Anesth Analg 2004;98:1724-1728
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000112317.68730.B0
ANESTHETIC PHARMACOLOGY
The Effect of Propofol on Cytotoxicity and Apoptosis of Lipopolysaccharide-Treated Mononuclear Cells and Lymphocytes
Ho-Kyung Song, MD*, and
Dae Chul Jeong, MD
Department of *Anesthesiology and
Pediatrics, Our Lady of Mercy Hospital, College of Medicine, The Catholic University of Korea, Inchon, South Korea
Address correspondence and reprint requests to Ho-Kyung Song, MD, Department of Anesthesiology, Our Lady of Mercy Hospital, College of Medicine, The Catholic University of Korea, #665 Pupyung-Dong, Pupyung-Gu, Inchon, S. Korea 403-720. Address e-mail to song{at}olmh.cuk.ac.kr
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Abstract
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IV anesthetics may inhibit proper immune responses and further compromise an already depressed defense system. To assess the possible role of propofol on human immune function in sepsis, we studied cytotoxicity, and apoptosis of mononuclear cells (MNCs). Peripheral blood MNCs were preincubated in 1 µg/mL of lipopolysaccharide (LPS) and then reincubated in different concentrations of propofol (1 µg/mL, 5 µg/mL, 10 µg/mL, or 50 µg/mL). To determine cytotoxicity, lactate dehydrogenase release was assayed by mixing MNCs (4 x 105/100 µL) with K-562 tumor cells as target cells (1 x 104/100 µL)(E: T ratio of 40:1). Apoptosis was determined by measuring the annexin positive cells using flow cytometry. Cytotoxicity and apoptosis of LPS-treated MNCs were unchanged by clinically acceptable concentrations of propofol (1 µg/mL, 5 µg/mL, and 10 µg/mL). However, significant differences were observed in cytotoxicity (P = 0.004) and apoptosis (P = 0.002) with propofol 50 µg/mL. By gating MNCs, we found that lymphocyte apoptosis was significantly increased at 50 µg/mL of propofol, but monocytes were unaffected (P = 0.02). In terms of cytotoxicity and apoptosis, propofol allowed MNCs to retain their cytotoxicity in septic conditions by protecting immune cells from apoptosis.
IMPLICATIONS: Propofol at acceptable therapeutic concentrations, and under experimentally contrived septic conditions, did not affect the cytotoxic activity of mononuclear cells or the apoptosis level of mononuclear cells, lymphocytes, and monocytes from peripheral blood.
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Introduction
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Endotoxemia and sepsis are common problems particularly in intensive care units, and where there is immune suppression due to surgery or anesthetics, which increase perioperative morbidity and mortality from infection in susceptible patients (1–3). Impairment of the immune response is generally a consequence of reduced cell-mediated immune responses, which are associated with reduced lymphocyte responsiveness to mitogen, natural killer (NK) cell cytotoxicity, mixed lymphocyte responses, or inhibited phagocytic mononuclear cells (MNCs) function (4–6). In septic conditions, numerous proinflammatory mediators unregulate the activation of apoptosis. Moreover, apoptosis-induced lymphocyte loss is implicated in cellular immune suppression because lymphocytes are essential components of the defense system against invading microorganisms (7).
Some anesthetics that have been reported to impair various aspects of immune function are nevertheless administered for several days when prolonged sedation is indicated. It is possible that the use of these drugs in critically ill patients may further compromise an already depressed host-defense mechanism and may contribute to secondary infections (8,9). Propofol is a lipid-formulated anesthetic with an attractive pharmacokinetic and safety profile. However, little information is available about its effects on immune function.
To assess the possible role of propofol on human immune function in sepsis, we investigated the cytotoxic activity of MNCs on K-562 target cells and evaluated the apoptosis of lymphocytes and monocytes after they had been preincubated with lipopolysaccharide (LPS).
