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
IV anesthetics may inhibit proper immune responses
and further compromise an already depressed defense
system. To assess the possible role of propofol on hu-
man immune function in sepsis, we studied cytotoxic-
ity, and apoptosis of mononuclear cells (MNCs). Pe-
ripheral blood MNCs were preincubated in 1 ␮g/mL of
lipopolysaccharide (LPS) and then reincubated in dif-
ferent concentrations of propofol (1 ␮g/mL, 5 ␮g/mL,
10 ␮g/mL, or 50 ␮g/mL). To determine cytotoxicity,
lactate dehydrogenase release was assayed by mixing
MNCs (4 ⫻ 105/100 ␮L) with K-562 tumor cells as target
cells (1 ⫻ 104/100 ␮L)(E: T ratio of 40:1). Apoptosis was
determined by measuring the annexin positive cells
E
ndotoxemia 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 re-
sponses, which are associated with reduced lympho-
cyte responsiveness to mitogen, natural killer (NK)
cell cytotoxicity, mixed lymphocyte responses, or in-
hibited phagocytic mononuclear cells (MNCs) func-
tion (4 – 6). In septic conditions, numerous proinflam-
matory mediators unregulate the activation of
apoptosis. Moreover, apoptosis-induced lymphocyte
loss is implicated in cellular immune suppression be-
cause lymphocytes are essential components of the
defense system against invading microorganisms (7).
Accepted for publication November 24, 2003.
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@olmh.cuk.ac.kr.
DOI: 10.1213/01.ANE.0000112317.68730.B0
1724 Anesth Analg 2004;98:1724–8
using flow cytometry. Cytotoxicity and apoptosis of
LPS-treated MNCs were unchanged by clinically ac-
ceptable concentrations of propofol (1 ␮g/mL, 5 ␮g/
mL, and 10 ␮g/mL). However, significant differences
were observed in cytotoxicity (P ⫽ 0.004) and apoptosis
(P ⫽ 0.002) with propofol 50 ␮g/mL. By gating MNCs,
we found that lymphocyte apoptosis was significantly
increased at 50 ␮g/mL of propofol, but monocytes were
unaffected (P ⫽ 0.02). In terms of cytotoxicity and apo-
ptosis, propofol allowed MNCs to retain their cytotox-
icity in septic conditions by protecting immune cells
from apoptosis.
(Anesth Analg 2004;98:1724 –8)
Some anesthetics that have been reported to impair
various aspects of immune function are nevertheless
administered for several days when prolonged seda-
tion 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 cyto-
toxic activity of MNCs on K-562 target cells and eval-
uated the apoptosis of lymphocytes and monocytes
after they had been preincubated with lipopolysaccha-
ride (LPS).
Methods
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
©2004 by the International Anesthesia Research Society
0003-2999/04
ANESTH ANALG
2004;98:1724 –8
equal volume of phosphate buffered saline (PBS).
MNCs were separated by density gradient centrifuga-
tion 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 ⫻ 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 ␮g/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 rein-
cubated with various concentrations of propofol, and
MNCs without the addition of propofol served as
control. In the experimental groups, propofol at 1
␮g/mL, 5 ␮g/mL, or 10 ␮g/mL was added to the
culture media for 20 h. These concentrations represent
the clinically acceptable therapeutic concentration. In
one group, propofol 50 ␮g/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 as-
say. MNCs as effector cells, at a concentration of 4 ⫻
105/100 ␮L in culture medium, were mixed with
K-562 cells, as target cells, at a concentration of 1 ⫻
104/100 ␮L, 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 ␮L of supernatant from each well, and a
microtiter plate reader (Molecular Devices Co., Sunny-
vale, CA) was used to evaluate changes in the absor-
bance. The percentage of cytotoxicity was calculated
after correcting for LDH release from MNCs using the
formula:
% cytotoxicity
LDHexperimental ⫺ LDHeffector cells ⫺ LDHspontaneous
⫽ ⫻ 100
LDHmaximal ⫺ LDHspontaneous
LDH release activity resulting from cells at each
defined concentration of propofol.
ANESTHETIC PHARMACOLOGY SONG AND JEONG 1725
EFFECT OF PROPOFOL ON IMMUNE RESPONSE
• LDHexperimental: release resulting from the co-
culturing of effector cells and target cells.
• LDHeffector cells: release resulting from the cultur-
ing effector cells separately.
• LDHspontaneous: release resulting from the cultur-
ing 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 apo-
ptosis of MNCs, lymphocytes, and monocytes at each
propofol concentration. The translocation of phospha-
tidylserine 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 meas-
ure of apoptosis. Moreover, dual staining with pro-
pidium 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 ⫻ 106 were then resuspended in 100 ␮L of staining
solution and incubated for 10 –15 min at 15–25°C.
Staining solution was prepared by prediluting 20 ␮L
of annexin V-fluorescein labeling reagent in 1000 ␮L of
HEPES buffer and adding 20 ␮L 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.
