage. We investigated how Fas/FasL levels changed after major surgery. The major aim of this study was to elucidate the involvement of the Fas/FasL system in postoperative inflammation. The investigation involved 10 patients admitted to the intensive care unit after surgery. Although the percentage of Fas⁺ cells and the amount of Fas expression tended to increase, there was no significant difference between pre- and post-operative samples. In contrast, the levels of FasL mRNA were dramatically up-regulated after operation. Post-operative C-reactive protein (CRP) levels increased and correlated well with FasL levels (r = 0.91, P < 0.01). Lymphocyte counts decreased after operation and were inversely proportional to FasL levels (r = 0.58, P < 0.05). These results suggest that the enhanced FasL expression is likely to be related to systemic inflammatory responses induced during the perioperative period. FasL up-regulation may be involved in the aggravation of tissue damage, including lymphocytopenia, in the early post-operative period.

INTRODUCTION

Fas is a cell membrane molecule belonging to the tumour necrosis factor (TNF) receptor superfamily. Fas is expressed on immature thymocytes, activated T cells and non-lymphoid cells in the liver, lung, and heart [1]. FasL is also a cell membrane molecule belonging to the TNF superfamily. This ligand is expressed mainly on cytotoxic T lymphocytes, natural killer (NK) cells, and neutrophils [2–5]. The interaction of Fas and FasL induces apoptotic signals in cells expressing Fas. Apoptosis occurs through the activation of a cell-intrinsic suicide program. During apoptosis, the nucleus and the cytoplasm condense, and the dying cell often fragments into membrane-bound apoptotic bodies that are rapidly phagocytosed and digested by macrophages [1]. The Fas/FasL interaction is primarily beneficial in maintaining immunological homeostasis by eliminating unnecessary cells, such as autoreactive lymphocytes [6]. Dysregulation of the interaction, however, could possibly lead to tissue damage by autoimmune processes. In this regard, numerous investigations have established the immunopathological relevance of the Fas/FasL system [2–9]. For example, the loss of Fas expression or of its ligand results in the development of immune disorders, such as an autoimmune disease that appears similar to human systemic lupus erythematosus (SLE), including autoantibody production and abnormal lymphocyte accumulation in lymphoid organs, in lymphoproliferation disorder (lpr) [10] or generalized lymphoproliferative disease (gld ) [11]. In humans, a rare autoimmune lymphoproliferative syndrome (ALPS) has been reported, which is characterized by massive non-malignant lymphadenopathy and expanded populations of abnormal lymphocytes [12]. These patients lacked the Fas/FasL interaction-mediated lymphocyte apoptosis associated with a large deletion in the gene which encodes Fas and no detectable cell surface expression of Fas.

Surgical stress has been shown to induce remarkable alterations in the immune system [13–16]. In general, major surgery induces not only systemic inflammatory responses but also immune suppression [13,14]. Proinflammatory cytokines, C-reactive protein (CRP), and the numbers of neutrophil are up-regulated by surgical stress. Serum levels of proinflammatory cytokines (e.g. IL-6) and CRP correlate with the magnitude of tissue injury [15]. On the other hand, lymphocytopenia is often observed in the early post-operative period [16]. Recently, it was reported that circulating lymphocytes in the early post-operative period are susceptible to Fas-mediated apoptosis, which may cause depletion of circulating lymphocytes after surgery [17]. The alteration of the Fas/FasL system during the perioperative period, however, remains unknown. Thus, the major aim of this study was to elucidate the involvement of the Fas/FasL system in inflammatory states after major surgery.

PATIENTS AND METHODS

Patients

Ten patients (five oesophageal carcinoma, five cardiac disease) who were consecutively admitted to the intensive care unit after surgery were recruited in this investigation. The demographic data of the patients are summarized in Table 1. In general, operations for oesophageal carcinoma and cardiac diseases are typical of major surgery, which induces a severe systemic inflammatory response. This investigation was approved by the Institutional Ethical Committee of Osaka University Hospital. Informed written consent was obtained from each patient. Peripheral blood samples were collected quickly from an indwelling arterial catheter before surgery (pre: morning of operation day at around 7:00 a.m.) and on post-operative days (POD: morning each day at around 7:00 a.m.) 1–4.

Table 1.