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Methods
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After obtaining approval from the Institutional Ethics Committee, informed consent was obtained from each participant. Twenty milliliters of peripheral venous blood was drawn from 10 healthy volunteers into heparinized tubes and immediately mixed with an equal volume of phosphate buffered saline (PBS). MNCs were separated by density gradient centrifugation after the blood sample had been layered on Ficoll-Paque solution (Pharmacia LKB Biotechnology Inc, Piscataway, NJ). The cells were washed twice with PBS and then once with tissue culture medium (RPMI 1640, GibcoBRL, Grand Island, NY). After checking cell viability using the trypan blue dye exclusion test, the MNCs (3–4 x 107 cells/mL) were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (GibcoBRL), 2 mM of L-glutamine, and 100 U/mL of streptomycin-excluding phenol red and sodium bicarbonate.
MNCs were then preincubated in 1 µg/mL of LPS (serotype: E. coli 055:B5, Sigma Co., St Louis, MO) for 5 h at 37°C in a humid atmosphere containing 5% CO2. After centrifugation, the separated MNCs were reincubated with various concentrations of propofol, and MNCs without the addition of propofol served as control. In the experimental groups, propofol at 1 µg/mL, 5 µg/mL, or 10 µg/mL was added to the culture media for 20 h. These concentrations represent the clinically acceptable therapeutic concentration. In one group, propofol 50 µg/mL was added for 20 h (toxic concentration).
Cytotoxic Study
To determine the cytotoxic activity of MNCs on the K-562 tumor cell line (ATCC, Rockville, MD), we used a modified lactate dehydrogenase (LDH) release assay. MNCs as effector cells, at a concentration of 4 x 105/100 µL in culture medium, were mixed with K-562 cells, as target cells, at a concentration of 1 x 104/100 µL, resulting in an effector-target ratio of 40:1. Each sample was determined in triplicate. The assay was performed in 96-hole well U-bottomed culture plates, which were incubated for 4 h at 37°C in a 5% CO2 humid atmosphere. Culture media was used for the spontaneous LDH assay, and Triton X-100 solution was added to the media for the maximum LDH assay. One-hundred microliters of LDH substrate mixture (Roche Diagnostics GmbM Mannheim, Germany) was added to 100 µL of supernatant from each well, and a microtiter plate reader (Molecular Devices Co., Sunnyvale, CA) was used to evaluate changes in the absorbance. The percentage of cytotoxicity was calculated after correcting for LDH release from MNCs using the formula:
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LDH release activity resulting from cells at each defined concentration of propofol.
- LDHexperimental: release resulting from the co-culturing of effector cells and target cells.
- LDHeffector cells: release resulting from the culturing effector cells separately.
- LDHspontaneous: release resulting from the culturing of K-562 cells separately (low control).
- LDHmaximal: release resulting from the total lysis of K-562 cells by Triton X-100 (high control).
Apoptosis Study
Flow cytometry was performed to estimate the apoptosis of MNCs, lymphocytes, and monocytes at each propofol concentration. The translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early event in apoptosis, and the binding of annexin V-fluorescein isothiocyanate (Roche Diagnostics GmbH) to phosphatidylserine in a Ca2+-dependent manner is used as a sensitive measure of apoptosis. Moreover, dual staining with propidium iodide (PI) enables membrane-disrupted cells to be readily distinguished because cells that have lost membrane integrity may also stain positively with annexin V. LPS-treated MNCs, which were cultured in different concentrations of propofol, were washed with PBS and centrifuged for 10 min at 1500 rpm. Cells 1 x 106 were then resuspended in 100 µL of staining solution and incubated for 10–15 min at 15–25°C. Staining solution was prepared by prediluting 20 µL of annexin V-fluorescein labeling reagent in 1000 µL of HEPES buffer and adding 20 µL of PI. According to the cell density, 0.4–0.8 mL of binding-buffer was added before analyzing the cells by flow cytometry (EPICS XL-MCL, Beckman Coulter, Miami, FL). MNCs were gated roughly into lymphocytes and monocytes. To discriminate between the two, gating was performed according to granularity and cell size using side and forward scattering characteristics (10).