Results
The cytotoxic activity of MNCs, as determined by
LDH release from K-562 tumor cells, was dependent
on propofol concentration (Fig. 1). Cytotoxicity per-
centage was unchanged at concentrations of 1 ␮g/mL,
5 ␮g/mL, and 10 ␮g/mL but was significantly de-
creased at 50 ␮g/mL versus the control (12.8% ⫾
11.4% versus 29.1% ⫾ 11.9%; P ⫽ 0.004).
1726 ANESTHETIC PHARMACOLOGY SONG AND JEONG ANESTH ANALG
EFFECT OF PROPOFOL ON IMMUNE RESPONSE 2004;98:1724 –8

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. 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 ␮g/mL
The apoptosis of MNCs, lymphocytes, and mono- of propofol was added to the culture media. Data are expressed as
cytes was evaluated by using quantitative flow cytom- mean ⫾ sd. *P ⬍ 0.05 versus ppf-0 and ppf-5.
etry. The percent of total MNCs death was signifi-
cantly increased at a propofol concentration of 50
␮g/mL (11.1% ⫾ 5.9%) as compared with the lower
propofol concentrations (0.07% ⫾ 0.5% at 5 ␮g/mL
and 1.6% ⫾ 3.0% at 10 ␮g/mL; P ⬍ 0.05) or with the
control that did not contain propofol (P ⫽ 0.001). The
percentage of apoptotic MNCs, defined as the percent-
age of annexin positive cells, was unchanged by 1–10
␮g/mL of propofol versus the control value. How-
ever, apoptosis was significantly increased by propo-
fol 50 ␮g/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 lympho-
cytes at 50 ␮g/mL versus the control (P ⬍ 0.05) (Fig.
3). 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 ␮g/mL
of propofol was added to the culture media. Data are expressed as
Discussion mean ⫾ sd. *P ⬍ 0.05 versus ppf-0 and ppf-5 of the lymphocytes
IV anesthetics may cause transient aberrations in the group.
immune systems of animals and humans (8 –11). In the
present study, we evaluated the immune responses of does not induce MNCs or lymphocyte loss in septic
MNCs with respect to the cytotoxic activity on K-562 conditions, providing it is administered in the clini-
cells and apoptosis under experimental septic condi- cally acceptable range.
tions induced by LPS. Because propofol was found to It has been postulated that adequate early cytotoxic
decrease the cytotoxic activity of MNCs only at a activity by the cell-mediated immune system against
concentration of 50 ␮g/mL, which is more than the infections is an essential feature of host-defense.
clinical range, our results suggest that propofol does Propofol is a relatively safe drug from the immuno-
not affect the cytotoxic activity of MNCs at clinically logical viewpoint. In addition to attenuating both or
acceptable concentrations. Moreover, MNCs and lym- either of pro- and antiinflammatory cytokine re-
phocytes apoptosis increased only at this higher sponses to inflammation (12), propofol did not de-
propofol concentration, demonstrating that propofol press T-lymphocyte proliferation (13) or leukocyte
ANESTH ANALG
2004;98:1724 –8
function (8,9) compared with thiopental and etomi-
date and maintained the microbicidal function of al-
veolar 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 treat-
ment against sepsis by protecting animals from meta-
bolic acidosis and thus reducing the mortality rate
(15,16). In the present study, we similarly demon-
strated that propofol, even after 20 hours of incuba-
tion, 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. Al-
though 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 re-
sponsible for immune depression. Host response to
sepsis represents a balance between proinflammatory
and compensatory antiinflammatory factors. More-
over, 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 bene-
ficial to the host by eliminating lymphocytes that pro-
duce excessive proinflammatory cytokines, thus im-
proving organ function and survival. Conversely,
lymphocyte apoptosis may be harmful in sepsis by
depleting the lymphocytes that are essential for de-
fense against invading microorganisms (7), which
may hamper the ability of the septic patient to eradi-
cate infection and predispose the individual to sec-
ondary infection, also leading to multiple organ dys-
function (7). In fact, a reduction in the number of
circulating lymphocytes is most frequently observed
in sepsis, which increases the risk of nosocomial in-
fection (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 ex-
plained by the potent antioxidant effect of propofol on
ANESTHETIC PHARMACOLOGY SONG AND JEONG 1727
EFFECT OF PROPOFOL ON IMMUNE RESPONSE
hydrogen peroxide, hydroxyl radicals, and superox-
ide, which are induced by tissue or cell injury (22).
In the present study, we treated MNCs with LPS
initially to activate them immunologically and reincu-
bated 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 phys-
iologic 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 peri-
operative 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 con-
sidered in any future study, include whether prevent-
ing loss of circulating MNCs or lymphocytes from
apoptosis, and thus maintaining cytotoxicity, could be
beneficial to host-defense in sepsis. Because the num-
ber 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 dura-
tion 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.
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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