Demographic data of the patients

Oesophageal carcinoma patients. All patients underwent radical operation with thoracotomy. Two patients had received chemotherapy and radiation therapy before the operation. The operation time was 575.0 ± 69.2 min.

Cardiac disease patients. Three patients underwent single valve replacement, one underwent coronary artery bypass grafting, and the other underwent correction of Fallot's tetralogy. The operation time was 371.0 ± 39.9 min.

No immunosuppressive agents, such as steroids, were administered to any of the patients during the perioperative period. All patients received blood transfusion during operation or post-operation. We have routinely used leucocyte depletion filter (PALL, Tokyo, Japan) to remove leucocytes after radiation. Thus, any influence on the results due to contamination would have been negligible. None of the patients displayed symptoms of infection before surgery. All values are expressed as means ± s.e.m.

Flow cytometry

Expression of Fas on CD4⁺ T cells was examined by flow cytometry. The following MoAbs were used in this study: PE-labelled mouse anti-human CD95 (Fas), FITC-labelled mouse anti-human CD4 MoAb (Becton Dickinson, San Jose, CA). For immunofluorescence staining, 50 μl of heparinized whole blood and 10 μl of MoAb (PE-labelled and FITC-labelled) were incubated for 15 min at room temperature in the dark. The samples were then incubated with 2 ml of erythrocyte-lysing solution (Ortho Diagnostic System, Tokyo, Japan) for 10 min at room temperature in the dark. After incubation, samples were centrifuged at 500 g for 10 min. After the supernatant was discarded, cells were suspended in PBS containing 2% of bovine calf serum and were centrifuged at 500 g for 10 min. Then the cells were resuspended in PBS containing 1% paraformaldehyde and stored in the dark prior to analysis. Flow cytometric measurements were carried out with a FACScan (Becton Dickinson). Analysis was performed using a software application (CELLQuest; Becton Dickinson). Lymphocytes were gated according to their forward and side scatter. FITC- and PE-labelled isotype control MoAbs were used to determine the positive and negative population.

Preparation of PBMC

Heparinized whole blood samples (10 ml) were diluted by the addition of an equal volume of PBS. Then the diluted blood was carefully layered over Lymphoprep (Nycomed, Oslo, Norway) and centrifuged at 1000 g for 20 min at 20°C in a swing-out rotor. After centrifugation, the mononuclear cells formed a distinct band at the medium interface. The harvested PBMC were suspended in cold PBS and washed twice. Then the PBMC samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

Semi-quantitative RT-PCR

The expression of FasL mRNA in mononuclear cells was examined by RT-PCR using a previously described method [18] with minor modifications. Total cellular RNA was extracted by the guanidinium-isothiocyanate method. Single-strand cDNA was synthesized with reverse transcriptase from poly A(+) mRNA and was used for the PCR reaction. Primer sequences were as follows: β-actin, 5′-GTGGGGCGCCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTC-3′; FasL, 5′-CTGGGGATGTTTCAGCTCTTC-3′ and 5′-CTTCACTCCAGAAAGCAGGAC-3′. Fifty nanograms of cDNAs were amplified by PCR under the following conditions: heating at 92.5°C for 2 min, then amplification of the cDNAs for 35 cycles, where each cycle consisted of 92.5°C for 1 min, 60°C for 1 min, and 60°C for 7 min, after which, storing of the samples at 4°C until analysed using a programmed thermal cycler (Gene Amp 9600; Perkin Elmer, Norwalk, CT). After amplification, PCR products were separated by electrophoresis on 1.8% agarose gel and visualized by UV light illumination after the samples had been stained with ethidium bromide. Band intensity was calculated using laser densitometry (ATTO densitograph; ATTO, Tokyo, Japan) and was expressed as the ratio of FasL to β-actin.

CRP, lymphocyte count

CRP in serum was measured by latex particle-enhanced turbidimetry [19]. The number of lymphocytes was measured in each sample by use of an electronic counter.

Statistical analysis

All results were expressed as means ± s.e.m. The Mann–Whitney U-test, one factor ANOVA test and Pearson's correlation coefficient were used to compare the intergroup values. Statistical significance was determined to be a P value of < 0.05.