Data are reported as mean ± SD and were analyzed by Friedman repeated-measures analysis of variance on ranks equipped with Sigma-Stat (version 2.03) from SPSS (St. Louis, MO) to compare the lymphocyte and monocyte groups. Cytotoxic or apoptotic differences between concentrations were analyzed by analysis of variance followed by the Dunnett method for multiple comparisons. Significance was accepted at P < 0.05.
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Results
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The cytotoxic activity of MNCs, as determined by LDH release from K-562 tumor cells, was dependent on propofol concentration (Fig. 1). Cytotoxicity percentage was unchanged at concentrations of 1 µg/mL, 5 µg/mL, and 10 µg/mL but was significantly decreased at 50 µg/mL versus the control (12.8% ± 11.4% versus 29.1% ± 11.9%; P = 0.004).
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Figure 1. The cytotoxic activity of mononuclear cells, as determined by lactate dehydrogenase release from K-562 tumor cells, showed that the values were dependent on propofol concentration (P = 0.004). Values are mean ± SD. *P < 0.05 versus control.
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The apoptosis of MNCs, lymphocytes, and monocytes was evaluated by using quantitative flow cytometry. The percent of total MNCs death was significantly increased at a propofol concentration of 50 µg/mL (11.1% ± 5.9%) as compared with the lower propofol concentrations (0.07% ± 0.5% at 5 µg/mL and 1.6% ± 3.0% at 10 µg/mL; P < 0.05) or with the control that did not contain propofol (P = 0.001). The percentage of apoptotic MNCs, defined as the percentage of annexin positive cells, was unchanged by 1–10 µg/mL of propofol versus the control value. However, apoptosis was significantly increased by propofol 50 µg/mL (P = 0.002)(Fig. 2). After differentiating between lymphocytes and monocytes by gating, it was found that apoptosis levels were different in the two cells types (P = 0.02). Propofol did not affect the apoptosis of monocytes at any propofol concentration, but apoptosis was significantly increased in lymphocytes at 50 µg/mL versus the control (P < 0.05) (Fig. 3).
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Figure 2. Effect of propofol on lipopolysaccharide-treated MNCs apoptosis at different concentrations. MNCs = mononuclear cells; ppf-0 = without addition of propofol for control; ppf-5 = 5 µg/mL of propofol was added to the culture media. Data are expressed as mean ± SD. *P < 0.05 versus ppf-0 and ppf-5.
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Figure 3. Effect of different concentration of propofol on lipopolysaccharide-treated monocytes and lymphocytes apoptosis. ppf-0 = without addition of propofol for control; ppf-5 = 5 µg/mL of propofol was added to the culture media. Data are expressed as mean ± SD. *P < 0.05 versus ppf-0 and ppf-5 of the lymphocytes group.
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Discussion
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IV anesthetics may cause transient aberrations in the immune systems of animals and humans (8–11). In the present study, we evaluated the immune responses of MNCs with respect to the cytotoxic activity on K-562 cells and apoptosis under experimental septic conditions induced by LPS. Because propofol was found to decrease the cytotoxic activity of MNCs only at a concentration of 50 µg/mL, which is more than the clinical range, our results suggest that propofol does not affect the cytotoxic activity of MNCs at clinically acceptable concentrations. Moreover, MNCs and lymphocytes apoptosis increased only at this higher propofol concentration, demonstrating that propofol does not induce MNCs or lymphocyte loss in septic conditions, providing it is administered in the clinically acceptable range.
It has been postulated that adequate early cytotoxic activity by the cell-mediated immune system against infections is an essential feature of host-defense. Propofol is a relatively safe drug from the immunological viewpoint. In addition to attenuating both or either of pro- and antiinflammatory cytokine responses to inflammation (12), propofol did not depress T-lymphocyte proliferation (13) or leukocyte function (8,9) compared with thiopental and etomidate and maintained the microbicidal function of alveolar macrophage during anesthesia comparing an inhaled anesthetic, such as isoflurane (14). Moreover, data from an endotoxin-induced septic shock model suggested that propofol might be a beneficial treatment against sepsis by protecting animals from metabolic acidosis and thus reducing the mortality rate (15,16). In the present study, we similarly demonstrated that propofol, even after 20 hours of incubation, does not have a harmful effect on the cytotoxic activity of MNCs in septic conditions.