RESULTS

Fas expression on CD4⁺ T cells

Although the percentage of CD4⁺ Fas⁺ T cells and the average mean fluorescence intensity (MFI) of Fas on CD4⁺ T cells did tend to increase, there was no significant difference between values from samples taken on Pre and POD 1, 2, and 3. Fas was expressed constitutively on CD4⁺ T cells and there was only a minimal increase after surgery (Fig. 1). The average MFI from 10 patients were 29.6 ± 4.5 (Pre), and 31.9 ± 4.0, 33.3 ± 3.5, and 34.3 ± 3.5 (POD 1–3), respectively. We also analysed Fas expression on peripheral CD4⁺ T cells in samples from 10 healthy volunteers. There were no significant differences between normal control and Pre data (data not shown). Judging from these results, oesophageal carcinoma and cardiac disease do not seem to have a great influence on Fas expression.

Fig. 1.

Fas expression on CD4⁺ T cells before and after surgery. The expression of Fas was analysed by flow cytometry. There are no significant variations in any of the values. The data from 10 patients are shown as the mean ± s.e.m.

The expression of mRNA for FasL in PBMC

The levels of FasL mRNA in PBMC after surgery (POD 2 and 3) were significantly higher than the Pre level (Figs 2 and 3). FasL mRNA was hardly expressed at all in PBMC isolated from the pre-operative (Pre) period, but was extremely up-regulated after major surgery. We analysed FasL mRNA expression in PBMC isolated from two healthy volunteers. The results showed hardly any expression of FasL mRNA (data not shown).

Fig. 2.

Levels of FasL mRNA were examined by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using β-actin as a standard. The levels of FasL mRNA in PBMC after surgery (POD 2 and 3) were significantly higher than the pre-operative level (Pre) The data from 10 patients are shown as the mean ± s.e.m. * P < 0.05 versus Pre level; ** P < 0.01 versus Pre level.

Fig. 3.

The representative alteration of FasL mRNA expression in PBMC during perioperative periods. PBMC samples were collected at Pre and POD 1, 2, and 3. The expression of mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).

CRP level

Serum CRP concentrations significantly increased after operation and the maximum value was observed on POD 2 (Fig. 4). The trends of CRP elevation correlated well with the changes in FasL mRNA after surgery (Pearson's correlation coefficient = 0.90, P < 0.01). There was a weak correlation between CRP and Fas (Pearson's correlation coefficient = 0.39, P < 0.05).

Fig. 4.

Post-operative changes in the levels of serum C-reactive protein (CRP). CRP levels increased significantly after surgery and the highest levels were observed on POD 2. The data from 10 patients are shown as mean ± s.e.m. * P < 0.05 versus Pre level; ** P < 0.01 versus Pre level.

Lymphocyte count

Peripheral lymphocyte counts significantly decreased after surgery, and the minimum value was observed on POD 2 (Fig. 5). There was a moderate inverse correlation between the lymphocyte count and FasL mRNA expression in PBMC (Pearson's correlation coefficient = 0.67, P < 0.05). There was no significant correlation between lymphocyte count and Fas expression.

Fig. 5.

The numbers of lymphocytes decreased significantly after surgery. The lowest values were observed on POD 2. Data from 10 patients are shown as mean ± s.e.m. * P < 0.01 versus Pre level.

Comparison of the oesophageal carcinoma group and the heart disease group

In the oesophageal carcinoma group, the percentage of CD4⁺ Fas⁺ T cells and the average MFI of Fas on CD4⁺ T cells was inclined to be high compared with the heart disease group, but there were no significant differences in any of the values between the two groups (data not shown). Compared with the heart disease group, the oesophageal carcinoma group showed significantly higher values, including the expression of mRNA for FasL in PBMC on Pre, and POD 2 (Fig. 6) and in the serum CRP concentration on POD 1 and 2 (data not shown).

Fig. 6.

The comparison of FasL expression on mononuclear cells from the oesophageal carcinoma (•) and heart disease groups (○). Significant differences between the two groups were found in the expression of FasL. The values of the oesophageal carcinoma group in Pre and POD 2 were significantly higher than the corresponding values for the heart disease group. Data from 10 patients are shown as mean ± s.e.m. * P < 0.05; ** P < 0.01.

DISCUSSION

In the present study, we focused our scientific interest on the involvement of Fas/FasL in the inflammatory responses induced by surgical stress. Our study shows that a dramatic up-regulation of FasL mRNA levels in PBMC occurs after major surgery. Although the mononuclear cells isolated from the peripheral blood of patients showed hardly any expression of FasL mRNA before surgery, the results were similar to those for samples obtained from healthy volunteers: post-operatively the expression dramatically increased. The changes in the FasL mRNA levels correlated well with CRP levels. CRP is an important inflammatory protein with as much potency as some of the other inflammatory cytokines, including IL-1, IL-6, and TNF-α, and its production is regulated by these cytokines [20]. Proinflammatory cytokine IL-6 and CRP correlate well with the extent of tissue injury induced by major surgery [15]. Although the induction mechanisms for Fas/FasL expression have not been sufficiently clarified, our results provide evidence that the dramatic up-regulation of FasL could be related to the inflammation induced by surgical stress, because FasL is induced on the surface of activated cytotoxic T lymphocytes [2–4].

In the oesophageal carcinoma group, the expression of FasL and the serum concentration of CRP after surgery (POD 1 and 2) were significantly higher than in the heart disease group. Greater changes were observed in the oesophageal carcinoma group, probably because they were subject to a more prolonged period of operation and anaesthesia. The difference could be attributed to more sustained and more extensive perioperative invasion. These facts support the hypothesis that the magnitude of the increase in the expression of FasL may reflect the severity of the inflammatory responses. The oesophageal carcinoma group tended to have high Fas expression and also had a significantly higher FasL expression than the heart disease group, even in the preoperative period. One plausible explanation for high levels of Fas and FasL expression in preoperative periods of oesophageal carcinoma patients is that the presence of carcinoma itself stimulates the Fas and FasL system [21]. Fas-induced apoptosis has been found to occur spontaneously in malignant tumour cells, often markedly retarding their growth [21]. Additionally, two of the oesophageal carcinoma patients had received chemotherapy and radiation therapy in the preoperative period. There is evidence that these treatments can increase the numbers of apoptotic tumour cells [22]. These two factors probably account for the greater up-regulation of the Fas/FasL system of lymphocytes in the oesophageal carcinoma group than in the heart disease group.

A depletion of circulating lymphocytes is often observed in sepsis, severe thermal injury, and during the post-operative period [23]. There are two plausible explanations to account for the mechanism of lymphocytopenia. One is that lymphocytes are redistributed. Severe thermal injury produces a lymphocytopenia which features extensive lymphocyte activation. The depletion of circulating lymphocytes could be related to the expression of surface adhesion molecules and cell redistribution from blood to the tissues [23]. The other explanation is based on the clonal deletion of lymphocytes. It was recently reported that apoptosis is responsible for the depletion of circulating lymphocytes after surgery [17]. Thus, our result supports the hypothesis that up-regulated FasL induces excessive apoptosis and subsequent clonal deletion in lymphocytes expressing Fas.

Recent investigations have demonstrated that apoptosis due to Fas/FasL interaction is involved in the pathogenesis of tissue damage [24–27]. The transfer of lpr bone marrow, which is deficient in Fas on the cell surface, into wild-type hosts, which constitutively express Fas on cell surfaces, has evoked a graft-versus-host disease-like wasting syndrome [24,25]. The treatment of adult mice with anti-Fas antibody caused rapid hepatic failure and death, suggesting Fas/FasL interaction may mediate fulminant hepatitis in humans [26]. FasL was up-regulated in liver-infiltrating mononuclear cells and PBMC from patients with chronic active hepatitis C [27]. This finding suggests that the Fas/FasL system may play an important role in cellular injury of hepatocyte during hepatitis C viral infection.

The possible involvement of FasL in damaging the tissue of these organs during surgery requires more extensive study at both the molecular and cellular levels. To clarify these relationships, further studies utilizing lpr and gld mice in a post-operative inflammatory model would be useful.

In conclusion, this study demonstrates that rapid up-regulation of FasL occurs after major surgery, and one possibility suggested, although not proven, by our findings is that its induction might be related to inflammatory responses induced during the perioperative period. The increased expression of FasL might induce excessive apoptosis in host tissues following surgery. These reactions may be involved in the aggravation of tissue damage, including lymphocytopenia, in early post-operative period.