MNCs are a heterogeneous population that includes NK cells, lymphocytes, and monocytes. The effector cells of cell-mediated natural cytotoxicity are NK cells, and K-562 tumor cells are the most sensitive target cells of NK cells (17). However, in the present study, we evaluated the cytotoxic activity of MNCs without separating NK cells from NK-like cells because the goal of this study was to assess the cytotoxicity of NK and NK-like cells on K-562 tumor cells after they had been exposed to propofol under septic conditions. This technique also has the benefit of retaining cells and preventing unintended stimulations during the procedure, i.e., disease-related or medications that might have affected the results (4).
In septic conditions, apoptosis has been identified as an important cause of lymphocyte cell death. Although it is not possible to know whether apoptosis is beneficial or detrimental to host survival in sepsis, the apoptosis-induced loss of lymphocytes may be responsible for immune depression. Host response to sepsis represents a balance between proinflammatory and compensatory antiinflammatory factors. Moreover, the loss of a proper balance between these two can result in organ dysfunction or death. In the case of the hyperinflammatory state, apoptosis may be beneficial to the host by eliminating lymphocytes that produce excessive proinflammatory cytokines, thus improving organ function and survival. Conversely, lymphocyte apoptosis may be harmful in sepsis by depleting the lymphocytes that are essential for defense against invading microorganisms (7), which may hamper the ability of the septic patient to eradicate infection and predispose the individual to secondary infection, also leading to multiple organ dysfunction (7). In fact, a reduction in the number of circulating lymphocytes is most frequently observed in sepsis, which increases the risk of nosocomial infection (18) and mortality rate in intensive care units (19). Also, the prevention of lymphocyte apoptosis is associated with improved survival in a murine model (20). However, the protection from apoptosis afforded by propofol showed a time-dependent decrease (21). The mechanism of its beneficial effect has been explained by the potent antioxidant effect of propofol on hydrogen peroxide, hydroxyl radicals, and superoxide, which are induced by tissue or cell injury (22).
In the present study, we treated MNCs with LPS initially to activate them immunologically and reincubated the cells in propofol-containing culture media for 20 hours. As a result, we found that the clinically acceptable range of propofol concentration did not induce further lymphocyte apoptosis or did not affect the MNCs cytotoxic activity versus the control cells. Therefore, the reduced cytotoxic activity of MNCs at the toxic range of propofol concentration could be explained by the increased apoptosis of lymphocytes.
Patients in sepsis experience high levels of stress because of pain and anxiety in addition to their physiologic and pathologic responses to sepsis. The need for an adequate level of sedation in these patients has been recently accepted, but there are no answers as to which drugs should be used. From the point of perioperative immune depression, our study suggests that propofol is as a relatively safe drug for the sedation of intensive care unit patients and for those who require assisted ventilation while in an already immune-compromised state.
The limitations of this study, which should be considered in any future study, include whether preventing loss of circulating MNCs or lymphocytes from apoptosis, and thus maintaining cytotoxicity, could be beneficial to host-defense in sepsis. Because the number of peripheral blood cytotoxic NK and NK-like cells would be rapidly increased due to recruitment from noncirculation sites, there are many factors that could be related to the total immune responses in septic patients. In addition, the mechanisms and the duration of the cellular protection afforded by propofol with respect to its antioxidant effects are unknown. In conclusion, this study demonstrated that propofol, at clinically acceptable concentrations, did not alter the extents of apoptosis of lymphocytes and MNCs and thus maintained their cytotoxic potencies in septic conditions.
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Acknowledgments
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The authors are grateful to Chul Hee Lee and Mi Young Yum, Department of Clinical Research Laboratory, Our Lady of Mercy Hospital, Inchon, South Korea, for their technical assistance.
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Accepted for publication November 24, 2003.
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Abstract
Introduction
